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This is to certify that the
dissertation entitled

IMPACT OF BIOFILM FORMATION AND SUBLETHAL
INJURY OF LISTER/A MONOCYTOGENES ON TRANSFER
TO DELICATESSEN MEATS

presented by

LINDSEY ANN KESKINEN

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IMPACT OF BIOFILM FORMATION AND SUBLETHAL INJURY OF LISTERIA
MONOC YT OGENES ON TRANSFER TO DELICATESSEN MEATS

By

Lindsey Ann Keskinen

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY
Department of Food Science and Human Nutrition

2006

ABSTRACT

IMPACT OF BIOFILM FORMATION AND SUBLETHAL INJURY OF LISTERIA
MONOCYTOGENES ON TRANSFER TO DELICATESSEN MEATS

By
Lindsey Ann Keskinen

Presence of Listeria monocytogenes strains endemic to food processing
environments is presumably related to biofilm formation. Following exposure to various
environmental stresses, Listeria cells may be more prone to attach to surfaces. Due to
concerns regarding the potential impact of biofilm formation on Listeria cross-
contamination of ready-to-eat meats in delicatessens, a series of studies was conducted
to: (1) determine the ability of L. monocytogenes to form biofilms under various
temperatures and stress conditions present in food processing and retail environments, (2)
determine the effects of biofilm-forming ability on direct and sequential transfer rates for
L. monocytogenes from delicatessen slicers to ready-to-eat meats, (3) determine the
effects of environmental stress on direct and sequential transfer rates for L.
monocytogenes from delicatessen slicers to ready-to-eat meats, and (4) develop one or
mathematical models that can be used to predict the transfer rates for L. monocytogenes
during retail slicing of ready-to-eat meats. A total of 196 L. monocytogenes isolates were
assessed for biofilm formation at 22 and 4°C in Modified Welshimer’s Broth, as
measured by optical density (OD) of stained biofilms, while a subset of 26 food,
environmental and human clinical isolates were further assessed for biofilm formation
after exposure to common environmental stressors (starvation, cold-shock, chlorine
injury and acid injury). Only 5% of all isolates were strong biofilm-formers, forming

’biofilms with OD values two standard deviations above the mean, with 81% of strains

failing to produce detectable biofilms at 4°C. Prior injury of L. monocytogenes by
starvation and cold resulted in enhanced biofilm formation, while exposure to acid and
chlorine diminished subsequent biofilm formation. Cold— and chlorine-shock produced
statistically similar levels of injury, however the cultures were significantly different in
their abilities to form biofilms (mean OD chlorine-shock = 0.309, mean OD cold-shock =
1.457), showing that non-oxidative stresses common in the environment increase
likelihood of biofilm formation.

Thereafter, six of the identified strong and weak biofilm-forrning strains were
combined into two 3-strain cocktails. The cocktails (healthy, cold-shocked or chlorine-
injured) were used to inoculate stainless steel delicatessen slicer blades (106 CFU/blade).
After incubation for 6 and 24 h (22°C/~78% RH), the inoculated blades were attached to
a gravity-fed delicatessen slicer and used to generate 30 slices from retail chubs of roast
turkey breast or Genoa salami. Biofilm-forming ability, length of incubation on stainless
steel, and prior injury had no significant affect on transfer. Listeria was able to survive
physiological stress and contaminate at statistically similar levels to healthy cells.
Overall, significantly greater cumulative transfer to turkey (cumulative transfer = 4.2 log
CFU) than salami (cumulative transfer = 3.5 log CFU) was observed. Under all
conditions, L. monocytogenes was still present on the slicer after slicing.

These findings were then used to validate a predictive model in the form [CFU
(X) = kax] along with a program written in GWBasic. This model can be used if any two
of the following three values are known: (a) initial inoculum, (b) total bacteria transfer,
(c) bacteria fraction remaining on the blade after consecutive slicing, solving for each

‘ model parameter CFU (X), k, or a. The fit of the model ranged from R2 = 0.65 — 0.94.

ACKNOWLEDGEMENTS

I have been fortunate to have the support and guidance of a wonderful advisor,
Dr. Elliot Ryser, who has taken the time and effort to try to teach me to be a competent
researcher and writer. Without his encouragement, I would have nothing to show for the
last few years of research, because I would still be sitting in front of my computer trying
to figure out how to write my dissertation.

I also want to thank my committee members, past and present: Dr. Alden Booren,
Dr. Gary Burgess, Dr. Wesley Osburn, Dr. James Pestka, and Dr. Ewen Todd. Dr.
Booren was kind enough to step in as a committee member after Dr. Osburn left MSU,
and I am grateful that he was willing to take on the task. In addition to this, he allowed
me to conduct a portion of my research in the Meat Lab on 3rd floor Anthony—I know
that this was a concern for a number of people, and I appreciate his time and effort in
making sure that my research could be completed. Dr. Burgess stepped in at the last
minute to complete the modeling portion of this research project, and his assistance was
invaluable. Dr. Pestka has been particularly helpful at key times in my education—he
helped me find my first job at Neogen Corporation, and when I decided to apply for
graduate school he provided me with advice and encouraged me to pursue a Ph.D. Dr.
Todd played a key role in guiding this research, particularly back in the planning phases.
His time and wisdom are greatly appreciated.

My labmates and classmates have provided a great support system. I can’t
imagine a better group of people, and I am thankful to have walked away from graduate

' school with so many fiiends.

iv

Finally, I owe a huge debt of gratitude to my friends and family. Mom, Bill, C.J.,
Joe, Leslie, Vanessa, Raina and Dad are wonderful people, and I am glad that I was able
to attend graduate school so close to home and spend this time with them. Their help and
support was vital. My grandparents (Klecklers and Keskinens), my aunts, uncles and
cousins have given me so much encouragement—I have such a wonderful family. My
childhood friends helped me in so many ways through the past few years, especially
Sarah, Stephanie and Jay. My life over the past few years would have been very difficult

without all of their help and support.

TABLE OF CONTENTS

LIST OF TABLES ................................................................................... x
LIST OF FIGURES .............................................................................. xiii
KEY TO SYMBOLS AND ABBREVIATIONS ......................................................... xvii
INTRODUCTION .................................................................................. 1
CHAPTER 1 LITERATURE REVIEW ......................................................... 5
1.1 Listeria and Listeriosis ................................................................... 5
1.2 Listeriosis Outbreaks from Ready-to-Eat Meats ...................................... 8
1.3 Listeria Recalls ........................................................................... 9
1.4 Listeria monocytogenes in RTE Meats ............................................... 1 1
1.5 Listeria Risk Assessments for RTE Meats .......................................... 16
1.6 Strategies and regulations for decreasing the incidence of Listeriosis ........... 20
1.7 Listeria survival in the environment and on delicatessen slicers .................. 23
1.8 Prevalence of Listeria monocytogenes in food processing environments ....... 26
1.9 Bacterial transfer during food processing ............................................ 29
1.10 Bacterial transfer during retail food handling ....................................... 31
1.11 Bacterial transfer in the home ......................................................... 36
1.12 Listeria persistence on surfaces and biofilm formation ............................ 39
1.13 Predictive modeling ..................................................................... 45
1.14 Predictive modeling of bacterial growth ............................................. 45
1.15 Predictive modeling of bacterial transfer ............................................. 47
1.16 Goals of the current study .............................................................. 49
CHAPTER 2 VARIATION IN BIOFILM FORMATION BY LIST ERIA
MONOCYTOGENES STRAINS AT 4°C AND 22°C ....................................... 51
2.1 ABSTRACT .............................................................................. 52
2.2 INTRODUCTION ...................................................................... 53
2.3 MATERIALS AND METHODS ...................................................... 55
2.3.1 Listeria monocytogenes strains .................................................. 55
2.3.2 Culture preparation ............................................................... 55
2.3.3 Microtiter plate assay for biofilm formation .................................. 56
2.3.4 Measurement of cell surface hydrophobicity by hydrophobic interaction
chromatography ................................................................... 57
2.3.5 Statistical Analysis ................................................................ 58
2.4 RESULTS ................................................................................ 58
2.4.1 Biofilm formation by Listeria monocytogenes ................................. 58
2.4.2 Surface hydrophobicity of weak and strong biofilm formers ............... 73
2.5 DISCUSSION ........................................................................... 74

vi

CHAPTER 3 VARIATION IN BIOFILM FORMATION BY HEALTHY AND COLD-,

STARVE-, ACID-, AND CHLORINE-INJURED LISTERIA MONOCYTOGENES ....... 77
3.1 ABSTRACT .............................................................................. 78
3.2 INTRODUCTION ...................................................................... 79
3.3 MATERIALS AND METHODS ...................................................... 82
3.3.1 Listeria monocytogenes strains ..................................................... 82
3.3.2 Culture preparation .................................................................. 82
3.3.3 Acid injured ........................................................................... 85
3.3.4 Cold injured ........................................................................... 85
3.3.5 Cold starved ........................................................................... 85
3.3.6 Chlorine injured ...................................................................... 85
3.3.7 Quantification of injury .............................................................. 86
3.3.8 Microtiter plate assay for biofilm formation .................................................. 86
3.3.9 Statistical analysis .................................................................... 87

3.4 RESULTS ................................................................................ 87

3.5 DISCUSSION ........................................................................... 93

CHAPTER 4 IMPACT OF BIOFILM FORMING ABILITY ON TRANSFER OF
SURFACE-DRIED LISTERIA MONOC YT OGENES FROM KNIFE BLADES TO

ROAST TURKEY BREAST ...................................................................... 95
4.1 ABSTRACT .............................................................................. 96
4.2 INTRODUCTION ...................................................................... 97
4.3 MATERIALS AND METHODS ...................................................... 98

4.3.1 Listeria monocytogenes strains .................................................. 98
4.3.2 Preparation of turkey slurry ...................................................... 99
4.3.3 Culture preparation ............................................................... 99
4.3.4 Knife blades ...................................................................... 100
4.3.5 Knife blade inoculation ......................................................... 100
4.3.6 Standardization of cutting force and speed ................................... 101
4.3.7 Restructured roast turkey breast ................................................ 101
4.3.8 Transfer of L. monocytogenes from an inoculated grade 304 stainless steel
knife blades to uninoculated restructured roast turkey breast ............. 102
4.3.9 Quantification of L. monocytogenes on used and unused knife blades... 102
4.3.10 Cleaning and decontamination of knife blades .............................. 103
4.3.11 Evaluation of survival of L. monocytogenes on knife blades using confocal
scanning laser microscopy ...................................................... 103
4.3.12 Statistical analysis ............................................................... 105
4.4 RESULTS .............................................................................. 105
4.4.1 Listeria transfer from knife blades over time ................................ 105
4.4.2 Survival of L. monocytogenes on knife blades over time .................. 108
4.5 DISCUSSION .................................................................... 109

vii

CHAPTER 5 IMPACT OF BACTERIAL STRESS AND BIOFILM FORMING
ABILITY ON TRANSFER OF SURFACE-DRIED LISTERIA MONOC YT OGENES

DURING SLICING OF DELICATESSEN MEATS ...................................... 112
5.1 ABSTRACT ........................................................................... 113
5.2 INTRODUCTION ..................................................................... 1 14
5.3 MATERIALS AND METHODS ................................................... 116
5.3.1 Listeria monocytogenes strains ................................................ 116
5.3.2 Preparation of turkey slurry .................................................... 117
5.3.3 Culture preparation, uninjured cocktails ...................................... 117
5.3.4 Culture preparation, cold-injured cocktails .................................. 118
5.3.5 Culture preparation, chlorine-injured cocktails .............................. 118
5.3.6 Delicatessen slicer inoculation ................................................ 119
5.3.7 Delicatessen meats .............................................................. 120
5.3.8 L. monocytogenes transfer from an inoculated delicatessen blade to
uninoculated product ............................................................ 120
5.3.9 Quantification of L. monocytogenes on used and unused slicer blades... 121
5.3.10 Cleaning and decontaminating the slicer ..................................... 122
5.3.11 Evaluation of survival of L. monocytogenes on slicer blades using
confocal scanning laser microscopy .......................................... 122
5.3.12 Statistical analysis ............................................................... 123
5.4 RESULTS .............................................................................. 124
5.4.1 Transfer of surface-dried L. monocytogenes from an inoculated
delicatessen slicer blade to uninoculated product ........................... 124
5.4.2 Affect of biofilm forming ability, injury, incubation time and production
transfer of L. monocytogenes .................................................... 134
5.4.3 Survival of L. monocytogenes on slicer blades over time .................... 137
5.5 DISCUSSION .......................................................................... 138

CHAPTER 6 VALIDATION OF A PREDICTIVE MODEL FOR LISTERIA
MONOC YT OGENES TRANSFER DURING SLICING OF DELICATESSEN MEATS

.................................................................................................... 142
6.1 ABSTRACT ............................................................................. 143
6.2 INTRODUCTION ..................................................................... 144
6.3 MATERIALS AND METHODS ................................................... 146

6.3.1 Transfer coefficients for surface-dried, uninjured and injured L.
monocytogenes during slicing of turkey and salami ........................ 146
6.3.2 Predictive modeling of L. monocytogenes transfer during slicing of roast
turkey breast and salami ......................................................... 146
6.3.3 Predicting CF U on meat as a function of slice number (X) ................ 148
6.3.4 Fitting the equation to data (finding “k” and “a”) ........................... 148
6.3.5 Interpretation of fit results ...................................................... 149
6.4 RESULTS .............................................................................. 150
6.4.1 Predictive model for L. monocytogenes transfer during slicing of turkey
and salami using a mechanical slicer .......................................... 153
6.5 DISCUSSION .......................................................................... 160

viii

CONCLUSIONS AND FUTURE RECOMMENDATIONS ............................ 163

APPENDIX I: KNIFE TRANSFER DATA ................................................ 167
APPENDIX II: SLICER TRANSFER DATA ............................................. 178
APPENDIX III: SAMPLE MICROGRAPHS ............................................. 203
APPENDIX IV: GWBasic SCREENSHOTS ............................................... 206
BIBLIOGRAPHY ............................................................................... 208

ix

Table 1.1

Table 1.2

Table 1.3

Table 1.4

Table 2.1

Table 2.2

Table 2.3

Table 2.4

Table 3.1

Table 3.2

Table 3.3

Table 4.1

Table 5.1

Table 5.2

LIST OF TABLES

Prevalence (%) of L. monocytogenes in RTE meat and poultry products,

1990 — 2000 ..................................................................... 12
L. monocytogenes-positive luncheon meat samples .......................... 13
Growth of L. monocytogenes on RTE delicatessen meat .................... 15

Predicted relative risk rankings of Listeriosis among food categories for
three US. age-based subpopulations using median estimates of relative

predicted risks for Listeriosis on a per annum basis .......................... 18
L. monocytogenes strain information ........................................... 60
L. monocytogenes biofilm formation at 22°C by source ..................... 71
L. monocytogenes biofilm formation at 22°C by lineage .................... 72
L. monocytogenes biofilm formation at 22°C by serotype .................. 72
L. monocytogenes strains and sources .......................................... 84

Overall differences in L. monocytogenes injury and biofilm formation by
treatment ........................................................................... 88

Relative rankings of L. monocytogenes strains according to biofilm
forming ability ..................................................................... 92

Number of direct counts and positive enrichments for roast turkey breast
sliced with L. monocytogenes-contaminated knife blades after 6 and 24 h
................................................................................. 107

Number of direct counts and positive enrichments for salami sliced with
slicer blades contaminated with healthy, cold- or chlorine-injured L.
monocytogenes .................................................................. 1 32

Number of direct counts and positive enrichments for roast turkey breast
sliced with slicer blades contaminated with healthy, cold- or chlorine-
injured L. monocytogenes ...................................................... 133

Table 5.3

Table 6.1

Table Al.l

Table Al.2

Table A2.1

Table A2.2

Table A2.3

Table A2.4

Table A2.5

Table A2.6

Table A2.7

Table A2.8

Table A2.9

Table A2.l0

Cumulative log transfer of previously injured and uninjured L.
monocytogenes to delicatessen meat and percent injury at the time of
transfer ........................................................................... 134

Model predicted fraction of transfer of Listeria monocytogenes from
delicatessen slicers to delicatessen meat (fl) and environment (,5) by
product, biofilm forming ability, injury, and incubation time on stainless
steel blade ..................................................................... 155

Listeria monocytogenes transfer from knife blades to turkey (6 log
CF U/blade) ................................................................... 168

Listeria monocytogenes transfer from knife blades to turkey (8 log
CFU/blade) ................................................................... 173

Listeria monocytogenes transfer from slicer to turkey (strong biofilm

former/uninjured/6 h incubation) ........................................... 179
Listeria monocytogenes transfer from slicer to turkey (weak biofilm
former/uninjured/6 h incubation) .......................................... 180
Listeria monocytogenes transfer from slicer to turkey (strong biofilm
former/uninj ured/24 h incubation) ........................................ 181
Listeria monocytogenes transfer from slicer to turkey (weak biofilm
former/uninjured/24 h incubation) ........................................ 182
Listeria monocytogenes transfer from slicer to turkey (strong biofilm
former/cold-injured/6 h incubation) ....................................... 183
Listeria monocytogenes transfer from slicer to turkey (weak biofilm
former/cold-injured/6 h incubation) ....................................... 184
Listeria monocytogenes transfer from slicer to turkey (strong biofilm
former/cold-injured/24 h incubation) ..................................... 185
Listeria monocytogenes transfer from slicer to turkey (weak biofilm
former/cold-injured/24 h incubation) ..................................... 186
Listeria monocytogenes transfer from slicer to turkey (strong biofilm
former/chlorine-injured/6 h incubation) .................................. 187
Listeria monocytogenes transfer from slicer to turkey (weak biofilm
former/chlorine-injured/6 h incubation) .................................. 188

xi

Table A2.11

Table A2.12

Table A2.13

Table A2.14

Table A2.15

Table A2.16

Table A2.17

Table A2.18

Table A2.19

Table A2.20

Table A2.21

Table A2.22

Table A2.23

Table A2.24

Listeria monocytogenes transfer from slicer to turkey (strong biofilm
former/chlorine-injured/24 h incubation) ................................

Listeria monocytogenes transfer from slicer to turkey (weak biofilm
former/chlorine-injured/24 h incubation) ................................

Listeria monocytogenes transfer from slicer to salami (strong biofilm
former/uninjured/6 h incubation) ..........................................

Listeria monocytogenes transfer fi'om slicer to salami (weak biofilm
former/uninjured/6 h incubation) ..........................................

Listeria monocytogenes transfer from slicer to salami (strong biofilm
former/uninjured/24 h incubation) ........................................

Listeria monocytogenes transfer from slicer to salami (weak biofilm
former/uninjured/24 h incubation) ........................................

Listeria monocytogenes transfer from slicer to salami (strong biofilm
former/cold-injured/6 h incubation) .......................................

Listeria monocytogenes transfer from slicer to salami (weak biofilm
former/cold-injured/6 h incubation) .......................................

Listeria monocytogenes transfer from slicer to salami (strong biofilm
former/cold-injured/24 h incubation) .....................................

Listeria monocytogenes transfer from slicer to salami (weak biofilm
former/cold-injured/24 h incubation) .....................................

Listeria monocytogenes transfer from slicer to salami (strong biofilm
former/chlorine-injured/6 h incubation) ..................................

Listeria monocytogenes transfer from slicer to salami (weak biofilm
former/chlorine-injured/6 h incubation) ..................................

Listeria monocytogenes transfer from slicer to salami (strong biofilm
former/chlorine-injured/24 h incubation) ................................

Listeria monocytogenes transfer from slicer to salami (weak biofilm
former/chlorine-injured/24 h incubation) ................................

xii

189

190

191

192

193

194

195

196

197

198

199

200

201

202

Figure 1.1

Figure 1.2

Figure 1.3
Figure 1.4
Figure 1.5

Figure 2.1

Figure 2.2

Figure 2.3

Figure 3.1
Figure 3.2
Figure 3.3
Figure 3.4
Figure 3.5

Figure 4.1

Figure 4.2

LIST OF FIGURES

Listeria monocytogenes-related Class I recalls of delicatessen meat

products, 1994 — 2004 ........................................................... 10
Regulatory testing for Listeria monocytogenes in RTE products by
calendar year, 1990 — 2005 ...................................................... 22
Example of delicatessen meat slicer ........................................... 33
Example of delicatessen meat slicer designed for easier sanitation ....... 33
Stages in biofilm development ................................................. 40
Distribution of optical densities at 4°C for 196 L. monocytogenes isolate;
Distribution of optical densities at 22°C for 196 L. monocytogenes isolate:
Log relative hydrophobicity of weak and strong biofilm forming strains of
L. monocytogenes ................................................................ 73
L. monocytogenes biofilm formation by uninjured cells .................... 89
L. monocytogenes biofilm formation by acid injured cells ................. 89
L. monocytogenes biofilm formation by chlorine injured cells ............ 90
L. monocytogenes biofilm formation by cold injured cells ................. 90
L. monocytogenes biofilm formation by cold starved cells ................. 91

Instron 5565 electromechanical compression analyzer with modified upper
load cell for knife blades ........................................................ 101

Transfer of weak and strong biofilm forming strains of L. monocytogenes
from an inoculated knife blade (8 log CFU/blade; incubation = 6 and 24 h,
78 :t 2% RH/22°C) to roast turkey breast .................................... 106

xiii

Figure 4.3

Figure 5.1

Figure 5.2

Figure 5.3

Figure 5.4

Figure 5.5

Figure 5.6

Figure 5.7

Figure 5.8

Figure 5.9

Figure 5.10

Figure 5.11

Transfer of weak and strong biofilm forming strains of L. monocytogenes
from an inoculated knife blade (6 log CFU/blade; incubation = 6 and 24 h,
78 i 2% RH/22°C) to roast turkey breast .................................... 106

Contact areas of gravity fed delicatessen slicer .............................. 119

Transfer of healthy, strong biofilm forming L. monocytogenes from an
inoculated slicer blade (6 log CFU/blade; 6 and 24 h/78 :t 2% RH/22°C) to
uninoculated turkey .............................................................. 125

Transfer of healthy, weak biofilm forming L. monocytogenes from an
inoculated slicer blade (6 log CF U/blade; 6 and 24 h/78 :t 2% RH/22°C) to
uninoculated turkey .............................................................. 125

Transfer of healthy, strong biofilm forming L. monocytogenes from an
inoculated slicer blade (6 log CFU/blade; 6 and 24 h/78 i 2% RH/22°C) to
uninoculated salami ............................................................. 126

Transfer of healthy, weak biofilm forming L. monocytogenes from an
inoculated slicer blade (6 log CFU/blade; 6 and 24 h/78 i 2% RH/22°C) to
uninoculated salami ............................................................. 126

Transfer of chlorine-inj ured, strong biofilm forming L. monocytogenes
from an inoculated slicer blade (6 log CFU/blade; 6 and 24 h/78 i 2%
RH/22°C) to uninoculated turkey ............................................. 127

Transfer of chlorine-injured, weak biofilm forming L. monocytogenes
from an inoculated slicer blade (6 log CFU/blade; 6 and 24 h/78 :I: 2%
RH/22°C) to uninoculated turkey ............................................. 127

Transfer of chlorine-inj ured, strong biofilm forming L. monocytogenes
from an inoculated slicer blade (6 log CFU/blade; 6 and 24 h/78 :I: 2%
RI-I/22°C) to uninoculated salami ............................................. 128

Transfer of chlorine-injured, weak biofilm forming L. monocytogenes
from an inoculated slicer blade (6 log CF U/blade; 6 and 24 h/78 d: 2%
RH/22°C) to uninoculated salami ............................................. 128

Transfer of cold-injured, strong biofilm forming L. monocytogenes from
an inoculated slicer blade (6 log CFU/blade; 6 and 24 h/78 i 2%
RH/22°C) to uninoculated turkey ............................................. 129

Transfer of cold-injured, weak biofilm forming L. monocytogenes from an
inoculated slicer blade (6 log CFU/blade; 6 and 24 h/78 i 2% RI-I/22°C) to
uninoculated turkey .............................................................. 129

xiv

Figure 5.12

Figure 5.13

Figure 5.14

Figure 5.15

Figure 5.16

Figure 5.17

Figure 6.1

Figure 6.2

Figure 6.3

Figure 6.4

Figure 6.5

Figure 6.6

Figure 6.7

Figure 6.8

Transfer of cold-injured, strong biofilm forming L. monocytogenes from
an inoculated slicer blade (6 log CFU/blade; 6 and 24 h/78 :1: 2%
RH/22°C) to uninoculated salami ............................................. 130

Transfer of cold-injured, weak biofilm forming L. monocytogenes from an
inoculated slicer blade (6 log CFU/blade; 6 and 24 h/78 d: 2% RH/22°C) to
uninoculated salami ............................................................. 130

Cumulative log transfer of L. monocytogenes to delicatessen meat by
product and incubation time .................................................... 135

Cumulative log transfer of L. monocytogenes to delicatessen meat by
product and injury treatment ................................................... 135

Cumulative log transfer of L. monocytogenes to delicatessen meat by
product and cocktail ............................................................. 136

Percent injury of L. monocytogenes at the time of transfer to delicatessen
meat by previous injury treatment and cocktail .............................. 136

Cumulative L. monocytogenes transfer from an inoculated slicer blade (6
log CFU/blade) to turkey and salami .......................................... 151

Cumulative transfer by strong and weak biofilm forming L.
monocytogenes from an inoculated slicer blade (6 log CFU/blade) to
turkey and salami ................................................................ 151

Cumulative L. monocytogenes transfer from an inoculated slicer blade (6
log CFU/blade; 6 and 24 h/78 i 2% RH/22°C) to turkey and salami. . ..152

Cumulative transfer by uninjured and cold-injured L. monocytogenes from
an inoculated slicer blade (6 log CF U/blade) to turkey and salami ....... 152

Example: GWBasic output for turkey and salami sliced using a slicer
blade inoculated with weak biofilm forming L. monocytogenes (106
CFU/blade) ........................................................................ 154

Plotted output using GWBasic for assessing L. monocytogenes transfer
from an inoculated slicer blade (6 log CFU/blade) to salami... ............156

Plotted output using GWBasic for assessing L. monocytogenes transfer
from an inoculated slicer blade (6 log CF U/blade) to turkey .............. 156

Plotted output using GWBasic for assessing transfer of strong biofilm-

forming L. monocytogenes from an inoculated slicer blade (6 log
CFU/blade) to turkey and salami .............................................. 157

XV

Figure 6.9

Figure 6.10

Figure 6.11

Figure 6.12

Figure 6.13

Figure A3.1

Figure A3.2

Figure A3.3

Figure A3.4

Figure A4.1

Figure A4.2

Plotted output using GWBasic for assessing transfer of weak biofilm-
forming L. monocytogenes from an inoculated slicer blade (6 log
CFU/blade) to turkey and salami .............................................. 157

Plotted output using GWBasic for assessing transfer of uninjured L.
monocytogenes from an inoculated slicer blade (6 log CFU/blade) to
turkey and salami ................................................................ 158

Plotted output using GWBasic for assessing transfer of cold-injured L.
monocytogenes from an inoculated slicer blade (6 log CF U/blade) to
turkey and salami ................................................................ 158

Plotted output using GWBasic for assessing transfer of L. monocytogenes
from an inoculated slicer blade (6 log CFU/blade; 6 M78 1 2% RH/22°C)
to turkey and salami ............................................................ 159

Plotted output using GWBasic for assessing transfer of L. monocytogenes
from an inoculated slicer blade (6 log CFU/blade; 24 h/78 d: 2% RH/22°C)
to turkey and salami ............................................................. 159

Live/Dead micrograph of Listeria monocytogenes (strong biofilm formers,
cold-injured) afier 6 h of incubation on dry stainless steel ................ 204

Live/Dead micrograph of L. monocytogenes (strong biofilm formers,
chlorine-injured) after 1 h of incubation on dry stainless steel ............ 204

Live/Dead micrograph of L. monocytogenes (strong biofilm formers, cold-
injured) after 6 h of incubation on dry stainless steel ....................... 205

Live/Dead micrograph of L. monocytogenes (weak biofilm formers, cold-
injured) after 24 h of incubation on dry stainless steel ...................... 205

An example of the GWBasic modeling program ............................ 207
GWBasic modeling program output when used to model transfer of

Listeria monocytogenes (108 CF U/blade initial inoculum level) to
delicatessen meat ................................................................ 207

xvi

aw

AISI

AN OVA

ASTM

BATH

BPB

CDC

CFSAN

CFU

CLSM

cm

CT

CY

DNA

F A0

FDA

FSIS

GLM

KEY TO SYMBOLS AND ABBREVIATIONS

Water activity

American Iron and Steel Institute

Analysis of Variance

American Society for Testing and Materials
Bacterial Adhesion to Hydrocarbons
Butterfield’s Phosphate Buffer

Centers for Disease Control and Prevention
Center for Food Safety and Applied Nutrition
Colony Forming Unit(s)

Confocal scanning laser microscopy
centimeter(s)

Composite tissue

Calendar Year

day(s)

Deoxyribonucleic Acid

Food and Agriculture Organization of the United Nations
Food and Drug Administration

Food Safety Inspection Service

graIn(S)

General Linear Model

xvii

HACCP

HIC

min

MOX

mTPA

mTPAN

OD

PBS

PFGE

PPm
RAPD

RTE

SAS

TPA

TPAN

TSA-YE

hour(s)

Hazard Analysis and Critical Control Point
Hydrophobic Interaction Chromatography
minute(s)

milliliter(s)

millimeter(s)

Modified Oxford Agar

Modified Welshimer’s Broth

Modified Tryptose Phosphate Agar

Modified Tryptose Phosphate Agar with 4.5% Sodium Chloride
(N aCl)

Optical Density

Phosphate Buffered Saline

Pulsed-field Gel Electrophoresis

parts per million

Random Amplicification of Polymorphic DNA
Relative Humidity

Ready-to-eat

second(s)

Statistical Analysis Systems

Tryptose Phosphate Agar

Tryptose Phosphate Agar with 4.5% Sodium Chloride (NaCl)

Trypticase Soy Agar with 0.6% Yeast Extract

xviii

TSB-YE
US
US-DHHS
USDA
WHO

pl

um

Trypticase Soy Broth with 0.6% Yeast Extract

United States of America

United States Department of Health and Human Services
United States Department of Agriculture

World Health Organization

microliter(s)

micron(s)

xix

INTRODUCTION

Listeria monocytogenes is the leading microbiological cause of Class I recalls of
cooked or ready-to-eat (RTE) meat products in the United States. Contamination of these
products usually occurs afier processing, prior to packaging (Levine et al., 2001).
Delicatessen-sliced RTE turkey meat has been involved in 3 outbreaks of listeriosis since
2000, resulting in a total of 92 cases of illness, including 11 deaths and 6 miscarriages
(CDC, 2001; CDC, 2002; Olsen et al., 2005). In a subsequent survey of 31,705 RTE
products sampled from eight RTE product categories (fresh soft cheeses, bagged salads,
blue-veined cheeses, mold-ripened cheeses, seafood salads, smoked seafood, luncheon
meats and deli salads), 577 samples were positive for L. monocytogenes (Gombas et al.,
2003). Of the 9,199 luncheon meat samples taken as part of the study, 82 were L.
monocytogenes-positive, giving a prevalence rate of 0.89% (Gombas et al., 2003). Most
positive samples (75.6%) contained less than 1 CF U/g (Table 1.2). Most importantly,
luncheon meats that were store-packaged were more frequently contaminated with L.
monocytogenes (6.8 times as likely to be contaminated) than manufacturer-packaged
meats.

The higher prevalence of L. monocytogenes in delicatessen meat sliced at retail
strongly suggests that the delicatessen slicer is an important vehicle for cross-
contarnination of products. In order for cross-contamination to occur, L. monocytogenes
must survive on the surface for a period of time between the slicing of various products.
Prior to its introduction to the slicer surface, L. monocytogenes may be exposed to

refiigeration temperatures, low pH (fermented meats and cheeses), limited available

water, and sanitizers. While on the slicer surface, L. monocytogenes exposure to
desiccation and sanitizers is likely. These stresses have been shown to alter the
sensitivity of L. monocytogenes to other subsequent stresses, sometimes making it more
difficult to eradicate from the environment (Lou and Yousef, 1997; Koutsoumanis et al.,
2003; Koutsorunanis and Sofos, 2004; Gravesen et al., 2005; Moorman etal., 2005).
Listeria monocytogenes is able to become established in niches in food processing
environments, where certain strains have been found to persist for years (Tompkin, 2002;
Lunden et al., 2001). Equipment such as peelers, slicers, dicers, and conveyor belt lines
are not always designed in a way that facilitates effective cleaning and sanitizing. In a
survey of L. monocytogenes contamination in poultry processing environments, several
food contact surfaces were persistently contaminated with the same strains, including
slicer blades and blade covers, dicing machine blades and blade covers, a conveyor belt,
and a spiral conveyor in a freezer (Lunden et al., 2003). This inability to adequately
clean surfaces allows L. monocytogenes to persist in the environment and form biofilms
on food contact surfaces where the pathogen can be potentially transferred to RTE foods.
Persistent strains play an important role in contamination of RTE foods with such
strains 8 times more likely to contaminate finished product than transient strains (Lunden
et al., 2003). Lunden et al. (2000) reported that the same pulsed-field gel electrophoresis
(PFGE) type of L. monocytogenes was transferred to three different processing plants in a
dicing machine used in the three plants. The persistent strain was then tested for
adherence to stainless steel in broth culture at 25°C for 1, 2, and 72 h, along with three
non-persistent strains of L. monocytogenes isolated from the third plant. The persistent

strain was significantly more adherent than the non-persistent strains, a trend that has also

been correlated with biofilm formation in other studies (Lunden et al., 2000; Norwood
and Gilmour, 1999; Borucki et al., 2003).

Increased awareness of the potential cost and risk of multi-state listeriosis
outbreaks spurred the development of risk assessments for Listeria by United States
government agencies (FDA/USDA/CDC, 2003). Although 3 categories of RTE foods
showed a higher prevalence of L. monocytogenes contamination (patés, smoked seafoods,
and fresh, soft cheeses), deli meats ranked first in relative risk, due to their higher per
capita consumption which, in turn, leads to a wider exposure of the public to L.
monocytogenes (FDA/USDA/CDC, 2003). Overall, 14 cases of listeriosis are predicted
to occur for every 100 million servings of deli meat consumed (FDA/USDA/CDC, 2003).
While this may seem like a small number of cases, the mortality rate for listeriosis is
high, and listeriosis is the second most costly foodbome illness in the United States, with
an estimated annual cost of $2.3 billion in medical expenses and lost productivity,
including death (Frenzen, 2003). Listeriosis has the highest hospitalization rate and the
second highest number of fatalities of any foodborne illness tracked by the Centers for
Disease Control and Prevention in FoodNet (F DA/FSIS/CDC, 2003). The Healthy
People 2010 national health objective for listeriosis was to reduce the number of cases to
2.5 per 1,000,000 people by 2005. However, the number of listeriosis cases was 3.0 per
1,000,000 people in 2005 with the targeted goal not yet achieved (Reuters, 2006).

The research presented in this dissertation was conducted in response to Listeria
transfer rates being identified as a key informational gap in the Listeria Risk Assessment
published by the US federal government (FDA/USDA/CDC, 2003). Data obtained from

the research was used to validate the utility of a model developed by Vorst et al. (2005—

Ch.5) in predicting transfer of L. monocytogenes after exposure to bacterial stress (cold-
injury and chlorine-injury) and prolonged (6 and 24 h) desiccation on stainless steel to
turkey and salami. Additionally, the model was also tested for its ability to predict L.
monocytogenes transfer based on strain persistence and biofilm formation. The
underlying hypothesis for this study was that strain persistence would have an affect on
the survival and transfer of L. monocytogenes to delicatessen meats, particularly after

prolonged desiccation on stainless steel.

CHAPTER 1

LITERATURE REVIEW

1.1 Listeria and Listeriosis

Listeria is a genus of Gram-positive, non-spore forming, short rod-shaped,
facultatively anaerobic bacteria that are catalase positive, oxidase negative, methyl red
positive, and Voges-Proskauer positive (Swaminathan, 2001 ). The genus is comprised of
six species: L. monocytogenes, L. innocua, L. ivanovii, L. seeligeri, L. welshimeri, and L.
grayi. Species can be differentiated by hemolytic activity—L. ivanovii is strongly beta-
hemolytic, L. monocytogenes and L. seeligeri are weakly beta-hemolytic; and L. innocua,
L. welshimeri and L. grayi are non-hemolytic. Listeria monocytogenes can be
differentiated from L. ivanovii and L. seeligeri by its positive result for the CAMP-test
with Staphylococcus aureus and negative reaction with Rhodococcus equi on sheep blood
agar, and its inability to produce acid from D-xylose (Rocourt, 1999; Swarninathan,
2001). Listeria monocytogenes is the primary human pathogen within the genus and is of
concern to food processors due to its high fatality rate, growth at refrigeration
temperatures, resistance to salt and acid, and ability to persist in food processing
environments for up to 12 years (Lunden et al., 2001).

Listeriosis is the disease caused by infection with L. monocytogenes. Groups at
particular risk for listeriosis include the elderly, immunocompromised adults, pregnant
women, and neonates. Listeriosis is rare—out of an estimated 76 million cases of
foodbome illnesses per year in the United States, only about 2500 are caused by L.

‘ monocytogenes. However, the mortality rate for listeriosis is high, with those 2500 cases

resulting in an estimated 500 deaths every year. Nearly 90% of all reported cases of
listeriosis result in hospitalization (Mead et al., 1999). Due to its severity, invasive
listeriosis is the second most costly foodbome illness in the United States, with an
estimated annual cost of $2.3 billion in medical expenses and lost productivity, including
death (F renzen, 2003). Listeriosis has the highest hospitalization rate and the second
highest number of fatalities of any foodbome illness tracked by the Centers for Disease
Control and Prevention in FoodNet (FDA/FSIS/CDC, 2003). The Healthy People 2010
national health objective for listeriosis was to reduce the number of cases to 2.5 per
1,000,000 people by 2005. Partly as a result of new regulations, the number of cases
dropped from 4.7 per 1,000,000 people in 1997 to 2.6 cases per 1,000,000 in 2002, nearly
reaching the stated goal (US-DHHS, 2004). However, the number of listeriosis cases has
since increased to 3.0 per 1,000,000 people in 2005 with the targeted goal not yet
achieved (Reuters, 2006).

Humans acquire listeriosis through ingestion of contaminated food in 90% of
listeriosis cases (Mead et al., 1999). Listeriosis results in flu-like symptoms, meningitis,
spontaneous abortion, fetal death, or neonatal septicemia (Slutsker and Schuchat, 1999).
Febrile gastroenteritis, a less common and poorly characterized form of listeriosis,
usually occurs in previously healthy adults who ingest unusually large quantities of the
pathogen (Schlech, 2000). The incubation period for listeriosis ranges from 24-48 hours
for febrile gastroenteritis, to 14 to 70 days for the more typical invasive form of listeriosis
(Schlech, 2000).

Listeria monocytogenes contains 13 serotypes based on somatic (O) and flagellar

(H) antigens. Four of these serotypes—l/Za, 1/2b, 1/2c and 4b, account for over 95% of

human listeriosis cases with serotype 4b strains predominating (Graves et al., 1999;
Nightingale et al., 2005). Conventional strain typing methods, such as serotyping and
phage typing, result in poor discrimination and reproducibility between strains.
Serotyping of L. monocytogenes strains is difficult due to the limited availability of high
quality antisera and the number of antigens shared by different serotypes. For instance,
serotypes 4a, 4b, 4c, 4d, 1/2b, and 3b, all contain the same H antigens, and multiple
common 0 antigens are present in different serotypes (Liu et al., 2006). Molecular
typing methods, such as multilocus enzyme electrophoresis, ribotyping, random
arnplicification of polymorphic DNA (RAPD), and pulsed-field gel electrophoresis
(PFGE) result in better reproducibility and discrimination between strains. PFGE, which
is the basis for CDCs PulseNet System, is now used throughout the United States, Canada
and elsewhere to identify potential common source outbreaks of listeriosis and other
foodbome illnesses (Graves et al., 1999).

Listeria monocytogenes can be divided into three distinct genetic lineages:
Lineage I (serotypes 1/2b, 3b, 3c and 4b), Lineage II (serotypes 1/23, U20, and 3a), and
Lineage III (serotypes 4a, 4b, and 4c) (Nightingale et al., 2005; Roberts et a1, 2006).
Lineage I strains are responsible for most human listeriosis cases, while Lineage II strains
are common environmental isolates that are infrequently implicated in human listeriosis
(Nightingale et al., 2005; Saunders et al., 2006). Lineage 111 strains are rare, with one
survey showing that fewer than 3% of 1800 L. monocytogenes strains belonged to
Lineage III (Roberts et al., 2006). While strains of any lineage have the potential to
cause listeriosis, most research to date indicates that Lineage 1 strains are better adapted

to survive and multiply in foods and have greater pathogenic potential (Nightingale et al.,

2005). In contrast, Lineage 11 strains are better adapted to survive in the environment,

and can outcompete Lineage I strains during selective enrichment (Bruhn et al., 2005).

1.2 Listeriosis Outbreaks from Ready-to-Eat Meats

In the United States, transmission of L. monocytogenes from ready-to-eat (RTE)
meat products was first documented in a 1988 when a breast cancer patient in Oklahoma
developed listeriosis after consuming turkey frankfurters. The opened frankfurter
package recovered from the patient’s refrigerator contained over 103 CFU/ g of L.
monocytogenes. Afier tracing the product back to the manufacturer, the initial
contamination level (as determined by most probable number) was < 0.3 CF U/g (Barnes
et al., 1989).

A number of listeriosis outbreaks have since been linked to consumption of fully
cooked RTE meat products. These outbreaks involved multiple states in the US and
consequently attracted considerable public attention. The first of these multi-state
outbreaks, in 1998, involved turkey frankfurters contaminated with L. monocytogenes
serotype 4b—this outbreak caused 108 cases of listeriosis, 14 deaths and 4 miscarriages
or stillbirths in 24 states (Graves et al., 2005). Two major multi-state outbreaks of
listeriosis linked to the consumption of delicatessen-sliced RTE turkey meat occurred in
2000 and 2002, and involved 11 and 9 states, respectively. Both outbreaks resulted from
the consumption of turkey delicatessen meat contaminated with L. monocytogenes
serotype 4b. The 2000 outbreak was responsible for 30 cases of listeriosis, 4 deaths, 3
miscarriages/stillbirths and the recall of 16.9 million pounds of turkey (Olsen etal.,
2005). The outbreak in 2002 caused 46 cases of listeriosis, 7 deaths, and 3

' miscarriages/stillbirths in 9 primarily northeastern states. This outbreak prompted the

recall of 27.4 million pounds of delicatessen turkey meat (CDC, 2002). A third outbreak
of listeriosis linked to delicatessen-sliced turkey meat occurred in 2001 in Los Angeles
County, California, but was different from the others insofar as it caused 16 cases of
acute febrile gastroenteritis, resulting in no fatalities. It was caused by L. monocytogenes
serotype l/2a, found at levels of 1.6 x 109 CF U/g in the implicated turkey meat (Frye et

aL,2002)

1.3 Listeria Recalls

Listeria monocytogenes is the leading microbiological cause of Class I recalls of
cooked or RTE products. Contamination usually occurs after processing, prior to
packaging (Levine et al., 2001). From 1994 to April 2006, 85 recalls were issued for deli
meats containing L. monocytogenes with ham most frequently implicated (31 recalls),
followed by luncheon meats and sausages (category labeled as “other”—30), followed by
beef (13 recalls), turkey (8 recalls), and chicken (3 recalls) (Figure 1.1; USDA-FSIS,
2006).. Products were recalled if L. monocytogenes was present in a 25 g sample of the
meat product. Although most frequently contaminated, ham has not yet been linked to
any listeriosis outbreaks, whereas the infrequently recalled turkey has been involved in 3
outbreaks since 2000, resulting in a total of 92 cases of illness, including 11 deaths and 6
miscarriages (CDC, 2001; CDC, 2002; Olsen et al., 2005). Studies have shown that
growth conditions on RTE poultry are more favorable to L. monocytogenes than ham
(Glass and Doyle, 1989; Beumer et al., 1996; Burnett et al., 2005). This may explain
why, despite the higher number of ham recalls, only turkey has been linked to large

outbreaks of listeriosis.

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1.4 Listeria monocytogenes in RTE Meats

Given the likelihood for the presence of L. monocytogenes in the processing
environment, contamination of RTE meat products is a concern for both the meat
industry and regulatory agencies. A monitoring program for Listeria in cooked beef
products has been in place in the United States since 1987 and in 1993 the sampling
program was expanded to include meat/poultry products and meat/poultry spreads
(U SDA-FSIS, 2003). Based on government survey data from 1990-2000, L.
monocytogenes was more prevalent in ham and luncheon meats compared to the other
categories of RTE products (Table 1.1; Levine et al., 2001). In 1999, Hazard Analysis
and Critical Control Point plans (HACCP) were completely phased in for all meat and
poultry establishments, in accordance with the Pathogen Reduction: Hazard Analysis and
Critical Control Points final rule, also known as the “Mega-Reg” (USDA-F SIS, 1998).

In a subsequent survey of 31,705 RTE products sampled from eight RTE product
categories (fresh soft cheeses, bagged salads, blue-veined cheeses, mold-ripened cheeses,
seafood salads, smoked seafood, luncheon meats and deli salads), 577 samples were
positive for L. monocytogenes (Gombas et al., 2003). Of the 9,199 luncheon meat
samples taken as part of the study, 82 were L. monocytogenes-positive, giving a
prevalence rate of 0.89% (Gombas et al., 2003). Most positive samples (75.6%)
contained less than 1 CFU/g (Table 1.2). Luncheon meats that were store-packaged were
more frequently contaminated with L. monocytogenes (6.8 times as likely to be
contaminated) than manufacturer-packaged meats. However, the samples contaminated
at levels higher than 102 CF U/g were more likely to be manufacturer-packaged (Gombas

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Table 1.2. L. monocytogenes-positive luncheon meat samples from the Gombas et
al., survey (2003)

 

 

Contamination Level Number of Positive Percent of Positive Samples

(CFU/ g) Samples

0.04 — 0.1 42 51.2
>0.1 — 1 20 24.4
>1 —— 10 10 12.2

>10 — 102 2 2.4

>102 — 103 7 8.5

>103 — 10“ 1 1.2

 

In 2003, the Listeria monocytogenes final rule was put into effect, mandating
three alternative Listeria control strategies that are required for manufactures of RTE
meats. Currently, a multi-state survey is being conducted to determine the prevalence of
L. monocytogenes in RTE meat in the wake of the new regulation. In this study, 8,000
samples of RTE delicatessen meats from four F oodNet states (Georgia, California,
Minnesota, and Tennessee) are being purchased at retail and examined for both presence
and numbers of L. monocytogenes. Overall prevalence of L. monocytogenes in
delicatessen meats since the implementation of the final rule has decreased slightly to
0.77%, as opposed to the 0.89% observed by Gombas et al. (Draughon et al., 2006).
Delicatessen-sliced meats were again more likely to be L. monocytogenes-positive (1.4%)
than manufacturer-sliced meats (0.17%) (Draughon et al., 2006). Prior to the L.
monocytogenes final rule, 0.4% of manufacturer-sliced and 2.7% of delicatessen-sliced

meats were L. monocytogenes-positive (Gombas et al., 2003). In the current study, pork

l3

products had the highest prevalence (0.89%), followed by beef (0.79%), and poultry
(0.67%) (Draughon et al., 2006).

Numerous studies have shown that L. monocytogenes can grow on RTE meat at
refrigeration temperatures (Glass and Doyle, 1989; Grau and Vanderlinde, 1992; Beumer
et al., 1996; Burnett et al., 2005). Listeria contamination of RTE meats is typically low-
level (<0.03 CFU/g) (Gombas et al., 2003; Draughon et al., 2006). However, certain
products have been shown to support grth to higher levels during extended refrigerated
storage. In one study involving naturally contaminated RTE meats (luncheon meat, ham,
and chicken breast), L. monocytogenes populations increased to 104 CFU/ g after 4-6
weeks in products above pH 5 (Beumer et al., 1996). Burnett et al. (2005) studied growth
of L. monocytogenes on RTE turkey breast (uncured, pH 6.2, aW 0.98) and RTE ham
(cured, pH 6.2, aw 0.98) at 5, 7, and 10°C, and found that growth rates in RTE turkey
were higher than in ham (Table 1.3). These results are similar to earlier findings of Glass
and Doyle (1989) who showed that L. monocytogenes grew to 103-10S CFU/g on
vacuum-packaged processed poultry during 4 weeks of storage at 44°C, while
populations increased 103-104 CFU/ g over 6 weeks on ham, bologna and bratwurst.
Foods having a pH at or below 5 are generally unable to support growth of L.
monocytogenes. However, L. monocytogenes can survive in fermented meats including
hard salami, for at least three months, with greater survival observed in products as water

activity increases (Johnson et al., 1988).

14

Table 1.3. Growth of L. monocytogenes on RTE delicatessen meat (Burnett et al.,
2005)

 

 

Product Temperature (°C) Log CFU/g growth per
day

Turkey (turkey breast meat, 5 0.45
turkey broth, dextrose, salt,
sodium phosphate, garlic, 7 0.83
flavoring)

10 1.53
Ham (ham, water, salt, 5 0.42
sugar, dextrose, sodium
phosphate, monosodium 7 0.58
glutamate, sodium
erythorbate, sodium nitrite) 10 0,98

 

In addition to low pH or lower aw, certain additives can also suppress growth of L.
monocytogenes in RTE meat including nitrite at levels of 140-200 ppm (Grau and
Vanderlinde, 1992). A combination of sodium lactate (1.8%) and sodium diacetate
(0.25%) also completely inhibited L. monocytogenes growth in vacuum-packaged pork
frankfurters during 40 days of storage at 10°C, with an initial population reduction if the
contaminated frankfurters were dipped in 2.5% lactic or acetic acid prior to storage

(Barmpalia et al., 2004).

15

1.5 Listeria Risk Assessments for RTE Meats

Increased awareness of the potential cost and risk of multi-state listeriosis

outbreaks spurred the development of several risk assessments for Listeria by United

States government agencies (FDA/USDA/CDC, 2003). The Office of Management and

the Budget requires that risk assessments be conducted by US. federal government

agencies in order to assess the costs and benefits of planned regulations which will ensure

that an equally effective and equally beneficial alternative is not being overlooked

(Buchanan et al., 2004). As defined by the Codex Alimentarius, microbial risk

assessments should include the following four elements (Barraj and Petersen, 2004):

1)

2)

3)

4)

Hazard identification: The identification of biological, chemical and
physical agents that are capable of causing adverse health effects and
that may be present in a particular food or group of foods.

Hazard characterization: The qualitative or quantitative evaluation of
the nature of the adverse health effects associated with biological,
chemical and physical agents that may be present in food.

Exposure assessment: The qualitative or quantitative evaluation of the
likely intake of biological, chemical and physical agents via food and
exposures from other sources if relevant.

Risk characterization: The qualitative or quantitative estimation,
including attendant uncertainties, of the probability of occurrence and
severity of known or potential adverse health effects in a given
population based on hazard identification, hazard characterization and

exposure assessment.

16

Initially, an assessment of the relative risk to public health from foodbome L.
monocytogenes was conducted, in which the risks of contracting listeriosis from 23
categories of RTE foods were ranked (Table 1.4). Although 3 categories of RTE foods
showed a higher prevalence of L. monocytogenes contamination (pate's, smoked seafoods,
and fresh, soft cheeses), deli meats ranked first in relative risk, due to their higher per
capita consumption which, in turn, leads to a wider exposure of the public to L.
monocytogenes. Exposure can be high if the pathogen concentration in the food is high
or if large quantities are consumed, even if overall pathogen concentration is low (Barraj
and Petersen, 2004), as is the case for L. monocytogenes in deli meats. Overall, 14 cases
of listeriosis were predicted to occur for every 100 million servings of deli meat
consumed (FDA/USDA/CDC, 2003). Based on this initial risk assessment, another risk
assessment was carried out specifically for Listeria in RTE meat and poultry products
(USDA-F818, 2003). This risk assessment found that combined interventions including
various combinations of increased testing of food contact surface and sanitation, pre- and
post-packaging microbial reduction strategies, and product reformulation to include
growth inhibitors would be the most effective means of controlling the risk of listeriosis
(U SDA-FSIS, 2003). These strategies were incorporated into the Listeria monocytogenes

final rule implemented in 2003.

17

Table 1.4. Predicted Relative Risk Rankings for Listeriosis among Food
Categories for Three U.S. Age-Based Subpopulations Using Median Estimates
of Relative Predicted Risks for Listeriosis on a Per Annum Basis (U SDA-FSIS,
2003)

 

 

. a Subpopulatron
. Food Categorres . .. .,
g Intermediate Ageb 1 Elderly Perinatalb
SEAFOOD
ESmoked Seafood . 6 6 j 7
Raw Seafood : 17 20 17
Preserved Fish 3 l3 ' l3 13 '
Cooked Ready-to-Eat
9 8 9
E Crustaceans
l PRODUCE
1 Vegetables ’ 11 9 ; I 11 ’
Frurts ‘ 16 14 I 14
E DAIRY
{ Soft Mold-Ripened and Blue- . 14 j 15 - 15
EVeined Cheese . ~
Goat Sheep, and Feta Cheese 18 A 17 , 18 I
l Fresh Soft Cheese (e. g. queso
7 1 l 6
1 Icfresco)
l Heat-Treated Natural Cheese 10 10 10
E and Processed Cheese ‘ .
lAged Cheese 19 18 3 l9;
Fluid Milk, Pasteurizedd 3 2 i 2 I;
Flurd Milk Unpasteurrzed 15 0 16 I 16
‘ Ice Cream and Frozen Dairy 1 I U i A I» I . A I
EProducts 20 19 j 20
Miscellaneous Dairy Products . 5 I P 4 i I V 5 i

18

Table 1.4 (Cont’d)

I MEATS

{Qwem , .. _ . ,4 ., ,. . I ._ 5 . 4.
Eggggi-Dry Fermented , 12 _ 12 . 12
Dehhfigats . .. _ .. . _ .1 .. . . . .1. ’. 1.
:' Pate and Meat Spreads ' I 8 I 7 I 8
‘COMBINATION Foons * ’

Deli Salads 7 I. 2 3 ; 3

a Food categories are grouped by type of food but are not in any particular order.

b A ranking of 1 indicates the food category with the greatest predicted relative risk
of causing listeriosis and a ranking of 20 indicates the lowest predicted relative risk
5 of causing listeriosis.

° Data from soft ripened cheese made from unpasteurized milk were used 1n the
I,modeling to define the shape of the distribution of contamination data for fresh soft
Echeese.

dAll available data for this food category were used 1n the modeling to define the
Ishape of the distribution for this food category but only contamination data from
1 North America were used to determine the frequency of contamination. Also see text
I for discussion of the effects of uncertainty on the ranking for pasteurized milk and
I other foods that are consumed in high amounts.

c This ranking is based on the assumption that 1% to 14% of frankfurters are
consumed without reheating and the remainder are adequately heated before
consumption.

~--.-..--o,t .. . _..

19

1.6 Strategies and Regulations for Decreasing the Incidence of Listeriosis

In the wake of the multi-state listeriosis outbreaks linked to consumption of
delicatessen-sliced RTE turkey meat, the USDA implemented new regulations for the
control of L. monocytogenes in RTE meat processing facilities, in addition to the “zero-
tolerance” regulation, and the requirement that HACCP plans address potential L.
monocytogenes contamination problems. The major requirement instituted was that
facilities producing high-risk RTE meat products must develop scientifically validated L.
monocytogenes control programs. Meat processing facilities are currently required to

choose from the following three alternative control strategies (USDA-FSIS, 2003):

. Alternative 1 — Employ both a post-lethality treatment and a growth inhibitor for
L. monocytogenes on RTE products. Establishments opting for this alternative
will be subject to FSIS verification activity that focuses on the post-lethality
treatment’s effectiveness. Sanitation is important but is built into the degree of

lethality necessary for safety.

. Alternative 2 — Employ either a post-lethality treatment or a growth inhibitor for
the pathogen on RTE products. Establishments opting for this alternative will be
subject to more frequent FSIS verification activity than those in Alternative 1, and
will be required to test for the presence of L. monocytogenes or Listeria spp. on

food contact surfaces.

0 Alternative 3 — Employ sanitation measures only. Establishments opting for this
alternative will be targeted with the most frequent level of FSIS verification

activity. Within this alternative, FSIS will place increased scrutiny on operations

20

that produce hotdogs and deli meats. In a 2001 risk ranking, F818 and FDA

identified these products as being high-risk products for listeriosis.

Within one year of the 2002 listeriosis outbreak traced to delicatessen turkey
meat, USDA-F SIS reported a 25% decrease in the number of regulatory samples testing
positive for L. monocytogenes (between January 2003-September 2003, compared with
the number of positive samples detected in 2002; Gottlieb et al., 2006) (Figure 1.2).
Preliminary national surveillance data showed a decrease of 40% in cases of human
listeriosis, compared with the average yearly number of cases detected between 1996 and
1998. By the beginning of 2004, there were 2.7 cases of listeriosis per million, which
was nearly at the national goal of 2.5 cases/million by 2005 (Gottlieb etal., 2006).
Unfortunately, in 2005 the number of cases increased to 3.0 per million, short of the
targeted goal (Reuters, 2006).

At retail, the FDA Food Code provides the only requirements and guidelines for
preventing contamination with L. monocytogenes, and its implementation is taught to
retail and restaurant managers via the ServSafe Food Safety Training and Certification
Program. Largely, these requirements are specific to sanitation of food contact surfaces,
particularly central contact points, such as delicatessen slicers, knives, countertops and
coolers. The most recent version of the Food Code specifies that any product which is
stored under a controlled temperature and for a specified length of time for safety reasons
is a potentially hazardous food and more frequent cleaning of contact surfaces is required
(at least once every 4 h, as opposed to once every 10 h for non-hazardous foods; FDA,

2005).

21

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22

1.7 Listeria survival in the environment and on delicatessen slicers

The higher prevalence of L. monocytogenes in delicatessen meat sliced at retail
strongly suggests that the delicatessen slicer is an important vehicle for cross-
contarnination of products. In order for cross-contamination to occur, L. monocytogenes
must survive on the surface for a period of time between the slicing of various products.
Prior to its introduction to the slicer surface, L. monocytogenes may be exposed to
refrigeration temperatures, low pH (fermented meats and cheeses), limited available
water, and sanitizers. While on the slicer surface, L. monocytogenes exposure to
desiccation and sanitizers is likely. These stresses have been shown to alter the
sensitivity of L. monocytogenes to other subsequent stresses, sometimes making it more
difficult to eradicate from the environment (Lou and Yousef, 1997; Koutsoumanis et al.,
2003; Koutsoumanis and Sofos, 2004; Gravesen et al., 2005; Moorman et al., 2005).

Metal may have certain bactericidal properties. Stainless steel in contact with the
air will expose bacteria to desiccation, but food soils ofien prolong bacterial survival in
this otherwise inhospitable environment (Robine et al., 2002; Kusumaningram et al.,
2003). Composition of the metal alloy and its surface finish may also impact the
availability of harborage sites for bacteria and their subsequent survival. Massive copper
(99.99% copper) resulted in poorer survival of Enterococcusfaecalis over 96 h than 304
A181 stainless steel and copper-rich stainless steel (A181 211 stainless steel plus copper;
Robine et al., 2002). However, applying the inhibitory metal to stainless steel (as in the
case of the copper-rich stainless steel) did not result in significantly better inhibition of E.

I faecalis than was seen with 304 A181 stainless steel (Robine et al., 2002). Silver ion-

23

treated stainless steel can reportedly inhibit biofilm growth of Staphylococcus spp. on
catheters of kidney transplant recipients; however it has not been studied for its
effectiveness in food processing environments (Loertzer et al., 2006).

A typical stress that L. monocytogenes may encounter on a delicatessen slicer is
desiccation. Depending on temperature and RH of the environment, L. monocytogenes
can survive 82 (22°C, 0% RH) to more than 151 days (10°C, 88% RH) on sand without a
nutrient source (DeRoin et al., 2003). Survival under desiccated conditions is better at
lower temperatures (10 vs. 22°C) and higher RH (88 vs. 40 or 0%; DeRoin et al., 2003).
Under osmotic stress, L. monocytogenes will try to accumulate osmolytes, particularly
glycine betaine and camitine, from the growth medium (Gardan et al., 2003). Listeria
monocytogenes increases the expression of 12 different stress proteins when exposed to
salt, and suppress the production of 21 proteins (Esvan et al., 2000). In the absence of
available osmolytes, Listeria also produces a general stress protein, etc that promotes
osmotic stress tolerance (Gardan et al., 2003).

Listeria is able to grow at 4°C, by altering its membrane composition in order to
maintain membrane fluidity and increase passive permeability. This is achieved through
changes in fatty acid composition (Neunlist et al., 2005). In addition, when exposed to
cold starvation conditions, L. monocytogenes undergoes shrinkage of the cytoplasm,
eventually resulting in holes in the cytoplasm (Dykes, 1999). Camitine, which L.
monocytogenes uses as an osmoprotectant, is also thought to aid in tolerance to cold-
stress (Dykes and Moorhead, 2000).

Acid tolerance of L. monocytogenes has been studied by several researchers.

Listeria is able to better withstand lethal acid concentrations (pH 3.5) afier habituation to

24

sublethal acid stress (pH 5 - 6), with maximum acid tolerance induced by habituation to
pH 5.5 (Koutsoumanis and Sofos, 2004; Koutsoumanis et al., 2005). Listeria
monocytogenes can also remain viable for at least 20 h at pH 4.0, and also at pH 3.5 in
the presence of glucose (Shabala et al., 2002) by maintaining a higher intracellular pH of
7.0-7.5. However, in the absence of glucose, the ability to maintain a higher intracellular
pH at pH 5.5 is lost (Shabala et al., 2002). Other physiological changes in response to
acid stress include changes in protein synthesis and fatty acid composition of the cell
membrane (Koutsoumanis and Sofos, 2004), the latter of which increases the surface
hydrophobicity of acid adapted cells (Lou and Yousef, 1997).

Exposure to the aforementioned stresses can alter Listeria sensitivity to
quaternary ammonium sanitizers. After exposure to acid or starvation stress, L. innocua
is less sensitive to the quaternary ammonium sanitizer, cetrimide (Moorman et al., 2005).
This cross-protection does not occur after exposure to cold and heat stress, which
increases L. innocua sensitivity to cetrimide (Moorman et al., 2005). Listeria
monocytogenes strains that are resistant to quaternary ammonium compounds, other
sanitzers and antibiotics have been found to contain a gene (mer) that encodes for an
efilux pump (Romanova et al., 2002). However, some resistant isolates do not appear to
rely on efilux pumps, and instead alter their cell membrane fatty acid profile in response
to sanitizer stress, which can prevent entry of foreign chemicals into the cytoplasm (To et
al., 2002). The same study found that upon initial (30 h) exposure to sublethal levels of
benzalkonium chloride, biofilm growth was favored (To et al., 2002). Exposure to
sublethal concentrations of ethanol and isopropanol can also increase L. monocytogenes

attachment at 10, 20 and 30°C (Gravesen et al., 2005) with ethanol-adaptation increasing

25

L. monocytogenes resistance to lethal levels of acid, ethanol, hydrogen peroxide, and
sodium chloride (Lou and Yousef, 1997).

Elimination of Listeria biofilms from food contact surfaces is particularly
difficult. In general, a high degree of mechanical action and friction is required to
remove biofilms (Gibson et al., 1999). Hydrogen peroxide is effective at eliminating
biofilms, particularly since its efficacy is unaffected by high organic loads (Robbins et
al., 2005). Exposure to a pH of 12 through the addition of sodium hydroxide, is also an
effective means to eliminate L. monocytogenes, either as a biofilm or planktonic cells,
particularly when used prior to exposure to quaternary ammonium santizers (Chavant et

al., 2004a; Chavant et al., 2004b).

1.8 Prevalence of Listeria monocytogenes in Food Processing Environments

Tompkin (2002) identified three scenarios for the occurrence of foodbome

listeriosis:

1) Isolated, sporadic cases in which no food origin is identified.

2) Outbreaks involving a single lot of contaminated food, as the result of errors
in handling that lead to contamination and growth of L. monocytogenes in the
product.

3) Outbreaks involving a few to several hundred cases scattered over time and
location as the result of contamination of the food processing environment by
a persistent and virulent strain of L. monocytogenes, which then contaminates

multiple lots of product for days or even months.

26

In order for the third scenario to occur, L. monocytogenes must find a niche in the
processing environment that is protected from normal cleaning and sanitizing procedures
(Tompkin, 2002). Unfortunately, there is no shortage of possible niches in the typical
food-processing environment, and L. monocytogenes has been found to persist in
processing environments for up to 12 years (Lunden et al., 2001). Equipment such as
peelers, slicers, dicers, and conveyor belt lines are not always designed in a way that
facilitates effective cleaning and sanitizing. In a study of 13 dried sausage-processing
plants, effective cleaning and sanitizing was prevented by the complexity of processing
lines and machines (Thevenot et al., 2005). Organic residues remaining on these pieces
of equipment were associated with samples positive for L. monocytogenes when the
equipment was “clean” prior to the beginning of the processing shift (Thevenot et al.,
2005). Another study of meat, poultry and seafood processing facilities found that the
cleaning procedures used were ineffective in eliminating Listeria spp. in certain areas of
the processing environment, particularly conveyor belts, carts, floors and drains, with
these areas accounting for nearly 95% of L. monocytogenes-positive samples. Following
sanitation, 23.4% of samples from these areas were still L. monocytogenes-positive
(Godbjomsdottir et al., 2004). In a survey of L. monocytogenes contamination in poultry
processing environments, several food contact surfaces were persistently contaminated
with the same strains, including slicer blades and blade covers, dicing machine blades
and blade covers, a conveyor belt, and a spiral conveyor in a freezer (Lunden et al.,
2003). This inability to adequately clean surfaces allows L. monocytogenes to persist in
the environment and form biofilms on food contact surfaces where the pathogen can be

potentially transferred to RTE foods.

27

Persistent strains play an important role in contamination of RTE foods with such
strains 8 times more likely to contaminate finished product than transient strains (Lunden
et al., 2003). While transient strains were likely to be found in both the incoming raw
product and environment prior to any lethal cooking process, they were not found in any
of the post-cooking processing lines (Lunden et al., 2003). Other studies have also
reported that it is unusual to have the same strain of L. monocytogenes contaminating
both incoming raw product and the RTE final product, leading to the conclusion that
persistent environmental strains are frequently responsible for recontarnination of RTE
products (Nesbakken et al., 1996; Lappi et al., 2004).

It has been calculated that ideally, a food and environmental Listeria control
program in a processing facility may be able to keep the prevalence of product
contamination of a cooked RTE product at < 0.5%, with post process pasteurization
yielding a contamination rate that is essentially zero. A single lot of product
contaminated at this level may still be accepted despite the zero-tolerance regulation,
since there is a 61% statistical likelihood of L. monocytogenes contamination at the 0.5%
level going undetected by end product testing (Tompkin, 2002). Intervention strategies
can be effective in controlling the prevalence of L. monocytogenes in meat processing
environments, but appropriate intervention strategies vary between facilities. In some
plants, increased compartmentalization of raw and cooked processing areas is required to
decrease L. monocytogenes prevalence in the environment, which often requires
structural changes to the facility (Lunden et al., 2003; Lappi et al., 2004). Changes in
equipment design in order to eliminate harborage sites, such as difficult to clean areas in

mechanical slicers, interlocking conveyor belts and hollow rollers in conveyors may also

28

play an important role in improving Listeria control (Tompkin, 2002; Lappi et al., 2004).
In some cases, changes in sanitation programs are required including the addition of
sanitizing footbaths and trench drains (Lappi etal., 2004). Employee training in the
importance of certain procedures to reduce the risk of cross-contamination may also be
effective in reducing Listeria transfer to different areas and products in the plant (Lappi et

al., 2004).

1.9 Bacterial transfer during food processing

Meat processing equipment is typically constructed out of stainless steel, which
shares similarities with nonporous plastic cutting boards such as the ability to become
scratched and scarred with use and the inability to irreversibly absorb bacteria. When
beef trim was surface-inoculated with E. coli 0157:H7 at a level of ~6.0 log CFU/g and
ground using a Hobart model 84142 grinder, populations of 3-4 log CFU/cm2 were
recovered from the stainless steel auger housing during grinding (Farrell et al., 1998).
Following cleaning and sanitizing the grinder with chlorine or peroxyacetic acid
sanitizers, E. coli OlS7:H7 was still recoverable from the stainless steel surface by
enrichment (Farrell et al., 1998). In a similar study involving the distribution of
contamination by in a table-top bowl chopper used to process beef inoculated with E. coli
0157 :H7 (2 log CFU/ g), the pathogen was always transferred to subsequent batches of
beef processed in the bowl chopper with E. coli 01 57:H7 also present on the comb/knife
guard and the knife after processing (Flores, 2004). These findings emphasize the
importance of thorough cleaning and sanitizing to minimize cross-contamination in the

processing environment.

29

Lin et al. (2006) conducted a study in which the blade of a commercial-scale meat
slicer used to slice roast turkey breast, salami and bologna was inoculated to contain L.
monocytogenes at levels of 1, 2, or 3 log CFU/blade (l and 2 log CFU/blade inoculum
used with turkey only). The slices of meat were subsequently deposited on a conveyor
belt (material unspecified), which were then tested in pooled samples of five slices, and
quantified via MPN. More slices of meat tested positive by enrichment at the 3 log
CFU/blade inoculum level than at 1 or 2 log CFU/blade (Lin et al., 2006). Of the 20
equipment samples (10 under the blade housing, 6 blade samples, and 4 on conveyor
belts) taken after each of two slicing replicates per product, 5 blade surface samples were
positive, 6 positives were found from the blade housing, and 2 conveyor belt samples
were positive (Lin et al., 2006). Additionally, Lin et al. (2006) found that more
equipment samples were positive for L. monocytogenes afier slicing salami (8 samples)
than turkey (3 positive samples) or bologna (1 positive sample), which supports a longer
residence time for L. monocytogenes on fat-coated slicers as suggested by Vorst et al.
(2006).

A study was conducted of L. monocytogenes transfer from biofilms (both pure
culture and mixed with processing plant isolates) developed in raw beef exudate pipetted
onto a stainless steel surface (AISI grade 304, 2RB finish) at 15 and 25°C/ 100% RH to
mimic meat industry biofilms (Midelet et al., 2006). Transfer to a trypticase soy agar
cylinder used as a model food product was quantified and a transfer rate of 55% was
observed from pure culture biofilms, while the presence of Kocuria varians, a gram-
positive organism isolated from a dairy processing environment, increased the L.

monocytogenes transfer rate to 78% (Midelet et al., 2006). In this study, transfer

30

increased throughout the first three contacts, and then declined with each successive
contact beyond the third contact (Midelet et al., 2006). Exposure to chlorine shock
increased the adhesiveness of L. monocytogenes to the stainless steel surface, resulting in
a smaller transfer coefficient. This led the authors to conclude that while cleaning and
sanitizing may mitigate immediate risk of high initial levels of Listeria, reducing the
delayed risk of Listeria contamination due to prolonged attachment to surfaces could only
be achieved by lowering the attachment strength of L. monocytogenes to the food contact
surface, which none of the tested sanitizers was able to do (Midelet et al., 2006).

In a study in which volunteers’ hands were inoculated with wild-type E. coli (at
levels of 4-6 log CFU recovered from the hands), it was found that flock-lined rubber
gloves (20 mil thickness) largely prevented transfer to subsequently handled raw beef
cuts (Gill and Jones, 2002). Knitted polyester or cotton gloves reduced transfer (0.30 —
1.6 log CF U/beef sample) as compared to transfer observed from bare hands (2.5 — 3.5
log CFU/beef sample), but were not as effective in inhibiting transfer as rubber gloves (5

0.90 CFU/beef sample; Gill and Jones, 2002).

1.10 Bacterial transfer during retail food handling

Delicatessen slicer designs vary, but all of them contain the following basic
components (Figure 1.3): a circular, stainless steel blade; a built-in blade sharpener; a
stainless steel blade cover, typically grooved; a table to hold the meat, also grooved for
drainage; a grooved back plate to hold the meat in position for slicing; a collection area
for sliced product; and a motor covered by a stainless steel case. Based on this design,

several areas of the slicer are difficult to clean and sanitize effectively (e.g., the blade, the

31

area behind the blade, non-removable components), thus providing several potential
niches for bacterial pathogens including L. monocytogenes. However, as slicer
manufacturers have become more aware of the increased difficulty in cleaning these
areas, delicatessen slicers are now being re-designed for easy disassembly, cleaning and

sanitizing with most of these microbial niche areas being eliminated. (Figure 1.4).

32

Figure 1.3. Example of Delicatessen meat slicer (Chefmate, 10” manual slicer)

 

Figure 1.4. Example of delicatessen meat slicer designed for easier sanitation
(Berkel Company; South Bend, IN; X13 Slicer)

 

33

In food retail food handling environments, bacterial contaminants including
Listeria are most often found in difficult to clean areas that contain food particulates and
adequate moisture. Bacteria within these harborage sites are typically exposed to
stressful conditions including sanitizers, dehydration, starvation, and extremes in both
temperature and pH. Under these extreme conditions, L. monocytogenes can become
sublethally injured with the pathogen then unable to grow on many commonly used
selective plating media. Complex substrates are needed for growth with many of same
stresses present in the human host also present in food, such as lack of iron, oxidative
stress, pH extremes and starvation (Archer, 1996). This may have the effect of triggering
the expression of virulence genes, increasing the ability of pathogens to induce illness
(Archer, 1996).

Even under these unfavorable environmental conditions, bacterial foodbome
pathogens can remain viable on common food contact surfaces for days or weeks and go
on to cross—contaminate other products. In one early report, 469 cases of typhoid fever
were traced to a single can of corned beef sliced at a delicatessen with Salmonella Typhi
transferred from the delicatessen slicer to other deli meats that were subsequently sold
and consumed (Howie, 1968). The greater prevalence of L. monocytogenes in
delicatessen- as opposed to manufacturer-sliced meat is at least partly due to cross-
contamination in the delicatessen with one of the most obvious contact points being the
delicatessen slicer (Gombas et al., 2003). A study of L. monocytogenes contamination
routes in smoked salmon processing plants also found that slicers were harborage sites in

both plants in the study (Vogel et al., 2001).

34

In recent bacterial transfer work with mechanical delicatessen slicers, L.
monocytogenes has been shown to readily transfer both to and from slicer blades and deli
meats. Based on the work of Vorst et al., (2006), L. monocytogenes transferred from a
blade inoculated at 8 log CFU/blade to 30 successive slices of roast turkey breast with
transfer decreasing logarithmically to 2 log CFU/slice by the 30th slice. At lower
inoculum levels (5 log CFU/blade and 3 log CFU/blade), transfer was not quantifiable
beyond the 5th slice, with negative enrichments after 27 and 15 slices, respectively (Vorst
etal., 2006). In the same study, transfer to salami was more continuous throughout the
30 slices than to turkey or bologna, both of which were higher in moisture and lower in
fat than the salami. The difference in transfer between the products was attributed to the
layer of fat that accumulated on the slicer blade during slicing of salami, which was not
seen for the other two products (Vorst et al., 2006). These findings resemble those of Ak
et a1. (1994) which showed that chicken fat protected bacteria from desiccation and
removal from cutting boards. Consequently, fat from the salami may help protect L.
monocytogenes from desiccation and dispersal from the slicer in a similar fashion.
Furthermore, this corroborates the aforementioned results obtained by Lin et al. (2006)
who found that commercial-scale slicer equipment used to slice salami yielded more
positive samples than that used to slice turkey or bologna.

A study was conducted using Enterobacter aerogenes as a surrogate for
Salmonella spp., and rates for bacterial transfer were compared for transfer to and from
hands with or without polyethylene, food service grade gloves during cutting of chicken
and lettuce (Montville et al., 2001). The chicken was inoculated with ~ 8 log/CF U per

portion, and participants were instructed to cut the chicken into cubes, transfer it to a

35

bowl and then slice lettuce. Participants were not instructed in the proper way to put on
gloves, in order to better simulate real-world conditions of use (Montville et al., 2001).
The resulting transfer rate from chicken to lettuce was 0.01% to and from gloved hands,
while the transfer rate between ungloved hands and food was 10% (Montville et al.,
2001). In another study evaluating transfer of E. aerogenes from chicken to hands (using
the aforementioned inoculation method), and then to hand-washing areas and lettuce, the
reported transfer rates were highly variable (Chen et al., 2001). Transfer rates ranged
from 0.3 — 100% from chicken to hands, 0.003 — 12.3% from hands to the spigot on a
handwashing sink, 0.001 — 45.7% from dirty hands to clean hands after handwashing
(implying that hands were improperly washed), and 0.01 — 100% from “clean” hands to

lettuce (Chen et al., 2001).

1.11 Bacterial transfer in the home

Food handling in home kitchens can also lead to multiple routes of cross
contamination. Several studies have assessed bacterial transfer between food contact
surfaces, cloths and sponges used for cleaning, hands and food products (Scott and
Bloomfield, 1990; Montville etal., 2001; Sattar et al., 2001; Gill and Jones, 2002). Two
of these studies found that moist surfaces, cloths and hands transferred higher numbers of
bacteria to one another than was observed when the aforementioned surfaces were dry
(Scott and Bloomfield, 1990; Sattar et al., 2001). According to Sattar et al., (2001)
transfer of Staphylococcus aureus between moist cloths and moist fingertips was always
higher than transfer between the two if they were dry, and that the addition of friction by

rubbing the cloth with the hands for 10 3 resulted in a five-fold increase in transfer.

36

However, even in the scenario with the greatest amount of bacterial transfer (moist cloths
to moist hands with friction), only 2.5% of the S. aureus (5 log CFU/ piece of fabric)
inoculum was transferred (Sattar et al., 2001). Furthermore, a study by Scott and
Bloomfield (1990) found that S. aureus, E. coli and Salmonella spp. (2 log CFU/cloth
inoculum) were able to survive on soiled cloths for up to 24 h and transferred to laminate
surfaces and fingertips, which allows for possible recontarnination of a domestic kitchen
during cleaning.

Several studies have attempted to quantify transfer between food contact surfaces
and food in the absence of friction common to food processing scenarios. According to
Kusmnaningram et al. (2003), 21-43% of the S. aureus, Campylobacterjejuni and
Salmonella enteriditis populations on inoculated (6.7 — 9.4 log CFU/sponge) wet sponges
were transferred to stainless steel (AISI grade 304), with no significant differences seen
between the organisms in their rate of transfer to stainless steel or to subsequently applied
food products. Subsequently, 25-100% of the available population transferred to roast
chicken when applied to the stainless steel for 10 s with greater transfer observed when a
500-g weight was added to the chicken. Increasing the product weight did not have the
same effect on transfer to cucumber slices, which was found to occur at 50-100% of the
available bacterial population (Kusumaningram et al., 2003). However, when compared
to several other existing studies, it seems unlikely that 100% transfer can be achieved by
simply placing a food on a surface with the unusually high transfer levels achieved in this
study likely caused by inaccurate estimation of the surface inoculum. In the
aforementioned study, an unorthodox contact plate method was used to quantify the

bacterial population on stainless steel with the agar from the contact plate then suspended

37

and homogenized in a peptone saline solution, and diluted to a countable level prior to
plating (Kusumaningram et al., 2003). This undermines the validity of the transfer rates.
Another study found that ground beef (75-100 g patties) with an average bacterial
load of 6.7 log CF U/g transferred 2.5-3.0 log CFU/cm2 of E. coli OlS7:H7 to cutting
boards (polyethylene and wood laminate, no significant difference) after a 30 minute
contact time (Miller et al., 1996). Although transfer to the different cutting board types
was not significantly different, subsequent cross-contamination fi'om cutting boards to
other surfaces was different. In an earlier study that examined the survival of L.
monocytogenes, Salmonella typhimurium and Escherichia coli 01 57:H7 on cutting
boards (cut into 5 cm2 blocks), porous wooden cutting boards more effectively inhibited
cross-contamination due to the capillary action of the wood (Ak et al., 2004). Both
wooden (species included: ash, basswood, beech, birch, butternut, cherry, hard maple,
oak, and American black walnut) and plastic (polyacrylic, polyethylene, foamed
polypropylene, polystyrene, and hard rubber) were examined. Using wooden boards,
bacteria were internalized in the board to a depth of at least 15 um within 3-10 min and
were unavailable for subsequent transfer to other foods or knives when the boards were
later used for food preparation (Ak et al., 1994). However, bacteria were able to persist
and even multiply on the surface on nonporous plastic boards over a 12 h period at 22°C,
particularly if the ambient humidity was sufficient to prevent the surface from drying,
with the greatest persistence and survival in knife-scarred areas of the board containing

chicken fat (Ak et al., 1994).

38

1.12 Listeria persistence on surfaces and biofilm formation

Up to this point, virtually all bacterial transfer work has been conducted using
bacterial cultures that have been inoculated onto a surface, briefly dried and then placed
in contact with other surfaces or foods to simulate various transfer scenarios. However,
in reality, bacterial transfer is far more complex with the extent of bacterial attachment to
these surfaces ranging fi'om loosely attached cells to biofilms. Bacterial attachment is
defined as an affiliation between a bacterium and a surface (Notermans et al., 1991).
Many types of bacteria, including L. monocytogenes, can attach and persist on equipment
over extended periods of time. Listeria monocytogenes has been shown to adhere to 17
approved food contact surfaces, consisting of rubbers, polymers and metals, including
stainless steel (Beresford et al., 2001). Attachment is the first step in biofilm formation

(Figure 1.5).

39

Figure 1.5. Stages in biofilm development (MSU-CBE, 2002)

 

Initial attachment occurs via electrostatic or hydrophobic interactions between the
bacterial surface and the contact surface (Arnold and Bailey, 2000). The literature is
contradictory concerning the nature of the L. monocytogenes cell surface as well as food
contact surfaces and their subsequent interactions. Listeria monocytogenes has a negative
surface charge (Dickson and Koohmaraie, 1989; Chavant et al., 2002) and some studies
indicate that the surface is hydrophobic (Dickson and Koohmaraie, 1989; Ukuku and
F ett, 2002), while others indicate that the surface is hydrophilic (Mafu et al., 1991;
Briandet et al., 1999; Chavant et al., 2002). These discrepancies in the measurement of
cell surface hydrophobicity or hydrophilicity may be due to surface variations between
strains or differences in the methods used to measure hydrophobicity. Three commonly
used methods for determining hydrophobicity of microorganisms — hydrophobic
interaction chromatography (HIC), bacterial adherence to hydrocarbons (BATH), and
contact angle measurement—often produce very different results for the same bacterial
strain (Dickson and Koohmaraie, 1989). HIC and BATH are used more frequently since

they require less specialized equipment than contact angle measurement, but there are

40

disadvantages to both methods. HIC may produce inconsistencies due to nonspecific
binding of bacteria to the column (Dickson and Koohmaraie, 1989). BATH results may
vary due cell lysis or the hydrocarbon used (Dickson and Koohmaraie, 1989).
Depending on the methodology and subsequent results, different predictions are
made as to whether L. monocytogenes attachment to various surfaces is favored or not.
Even if attachment is not favored, physicochemical interactions do not totally prevent
attachment with attachment still occurring, albeit at lower levels on surfaces that would
otherwise be predicted to naturally repel bacterial cells (Dickson and Koohmaraie, 1989;
Mafu et al., 1991; Cunliffe et al., 1999). Smoot and Pierson (1998) reported a faster L.
monocytogenes attachment rate to stainless steel than to Buna-N rubber, although cell
surface hydrophobicity and surface free energies predicted that adhesion to Buna-N
rubber would be favored. Another study also found that hydrophilic and negatively
charged L. monocytogenes cells adhered better to stainless steel, than to
polytetrafluoroethylene (Chavant et al., 2002). Briandet et al. (1999) reported that
although L. monocytogenes tended to be hydrophilic, strains that were slightly more
hydrophobic than others adhered better to stainless steel. However, Midelet and
Carpentier (2002) observed stronger attachment of L. monocytogenes biofilms to
polymers (polyvinyl chloride and polyurethane) than to stainless steel, and also noted that
all of these surfaces were hydrophobic, with these same surfaces becoming hydrophilic
after exposure to meat exudate based on contact angle measurement. Cunliffe et al.
(1999) found that hydrophilic uncharged surfaces were slightly repellent to L.
monocytogenes. Another study reported that L. monocytogenes Scott A was more

attracted to polypropylene and rubber surfaces than to glass and stainless steel (Mafu et

41

al., 1991). When studying the physicochemical characteristics of L. monocytogenes and
its attachment to glass, hydrophobicity and surface charge had no correlation to the
degree of cell attachment (Chae et al., 2006). The trends suggested by many of these
physicochemical measurements are influenced by conditions of the individual experiment
and the methods used to determine surface hydrophobicity. Studies have found that
surface soil (meat exudate, skim milk, various proteins) will enhance attachment, and
alter the contact surface physicochemical properties (Barnes et al., 1999; Midelet and
Carpentier, 2002). Physicochemical properties of the cell surface and contact surface
may enhance or inhibit the rate of initial attachment, but attachment was not completely
inhibited in any of the aforementioned studies.

After the reversible step of initial attachment, irreversible attachment can follow.
This is the process in which bacteria secrete exocellular polymeric substances or
exopolysaccharides (EPS) that function as a glue to hold the bacterial cell to the surface
and retain the organism in subsequently formed biofilm communities (Stoodley et al.,
2002). Development of biofilms requires water or a relative humidity (RH) above 84%,
with optimal biofilm grth at 100% RH (Else et al., 2003). Level and rate of L.
monocytogenes attachment can be used to predict the ability of strains to form biofilms
and persist in the environment. Lunden et al. (2000) reported that the same pulsed-field
gel electrophoresis (PF GE) type of L. monocytogenes was transferred to three different
processing plants in a dicing machine used in the three plants. The persistent strain was
then tested for adherence to stainless steel in broth culture at 25°C for 1, 2, and 72 h,
along with three non-persistent strains of L. monocytogenes isolated from the third plant.

The persistent strain was significantly more adherent than the non-persistent strains, a

42

trend that has also been observed in other studies (Lunden et al., 2000; Norwood and
Gilmour, 1999; Borucki et al., 2003). Kalmokoff et al. (2001) also reported variation in
the ability of different L. monocytogenes strains to adhere to stainless steel, and
subsequent biofilm formation by the strains varied as well, with only one strain capable
of forming a biofilm consisting of bacterial aggregates. Listeria was able to adsorb to
stainless steel within 2 h, and enhanced attachment was observed in strains that produced
extracellular fibrils. Enhanced attachment also has been observed by L. monocytogenes
grown at 20 - 25°C, which is optimal for flagella production (V atanyoopaisarn et al.,
2000)

Given the predominance of Lineage 11 L. monocytogenes strains in the
environment, these strains would be expected to show increased biofilm formation,
leading to greater persistence in the environment. However, results vary, with one study
reporting better biofilm formation by Lineage I strains (Dj ordjevic et al., 2002), while
another noted better biofilm formation by Lineage 11 strains (Borucki et al., 2003).

Mutations and environmental stress have an affect on L. monocytogenes biofilm
formation. The ability to respond to nutrient deprivation seems to be a requirement for
survival in a biofilm and also to cause illness. Mutants lacking in the ability to mount a
stringent response to amino acid deprivation were inhibited in their ability to attach to
surfaces, and were also avimlent in a mouse model (Taylor et al., 2002). Cell surface
proteins are also important for surface growth — the addition of 0.01% trypsin to growth
media can reduce the adherent cell population by 99.9%, as compared to control cultures
without trypsin (Smoot and Pierson, 1998). However, excess protein in growth media

inhibits cell attachment to surfaces, thus chemically defined minimal media results in

43

better biofilm formation by L. monocytogenes (Kim and Frank, 1994). This provides
further evidence that biofilm formation is a survival strategy for bacteria in stressful
environments, along with the observation that biofilm formation aids subsequent survival
upon exposure to sanitzers (Chavant et al., 2004; Lomander et al., 2004; Somers et al.,
2004; Robbins et al., 2005), and also improves resistance to dessication, possibly due to
retention of water by EPS or by limiting the surface area available to air, thus slowing
evaporation of water from the microbial community (Fleming and Wingender, 2002).
The presence of other microorganisms on a surface can also influence the level of
L. monocytogenes colonization. Listeria monocytogenes will attach in significantly
higher numbers (> 3 log CFU/cmz) to a condensate-covered surface containing a pre-
existing Pseudomonas putida biofilm (Hassan et al., 2004). The effect of interactions
with other resident flora can also be inhibitory to L. monocytogenes. In a study of 31
strains of resident microflora from a food processing environment, 16 strains inhibited
growth of L. monocytogenes on the surface, 11 strains had no effect, and 4 strains
enhanced L. monocytogenes biofilm formation, as opposed to pure culture (Carpentier
and Chassaing, 2004). Furthermore, one of the 4 synergistic strains, Comamonas
testosteroni CCL 24, a gram-negative organism which was isolated from a food
processing environment, released a metabolite into its biofilm growth medium
supernatant, which was sufficient to enhance L. monocytogenes biofilm growth in pure
culture (Carpentier and Chassaing, 2004). This interaction is similar to that from
quorum-sensing molecules, which have not been characterized in L. monocytogenes.
Quorum-sensing is the regulation of gene expression in response to changes in cell

population density via chemical signaling using oligopeptides produced by Gram-positive

44

bacteria, and N-acyl-homoserine lactones in Gram-negative bacteria (Miller and Bassler,
2001; Lazazzera, 2000). Thus, the problem of Listeria transfer becomes very complex if

strongly adhering cells in biofilms are to be studied under the most realistic scenarios.

1.13 Predictive modeling

Within the last decade, risk assessments have necessitated the development of
dynamic models that provide estimates of bacterial survival, growth, and distribution
throughout food processing and storage. Microbiological risk assessments depend upon
exposure assessments; however, these exposure assessments rely on existing data for
presence of bacteria in food products, the accuracy of which is limited by sample size and
test methods in existing prevalence surveys (Gardner, 2004). Predictive modeling can
account for variations in sample size and test method in existing prevalence surveys, and
can allow for estimations of microbial contamination levels, distribution and rate of

transfer in the environment.

1.14 Predictive modeling of bacterial growth

Predictive models for the growth and distribution of microorganisms can be
divided into three types as defined by Bemaerts et al. (2004):
1) Empirical models, which are derived from experimental data and are
essentially curve-fitting models;
2) Mechanistic models, which are a precise mathematical translation of

underlying biological mechanisms;

45

3) Semimechanistic models, which take elements of both empirical and
mechanistic models due to the complexity and knowledge gaps about all
possible underlying biological mechanisms resulting in the difficulty in
development of purely mechanistic models.

Numerous predictive growth models have been developed and compared by
researchers, including the Pathogen Modeling Program developed by the United States
Department of Agriculture (USDA; Buchanan and Phillips, 1990; Tamplin, 2002). The
more recent version of the USDA Pathogen Modeling Program has a pre-programmed
graphical user interface and generates graphs and tables for various growth parameters,
which can provide input based on the needs of each user (Tamplin, 2002). These models
have been developed to predict the growth of foodbome pathogens, including L.
monocytogenes, in foods based on pH, salt and sodium nitrite content, as well as storage
temperature (Houtsma etal., 1996; LeMarc et al., 2002; Tamplin, 2002). Recently, an
attempt has been made to cross-reference the raw data used to develop models with the
resultant predictive models, in order to increase transparency for the derivation of
models. This has resulted in the development of ComBase, which allows users to access

the growth curves upon which predictive models are based (Baranyi and Tamplin, 2004).

46

1.15 Predictive modeling of bacterial transfer

Schaffner (2004) has described the basic mathematical framework for modeling
L. monocytogenes cross-contamination in food processing plants, using the following
equations:
1) Raw product CFU x Cross-contamination rate = Environmental CFU
2) Environmental CF U x Persistence rate = Environmental reservoir CFU
3) Environmental reservoir CFU x Cross-contamination rate = Product
contact surface CFU
4) Product contact surface CFU x Persistence rate = Product contact surface
reservoir CFU
5) Product contact surface reservoir CFU x Cross—contamination rate =
Finished product CFU
Due to a lack of data for quantitative transfer of L. monocytogenes, the two models
Schaffner (2004) developed around this framework using a Monte Carlo simulation were
only able to track L. monocytogenes numbers and prevalence or L. monocytogenes
prevalence alone, but not L. monocytogenes concentrations within raw and finished

products. The model illustrates the additive effect, in which each fraction of transfer

“ f, ” is an additive fimction of the previous fraction or f, = fa * f ,, where
“ fa ” = raw product and
“ fb ” = cross contamination rate

Using the resultant models can help a processor determine whether an overall greater
reduction in L. monocytogenes prevalence in a plant could be achieved by requiring better

raw material quality or by improved sanitation efforts (Schaffner, 2004). Furthermore,

47

these models predict that low numbers of incoming persistent strains of L.
monocytogenes strains will eventually predominate in the finished product (Schaffner,
2004). In a model developed to determine the risk of cross-contamination of salads by
Salmonella spp. or Campylobacter spp. from chicken in domestic kitchens, a higher
probability of Campylobacter spp. entering salads was predicted, due to its higher
prevalence and level in chicken (Kusumaningrum et al., 2004).

A model with similar benefits, in terms of determining the best testing sites to
minimize contamination of ground beef produced using a commercial grinder, was
developed by Flores and Stewart (2004). According to the model, rather than random
sampling of a ground beef lot to determine E. coli 01 57:H7 contamination, a more
accurate determination of contamination of the lot could be obtained by testing the collar
that fixes the grinder die and blade to the meat grinder (Flores and Stewart, 2004). This
was based on the fact that samples from a lot made with a randomly selected piece of
beef trim contaminated with 2 log CFU of E. coli 01 57:H7 would test negative for E. coli
OlS7:H7. However, in each case, the collar tested positive for E. coli 01 57:H7 (Flores
and Stewart, 2004).

In models that have been specifically developed to assess transfer of L.
monocytogenes, one model was developed to determine the risk of L. monocytogenes
transfer and subsequent growth due to contact with bare hands or gloved hands (Perez-
Rodriguez et al., 2006). This model predicted that the highest risk of contamination
comes from handling raw and ready-to-eat meats with the same gloves. This risk was
higher than the risk of cross-contamination from bare, washed hands (Perez-Rodriguez et

al., 2006). According to calculations obtained from this model, L. monocytogenes on

48

hands would have to be reduced 80% by washing in order to achieve a 50% reduction in
L. monocytogenes on subsequently handled slices of ham with L. monocytogenes counts
exceeding the European Union Food Safety Objective for L. monocytogenes (2 log

CF U/ g at consumption) by the end of the potential storage period (Perez-Rodriguez et al.,

2006).

1.16 Goals of the current study

Thus far, Vorst (2005b) has developed the only model to predict L.
monocytogenes transfer during slicing of RTE delicatessen meats on a commercial
delicatessen slicer. The model is a linear model that predicts the number of CFU
transferred to any given slice, as well as the number CFU lost to the environment through
aerosols and bacterial death. Under the conditions tested by Vorst (2005b), this model
had a correlation coefficient varying from R2 = 0.40 when slicing salami, to over 0.90
when slicing turkey or bologna with a slicer blade inoculated at 8 log CFU/blade (Vorst,

2005b)

Empirical data obtained from four years of laboratory research in the current
study was used to validate the utility of the Vorst (2005b) model in predicting transfer of
L. monocytogenes after exposure to bacterial stress (cold-injury and chlorine-injury) and
prolonged (6 and 24 h) desiccation on stainless steel to turkey and salami. Additionally,
the model was also tested for its ability to predict L. monocytogenes transfer based on
strain persistence and biofilm formation. The underlying hypothesis for this study was
that strain persistence would have an affect on the survival and transfer of L.

monocytogenes to delicatessen meats, particularly after prolonged desiccation on stainless

49

steel. This research was conducted in response to Listeria transfer rates being identified
as a key informational gap in the Listeria Risk Assessment published by the US federal
government (FDA/USDA/CDC, 2003). Additional research is required in this area in
order to refine existing assessments of the risk to the public for contracting listeriosis

from the consumption of delicatessen-sliced RTE meats.

50

CHAPTER 2

VARIATION IN BIOFILM FORMATION BY LIS T ERIA MONOC Y T OGENES

STRAINS AT 4°C AND 22°C

51

2.1 ABSTRACT

Potential biofilm formation by Listeria monocytogenes on food contact surfaces
can lead to cross-contamination and further spread of Listeria in commercial and home
settings. Additional research on Listeria biofilm formation is needed to help better define
the impact of food preparation practices on listeriosis estimations being developed in
current risk assessments. This study characterized biofilm-forming capabilities of a
diverse set of 196 L. monocytogenes strains at 4 and 22°C. Listeria monocytogenes
isolates from food, environmental, veterinary and clinical sources comprised of 16
different ribotypes were assessed for biofilm formation in Modified Welshimer’s Broth
using 96-well untreated polystyrene microtiter plates (3 wells/strain x 3 replicates).
Following 4 and 60 days of incubation at 22 and 4°C, respectively, the microtiter plate
wells were emptied, rinsed and air-dried. Afler staining fixed cells with crystal violet, the
optical density (OD) of the resolubilized dye was read at 570 nm. At 22°C, 83% and
95% of the OD values were within one and two standard deviations of the mean -- 0.53 i
0.38 and 0.76, respectively. At 4°C, 92% and 97% of the OD’s were within one and two
standard deviations of the mean—0.12 : 0.10 and 0.20, respectively, with 109 of 196
(55%) strains failing to produce detectable biofilms at 4°C. Significant differences in
biofilm formation were observed between strains of the same ribotype. While most L.
monocytogenes strains formed biofilms at room temperature, appreciable biofilm
formation was typically absent at 4°C, thus suggesting the inability of most L.
monocytogenes strains to produce significant biofilms in otherwise clean cold storage

areas.

52

2.2 INTRODUCTION

Many bacteria, including Listeria monocytogenes, have the ability to attach and
persist on equipment over extended periods of time. Persistent strains may play an
important role in the contamination of ready-to-eat (RTE) foods. In one processing
facility, persistent L. monocytogenes strains were 8 times more likely to contaminate
finished product than transient strains (Lunden et al., 2003). According to Lunden et al.
(2003) transient strains were prevalent in both incoming raw product and the environment
before processing, but were not found in any post-cooking processing lines. Other
studies have reported that the same strain of L. monocytogenes are infrequently recovered
from both incoming raw products and final RTE products, leading to the conclusion that
persistent environmental strains are most often responsible for recontaminating fully-
cooked RTE products (N esbakken et al., 1996; Lappi et al., 2004).

Level and rate of L. monocytogenes attachment have been used to predict the
ability of strains to form biofilms and persist in the environment. Lunden et al. (2000)
reported that a persistent strain of L. monocytogenes was transferred to three different
processing plants via a dicing machine and was significantly more adherent than non-
persistent strains, a trend which also has been reported previously (Lunden et al., 2000;
Norwood and Gilmour, 1999; Borucki etal., 2003). However, Djordjevic et al. (2002)
found no significant difference in biofilm-forming ability of L. monocytogenes strains
according to genetic lineage or environmental persistence. Kalmokoff et al. (2001)
reported variation in the ability of L. monocytogenes strains to adhere to stainless steel
with the extent of subsequent biofilm formation also varying. However, only one of 36

strains was capable of forming a biofilm containing bacterial aggregates. Enhanced

53

attachment has been observed by L. monocytogenes grown at 20-25°C, which is optimal
for flagella production (Vatanyoopaisarn et al., 2000).

Initial bacterial attachment to surfaces occurs via electrostatic or hydrophobic
interactions between the bacterial cell surface and the contact surface (Arnold and Bailey,
2000). Despite having a negative surface charge (Dickson and Koohmaraie, 1989;
Chavant et al., 2002), some studies claim that the surface of L. monocytogenes is
hydrophobic (Dickson and Koohmaraie, 1989; Ukuku and F ett, 2002), while others
indicate that the surface is hydrophilic (Mafu et al., 1991; Briandet et al., 1999; Chavant
et al., 2002). These discrepancies in cell surface hydrophobicity may be due to strain-to-
strain variation as well as differences in the methods used to measure hydrophobicity.

The objective of this study was to first assess biofilm-forming ability by a diverse
collection of 196 L. monocytogenes strains comprised of veterinary, clinical, food and
environmental isolates. From this collection, a subset comprised of the weakest and
strongest biofilm formers was evaluated for differences in cell surface hydrophobicity via
hydrophobic interaction chromatography (HIC) to help identify the role of cell surface

hydrophobicity in biofilm formation.

54

2.3 MATERIALS AND METHODS

2.3.1 Listeria monocytogenes strains.

A diverse set of 196 L. monocytogenes strains, partially characterized by lineage,
serotype, ribotype and isolation source (Table 2.1), was assayed for biofilm formation at
22 d: 2°C and 4 :1: 2°C. Surface hydrophobicity was tested on a subset of 6 strains of L.
monocytogenes (CWD 33, CWD 182, CWD 205, CWD 578, CWD 730, and CWD 845).
All strains were maintained at -80°C in trypticase soy broth containing 0.6% (w/v) yeast
extract (TSB-YE; Becton Dickinson, Sparks, MD) and 10% (v/v) glycerol (Sigma

Chemical Company, St. Louis, MO).

2.3.2 Culture preparation.

All strains were grown in TSB-YE for 18 h at 37°C, and then streaked to plates of
trypticase soy agar containing 0.6% yeast extract (TSA-YE) (Becton Dickinson) to obtain
confluent growth after 18 h of incubation at 37°C. Thereafter, L. monocytogenes cells
were harvested from TSA-YE plates by flooding the agar surface with 10 ml of 0. 1%
sterile peptone, with the concentration of the resulting cell suspension estimated from
MacFarland Turbidity Standards (Acuff, 1992). The resuspended culture was serially
diluted to a final concentration of 102 CFU/ml in Modified Welshimer’s Broth (MWB),
which contained the following ingredients per liter: KH2P04 (6.56 g), NazHPO4 - 7H20
(30.96 g), MgSO4 - 7HzO (0.41 g), ferric citrate (0.088 g), glucose (10.0 g), L-leucine
(0.1 g), L-isoleucine (0.1 g), L-valine (0.1 g), L-methionine (0.1 g), L-arginine (0.1 g), L-

cysteine (0.1 g), L-glutamine (0.6 g), riboflavin (0.5 mg), thiamine (1.0 mg), biotin (0.5

55

mg) and thioctic acid (0.005 mg) (Premaratne et al., 1991). All components of MWB

were obtained from Sigma Chemical Company.

2.3.3 Microtiter plate assay for biofilm formation.

A modification of the assay described by Stepanovic et al. (2000) was used to
assess biofilm formation by L. monocytogenes. After vortexing, 200 pl of the diluted cell
suspension containing 102 CFU/ml was pipetted into three wells of a 96-well untreated
polystyrene microtiter tissue culture plate (BD Falcon MicrotestTM Flat Bottom; Becton
Dickinson, Franklin Lakes, NJ). Three wells per plate containing 200 pl of MWB served
as negative controls. Biofilm assays were carried out at 22 a; 2°C for 4 d and at 4 i 2°C
for 60 d. At the end of incubation, the microtiter plate wells were emptied, rinsed three
times with 0.85% physiological saline while being gently shaken to remove unattached
cells, and then allowed to air-dry. The remaining Listeria cells were fixed to the well by
adding 200 pl of 99% methanol (Fisher Chemicals, Fair Lawn, NJ) with the methanol
decanted 15 min later. After allowing the plates to air-dry, the microtiter wells were
stained with 200 pl of 2% crystal violet (Biochemical Sciences, Inc., Swedesboro, NJ) for
5 min. After decanting the crystal violet, the wells were rinsed five times with deionized
water and air-dried. The remaining dye was resolubilized in 160 pl of 33% (v/v) glacial
acetic acid (EM Science, Gibbstown, NJ) and optical densities were read at 570 nm using

a VmaxTM Kinetic Microplate Reader (Molecular Devices, Menlo Park, CA).

56

2.3.4 Measurement of cell surface hydrophobicity by hydrophobic interaction

chromatography.

After subculturing from the frozen stock cultures twice in TSB-YE, cultures were
centrifuged (9740 X g, 10 min) at 4°C. The resulting cell pellets were washed twice in a
salt peptone solution containing 0.85% NaCl and 0.05% Bacto Peptone (Becton
Dickinson). For hydrophobic interaction chromatography, capillary pipettes (16 cm long,
5 mm diameter) (Corning Labware, Corning, NY) were plugged with glass wool and
washed sequentially with 5 ml of 75% ethanol and 10 ml of 0.02 M NaPO4 buffer (pH
6.8) (Sigma). Columns were packed with octyl-sepharose CL-4B (Sigma) and
equilibrated overnight at 4°C in 12 ml of NaPO4 buffer. Washed bacterial cell
suspensions (0.1 ml) were loaded onto the surface of the column followed by 12 ml of
NaPO4 buffer and the eluent was collected as described by Dickson and Koohmaraie
(1989). Eluted solution was plated on Modified Oxford Agar (MOX; Becton Dickinson)
and incubated at 35°C for 24 h. The experiment was replicated three times for each
strain. Relative hydrophobicity was calculated according to Dickson and Koohmaraie

(1989) using following formula:

Relative hydrophobicity = (CFU retained by the column) / (CFU eluted from the

column).

When the log value of relative hydrophobicity was < 0, the cell surface was considered

hydrophilic.

57

2.3.5 Statistical Analysis.

All experiments were replicated three times. Statistical analysis of the OD values
from the complete set of 196 strains was performed using a general linear model with a
general randomized complete block design using SAS (SAS, Version 8, SAS Institute,
Inc. Cary, NC). Surface hydrophobicity data were analyzed using a general linear model.
Significance was determined at P < 0.05. Statistical significance for biofilm formation
by lineage, serotype, ribotype and source was determined using a linear mixed effects

model (significance at P < 0.05).

2.4 RESULTS

2.4.1 Biofilm formation by Listeria monocytogenes.

Overall, significant variations in biofilm forming ability were observed for this
diverse set of L. monocytogenes isolates. OD values at 22 and 4°C ranged from 0.061 to
2.61 and 0.05 to 0.92, respectively, (Table 2.1) and were skewed to the left at both
temperatures (Figures 2.1 and 2.2), indicating that most strains were relatively weak
biofilm formers, with a few outlying strains yielding higher optical densities indicative of
stronger biofilms. At 22°C, 83% and 95% of the OD values were within one and two
standard deviations of the mean—0.53 _t 0.38 and 0.76, respectively. Based on this
analysis, 64, 31, and 5% of the strains were classified as weak (OD < 0.53), medium (OD
0.53 - 1.28) and strong (OD > 1.28) biofilm formers at 22°C. At 4°C, 92% and 97% of
the OD values were within one and two standard deviations of the mean—0.12 : 0.10

and 0.20, respectively, with 159 of 196 (81%) strains failing to produce detectable

58

biofilms, defined as an OD value not significantly different from the MWB negative

control.

59

Table 2.1. L. monocytogenes strain information (strains listed in descending order

by biofilm forming ability at 22°C)

 

 

 

Mean OD
Strain ID Source Serotype Ribotype Lineage

4°C 22°C

CWD 845a dairy plant 1/2b 54081 NA” 0.10 2.61
CWD 730 dairy plant 1/2a 19092 NA 0.13 1.94
CWD 33 unknown 4b 19167 NA 0.14 1.91
CWD 1368 ground beef NA 54183 NA 0.08 1.80
CWD 1734 pork sausage 3b 54081 NA 0.05 1.62
CWD 338 dairy plant 1/2a 19092 NA 0.16 1.49
CWD 1440 unknown NA NA NA 0.08 1.37
CWD 764 hot dog 1/2b 28643 NA 0.15 1.31
CWD 1258 pork sausage NA 28623 NA 0.22 1.30
CWD 1632 ground beef NA 54184 NA 0.05 1.29
CWD 1032 pork sausage NA 54081 NA 0.42 1.28
CWD 600 dairy plant 1/2b 54081 NA 0.08 1.27
CWD 603 dairy plant NA 54081 NA 0.10 1.11
CWD 1520 ground turkey NA 19236 NA 0.07 1.10
CWD 1429 unknown NA NA NA 0.09 1.08
CWD 1733 pork sausage NA 54132 NA 0.06 1.07
CWD 1430 unknown NA NA NA 0.17 1 .06
CWD 766 hot dog 3a 19092 NA 0.13 1.02
CWD 580 dairy plant 1/2b 54081 NA 0.07 1.01
CWD 1011 pork sausage NA NA NA 0.06 0.97
CWD 25 unknown NA 19075 NA 0.09 0.97
CWD 831 dairy plant NA 19231 NA 0.12 0.97
CWD 1078 chicken NA 19161 NA 0.12 0.96

60

Table 2.1. (Cont’d)

own 1369
own 1728
cwo 1760
own 1634
own 1742
own 1038
own 1667
cwo 1157
own 371
own 1278
ems-3°
cwo 1061
ETR-7-1
ETR-2-4
ETR-7-2
own 1205
own 372
ETR-6-1
own 602
own 1118
own 1305
own 1648
CWD 701

FSL J1-116"

CWD 1094

ground beef
pork sausage
raw goat milk
ground beef
pork sausage
pork sausage
pork sausage
ground beef
dairy plant

pork sausage

pork processor

pork sausage

pork processor
pork processor
pork processor

ground turkey

dairy plant

pork processor

dairy plant
ground beef
chicken
chicken

cheese

human, epidemic,

UK, 1988-1990

chicken

NA
NA
NA
1/2b
NA
NA

NA

NA
NA
NA

NA

NA
1/2a
NA
1/28
NA
NA
NA

1/2b

40

NA

61

19071
19071
NA.
54081
19231
19071
19071
54132
NA.
54081
NA.
19231
NA.
NA.
NA.
19192
NA.
NAl
54183
54081
19071
19161
54135
DUP-
1042

54081

NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA

NA

NA

0.09
0.09
0.05
0.05
0.05
0.06
0.06
0.14
0.16
0.10
0.16
0.12
0.07
0.20
0.16
0.11
0.07
0.32
0.05
0.09
0.06
0.05
0.06

0.14

0.07

0.96
0.93
0.93
0.88
0.88
0.87
0.87
0.84
0.84
0.83
0.83
0.81
0.81
0.79
0.79
0.78
0.78
0.77
0.76
0.75
0.75
0.72

0.72

0.70

0.69

Table 2.1. (Cont’d)

CWD 271
ETR-7-3
CWD 1664
CWD 1724
CWD 1768
ETR-2-5
ETR-7-5
CWD 2087
CWD 1461
CWD 1776
CWD 878

FSL J1 -094

CWD 102

FSL J2-064

CWD 1120
cwo 1176
cwo 1438
cwo 210
ETR-6-2
cwo 1298
CWD 1706
CWD 273
Cwo 811

dairy plant
pork processor
pork sausage
chicken

raw goat milk
pork processor
pork processor
unknown
unknown

raw goat milk
human clinical
human, sporadic
case

silage

animal, cow

ground beef
ground turkey
unknown

raw milk

pork processor
chicken
ground beef
dairy plant

dairy plant

4b

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

1/20

NA

1/20

NA

1/2b

NA

NA

NA

NA

NA

NA

NA

62

19161
IVA
54132
19231
NA.
INA
IVA
NA.
NA.
NA.
19161
DUP-
1030
19075
DUP-

1052/

dd 1962

19071
19192
NA.

19092
NA.

19161
19071
19161

19092

NA
NA
NA
NA
NA
NA
NA
NA
NA
NA

NA

NA

NA
NA
NA
NA
NA
NA
NA
NA

NA

0.05
0.34
0.05
0.06
0.05
0.07
0.15
0.06
0.11
0.07
0.08

0.12

0.08

0.10

0.26
0.16
0.05
0.05
0.16
0.06
0.05
0.05
0.08

0.69
0.68
0.67
0.67
0.67
0.65
0.65
0.64
0.63
0.62
0.62

0.62

0.61

0.61

0.60
0.59
0.58
0.58
0.58
0.57
0.54
0.54

0.53

Table 2.1. (Cont’d)

CWD 939

FSL C1-122

CWD 1427

CWD 725

FSL J1-177

CWD 1002
CWD 1424
CWD 1528
CWD 1624
CWD 1 198
CWD 680
CWD 897
ETR-3—3
ETR-1-1
CWD 1 191
CWD 1 318
CWD 1433
CWD 184
CWD 224
CWD 317
CWD 631
ETR-2-3
I:SL J2-035
CWD 685

dairy plant
human, sporadic
case

unknown

cow brain
human sporadic
case

pork sausage
unknown
ground turkey
unknown
ground turkey
cow udder
dairy plant
pork processor
pork processor
ground turkey
chicken
unknown

raw milk

dairy plant
dairy plant
unknown

pork processor
animal, goat

cow udder

NA

40

NA

NA

1/2b

1/20

NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA

NA

1/20

NA

63

19186
DUP-
10383
NA.
IVA
DUP-
1024
19071
NA.
54183
19231
19231
19071
19103
NA.
NA.
19157
18647
NA.
19092
19167
19092
54081

NA

dd 3581

19078

NA

NA

NA

NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
|

NA

0.05

0.15

0.15

0.11

0.13

0.08
0.07
0.05
0.05
0.08
0.15
0.10
0.69
0.22
0.23
0.15
0.06
0.20
0.10
0.09
0.08
0.17
0.12
0.08

0.53

0.53

0.52

0.51

0.51

0.50
0.50
0.50
0.50
0.49
0.49
0.49
0.49
0.46
0.45
0.45
0.45
0.45
0.44
0.44
0.44
0.44
0.44

0.43

Table 2.1. (Cont’d)

CWD 1092
CWD 1224
CWD 1281
CWD 1566
CWD 1603
CWD 1709

ETR-5—3

FSL R2-500

CWD 1677
CWD 1807
CWD 531

ETR-1-4

FSL J2-020

CWD 1223
ETR-3-2
ETR-5—5

F SL R2-501

CWD 1420

CWD 1436
ETR-1-2

chicken

pork sausage
pork sausage
unknown
unknown
ground beef
pork processor
food, epidemic,
North Carolina
(2000)

pork sausage
raw goat milk
dairy plant

pork processor

animal, cow

pork sausage
pork processor
pork processor
human, epidemic,
North Carolina
(2000)

unknown
unknown

pork processor

NA
NA
NA
NA
NA
NA

NA

4b

NA
NA
NA

NA

1/26

1/2b

NA

NA

4b

NA

NA

NA

64

54183
19071
19071
19074
19161
54132

NA

DUP-

10423

1 9161
NA

1 9092
NA
DU P-
1 0390
28647
NA

NA

DUP-

1042B

NA
NA

NA

NA
NA
NA
NA
NA
NA

NA

NA
NA
NA
NA

NA
NA

NA

NA
NA

NA

0.15
0.07
0.09
0.08
0.06
0.05
0.92

0.13

0.05
0.15
0.11

0.25

0.10

0.09
0.35

0.18

0.13

0.05

0.06

0.21

0.42

' 0.42

0.42
0.41
0.41
0.41

0.41

0.41

0.39
0.39
0.39
0.39

0.38

0.37
0.37

0.37

0.37

0.36

0.36

0.36

Table 2.1. (Cont’d)

FSL J1-049

own 1790
ETR-3-5
ETR-6-4

FSL J1-169

CWD 1287

ETR-3-4

FSL R2-502

ETR-5-2

FSLJ1-119

CWD 1 108
CWD 1326
CWD 1448
ETR-2-1
ETR-2-2
ETR-7-4

FSL J1-126

FSL J2-063

human, sporadic
case

raw goat milk
pork processor

pork processor

human, sporadic

pork sausage
pork processor
food, epidemic,
Illinois (1994)
pork processor
human, epidemic,
LA, 1985
chicken

chicken

unknown

pork processor
pork processor
pork processor
human, epidemic,

Switzerland, 1987

animal, sheep

3c

NA
NA

NA

3b

NA

NA

1/2b

NA

4b

NA
NA
NA
NA
NA
NA

40

1/2a

65

DUP-
1042
NA.
NA.
NA.
DUP-
1052
19161
NA.
DUP-
1051B
NA.
DUP-
1038
54132
19192
NA.
NA.
NA.
NA.
DUP-
1038
DUP-
1047/ dd

1153

NA
NA

NA

NA

NA

NA

NA
NA
NA
NA
NA

NA

0.09

0.10
0.28
0.06

0.11

0.06

0.24

0.11

0.22

0.06

0.08
0.05
0.11
0.12
0.23

0.14

0.13

0.12

0.36

0.35
0.35

0.35

0.35

0.34

0.34

0.34

0.33

0.33

0.32
0.32
0.32
0.32
0.32
0.32

0.32

0.32

Table 2.1. (Cont’d)

FSL N1 -225

CWD 30
CWD 431
CWD 1656
CWD 180
CWD 1 81 7
CWD 575
ETR—5-1
CWD 1066
CWD 1418
CWD 1573

ETR-5-4

FSL N3-031

CWD 1 814
CWD 554
ETR-1-3
CWD 131 3
CWD 852
ETR-3—1

F SL R2-503

CWD 246
ETR-6-5
CWD 1525

human, epidemic
(US 1998-99)
French Brie
cow udder
chicken

human clinical
raw goat milk
dairy plant
pork processor
pork sausage
unknown
unknown

pork processor
food (hot dog),
sporadic, US
raw goat milk
dairy plant
pork processor
chicken

dairy plant

pork processor

human, epidemic,

Illinois (1994)
silage
pork processor

ground turkey

4b

NA
NA
NA
NA
NA
NA
NA
NA
NA
NA

NA

1/2a

NA

NA

NA

NA

NA

NA

1/2b

NA

NA

NA

66

DUP-

1044A
19106
19071

54084

19161

NA.
54081
NA.
54135
NA.
19074
NA.
pth
1053
NA.
54081
NA.
19231
19092
19A
ENJP-
10518
19193
IVA

54084

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

0.13

0.08
0.12
0.08
0.07
0.06
0.07
0.11
0.06
0.20
0.06

0.49

0.11

0.05
0.16
0.14
0.11
0.13
0.26

0.13

0.15
0.18

0.05

0.32

0.31
0.31
0.30
0.30
0.30
0.30
0.30
0.29
0.29
0.29
0.29

0.29

0.28
0.28
0.28
0.27
0.27

0.27

0.27

0.26
0.26

0.25

Table 2.1. (Cont’d)

CWD 1723 chicken NA 54084 NA 0.05 0.25
CWD 95 silage NA 19071 NA 0.16 0.25
human, sporadic DUP-
FSL J1-031 43 Ill 0.12 0.25
case 1059A
CWD 1521 ground turkey NA 19071 NA 0.09 0.24
CWD 1789 raw goat milk NA NA NA 0.05 0.24
CWD 1794 raw goat milk NA NA NA 0.05 0.24
CWD 475 dairy plant NA 19071 NA 0.05 0.24
food, epidemic, DUP-
FSL N3-013 40 I 0.07 0.24
UK, 1988-1990 1042
human, epidemic
DUP-
FSL 122-499 (sliced turkey) 1/2a II 0.11 0.24
1053
(2000)
CWD 1332 chicken NA 54084 NA 0.06 0.23
DUP-
FSL C1-115 human, sporadic 3a II 0.12 0.23
1039C
food, epidemic, DUP—
FSL N3-022 4b l 0.08 0.23
Switzerland, 1987 1038
unknown
FSL W1-112 4a dd 6824 III 0.07 0.23
(formerly X1-010)
CWD 561 dairy plant 1/2a 19071 NA 0.09 0.22
CWD 909 dairy plant NA 19092 NA 0.10 0.22
unknown
FSL W1-110 4c dd 3823 III 0.15 0.22
(formerly X1-008)
CWD 24 unknown NA 19071 NA 0.10 0.21
ETR-l-s pork processor NA NA NA 0.09 0.21

67

Table 2.1. (Cont’d)

FSL J1-108

FSL J1-225

FSL J2-054

CWD 570

FSL J1-158

FSL J1-168

CWD 179

CWD 923

FSL C1-056

FSL J1-023

FSL J2-031

FSL M1-004

CWD 182

CWD 578

human, epidemic,
Halifax, 1981
human, epidemic
(Mass, 1983, Scott

A)

animal, sheep

dairy plant

animal, goat

human, sporadic
case

cow brain

dairy plant
human, sporadic

case

unknown

animal, bovine

human, sporadic
case
unknown

dairy plant

4b

4b

1/2a

NA

4b

4a

NA

NA

1/28

38

1/2a

N/A

4b

4d

68

DUP-

1038

DUP-

1 042

DUP-

1045/dd

1067
19092
ENJP-
10142
[NJP-
1061
19075
54184
DUP-
1030
DLWL
10143

DUP-

1039/dd

6362
DUP-
10398
19078

19161

NA

NA

NA

NA

NA

0.10

0.11

0.12

0.12

0.15

0.13

0.06

0.08

0.08

0.09

0.19

0.07

0.06

0.08

0.21

0.21

0.20

0.19

0.19

0.19

0.18

0.18

0.18

0.18

0.18

0.18

0.17

0.17

Table 2.1. (Cont’d)

food, epidemic DUP-

FSL N1-227 4b I 0.08 0.17
(US 1998-99) 1044A
human (hot dog), DUP-

FSL J1-101 1/2a II 0.16 0.16
sporadic, US 1053

ATCC 19115 unknown 40 NA NA 0.06 0.14

CWD 205 unknown 4c 19078 NA 0.21 0.14

DUP-
FSL J2—066 animal, sheep 1/2a 1054/ dd II 0.14 0.13
3075

food, epidemic, DUP-

FSL J1-110 4b l 0.10 0.12
LA. 1985 1038
food, epidemic, DUP-

FSL N3-008 40 I 0.10 0.08
Halifax, 1981 1038
unknown

FSL W1-111 40 dd 6821 III 0.08 0.08
(formerly X1-009)

 

aCWD strains provided by Dr. Catherine Donnelly, University of Vermont

b NA = Not available

° ETR strains from Michigan State University, Department of Food Science and Hmnan
Nutrition Culture Collection

d FSL strains provided by Dr. Martin Wiedmann, Cornell University

69

Figure 2.1. Distribution of optical densities at 4°C for 196 L. monocytogenes isolates

 

70-

60‘

 

50‘

40‘

 

M

30

20“

Percent of total strains

10'

 

 

 

 

 

 

 

\%

0 I I I l T l l l J l

0.05 0.15 0.25 0.35 0.45 0.55 0.65 0.75 0.85 0.95

Least Squares Mean ODs-zonm

 

 

Figure 2.2. Distribution of optical densities at 22°C for 196 L. monocytogenes

isolates

 

50“

4O

 

\

 

30‘

20'

\

\

 

Percent of total strains

 

 

 

 

 

 

 

 

 

0 I I l l I I T l ' I

0.15 0.45 0.75 1.05 1.35 1.65 1.95 2.25 2.55
Least Squares Mean ODsyonm

70

Significant differences in biofilm-forming ability were observed based on source
(Table 2.2) and lineage (Table 2.3) of the isolate. Food and environmental isolates were
significantly better at forming biofilms than clinical and veterinary isolates. Lineage I
strains were significantly better at forming biofilms than Lineage 111 strains. Significant
differences were also observed based on serotype, with serotype l/2b forming
significantly stronger biofilms than serotypes 1/2a and 4b (Table 2.4). However for some
serotypes no significant differences were observed, possibly due to the small sample size
and large standard deviation. No significant differences were observed in biofilm
forming ability by ribotype when analyzing ribotypes of which there were three or more
strains.

Table 2.2. L. monocytogenes biofilm formation at 22°C by source

 

Source n Mean 00570 Std. D. Range

 

Clinical 22 0.34a 0.16 0.16 — 0.70
Environment 58 0.56bc 0.44 0.17 — 2.61
Food 74 0.61b 0.36 0.08 - 1.80

Veterinary 13 0.34“ 0.15 0.13 - 0.61

 

Means with different letters are significantly different (P < 0.05).

71

Table 2.3. L. monocytogenes biofilm formation at 22°C by lineage*

 

Lineage n Mean 00570 Std. D. Range

 

l 21 0.348 0.16 0.08 — 0.70
ll 12 0.26ab 0.14 0.13 - 0.62
Ill 7 0.19b 0.05 0.08 - 0.25

 

* FSL strains provided by Dr. Martin Wiedmann, Cornell University

Means with different letters are significantly different (P < 0.05).

Table 2.4. L. monocytogenes biofilm formation at 22°C by serotype

 

Serotype n Mean OD570 Std. D. Range

 

1/2a 14 0.52ad 0.55 0.13 — 1.94
1/2b 13 0.84be 0.63 0.27 — 2.61
1/2c 2 0.56“ 0.09 0.50 - 0.62
36 3 0.48 0.47 0.18 — 1.02
3h 2 0.99(‘3 0.90 0.35 — 1.62
3c 1 0.36 NA NA

4a 3 0.228 0.03 0.19 — 0.25
46 15 0.39ad 0.46 0.08 - 1.91
4c 3 0.15c 0.07 0.08 — 0.22
4d 1 0.17 NA NA

 

Means with different letters are significantly different (P < 0.05).

72

2.4.2 Surface hydrophobicity of weak and strong biofilm formers.

Based on these results, the three strongest and three of the weakest biofilm
formers were further evaluated for hydrophobicity using hydrophobic interaction
chromatography. The three weakest biofilm forming strains (CWD 182, CWD 205, and
CWD 578) were significantly more hydrophobic than the three strongest biofilm forming
strains (CWD 33, CWD 730, and CWD 845) (Figure 2.3). However, while one of the
weakest biofilm formers (CWD 578) was slightly hydrophilic, its cell surface
hydrophobicity was not significantly different from the other more hydrophobic weak
biofilm forming strains.

Figure 2.3. Log relative hydrophobicity of weak and strong biofilm forming strains
of L. monocytogenes

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

0.2 7
J "l'
E, 0.1 l _
.2 i
.D ..
3 or C3 " E:
3 i l-
5 I 578(W)b
E: -01 182;.(W)205(W)
>
'5 02
- . j
g ._
3’ 33(3) I
-I -0.3 e, a
i
730 ~-
414 l (S)b845(S)c

L. monocytogenes Strain

(S) = Strong biofilm forming L. monocytogenes strain
(W) = Weak biofilm forming L. monocytogenes strain
Means with different letters (a, b, or c) are significantly different (P < 0.05).

73

2.5 DISCUSSION

Microtiter plate assays provide an indirect means to screen many different strains
for quantitative differences in biofilm formation (Stepanovic et al., 2000; Djordjevic et
al., 2002; Borucki et al., 2003). While unable to precisely mimic conditions encountered
in food processing environments, these biofilm assays can be used to rapidly screen and
identify specific bacterial strains for further use in more labor-intensive studies.
Researchers have previously used microtiter plate assays to evaluate biofilm formation by
different strains of L. monocytogenes on polyvinyl chloride (PVC) at 32°C after 20 and
40 h (Djordjevic et al., 2002) and at 30°C after 40 h (Borucki et al., 2003). Both studies
compared biofilm formation by persistent and non-persistent strains. According to
Borucki et al. (2003), persistent strains were better able to form biofilms. However,
Djordjevic et al. (2002) observed no such difference between persistent and non-
persistent strains. Additionally, while Borucki et al. (2003) reported greater biofilm
formation by L. monocytogenes strains belonging to Lineage II, Djordjevic et al. (2002)
observed greater biofilm formation in Lineage I strains. Our results differed from both of
these studies. While we found no significant difference in biofilm formation between
Lineages I and 11, significant differences were observed between Lineage I and III strains,
with the latter producing significantly weaker biofilms. Given the predominance and
increased persistence of Lineage 11 strains in food processing environments, these strains
would be expected to produce stronger biofilms as shown from our data. In general,
significantly better biofilm formation was observed among environmental and food as
opposed to clinical and veterinary isolates, however, lineage data were only available for

the 40 strains obtained from Wiedmann. Unlike the study by Borucki et al. (2003),

74

significant differences in biofilm formation were observed between the three major
serotypes — 1/2a, 1/2b and 4b. In addition to the use of different strain sets varying
according to size, isolation source and genetic diversity, considerable strain-to-strain
variation in biofilm formation leading to large standard deviations complicates any direct
comparison of results from other studies. In this study, the same strains used by
Djordjevic et al. (2002) were analyzed, with different results seen in their biofilm
forming ability in relation to each another. In addition, the OD values were generally far
lower than those reported by Djordjevic et al. (2002), with this outcome likely due to
procedural differences in the microtiter plate assay and incubation temperatures.
However, even though the identical strains used by Djordjevic et al. (2002) generally
yielded lower OD values in our study, a far greater range in biofilm formation was
observed at 22°C for all 197 strains, as measured by OD (0.078 - 2.605).

For those strains at either extreme in biofilm formation, differences were observed
in cell surface hydrophobicity. Isolates that were particularly strong biofilm formers in
this study were significantly more hydrophobic than those strains forming extremely
weak biofilms. Reports from the literature vary greatly concerning hydrophobicity of the
L. monocytogenes cell surface, and the pathogen’s subsequent interactions with food
contact surfaces. Smoot and Pierson (1998) found that the rate of L. monocytogenes
attachment to stainless steel was faster than attachment to Buna-N rubber, although cell
surface hydrophobicity and surface free energies predict that adhesion to Buna-N rubber
is favored. Another study also found that hydrophilic, negatively-charged cells of L.
monocytogenes adhered better to stainless steel, than to polytetrafluoroethylene (Chavant

et al., 2002). Briandet et al. (1999) found that although L. monocytogenes tends to be

75

hydrophilic, strains that are slightly more hydrophobic than others adhere better to
stainless steel. However, Midelet and Carpentier (2002) observed stronger attachment of
L. monocytogenes biofilms to polymers (polyvinyl chloride and polyurethane) than to
stainless steel, and also noted that meat exudates changed these surfaces from
hydrophobic to hydrophilic as determined by contact angle measurement. According to
Cunliffe et al. (1999), hydrophilic uncharged surfaces were slightly repellent to L.
monocytogenes. In other work, L. monocytogenes Scott A was more attracted to
polypropylene and rubber surfaces than to glass and stainless steel (Mafu et al., 1991). In
a study of the physicochemical characteristics of L. monocytogenes and its attachment to
glass, hydrophobicity and surface charge had no correlation to the extent of cell
attachment (Chae et al., 2006).

Differences seen in the previous studies may be due to the choice of strains tested.
If these strains were randomly chosen rather than because they frequently appear in the
existing literature (e.g., L. monocytogenes L028, and Scott A), interpretation of these
findings may be difficult without first knowing how the strains behave relative to one
another on surfaces. In this study, Scott A was not a strong biofilm former, which may
be correlated to a more hydrophilic cell surface compared to the strong biofilm formers.
However, extrapolating the impact of cell surface hydrophobicity to biofilm-forming
ability by all L. monocytogenes based on a few well-studied strains may lead to

inaccurate assumptions about L. monocytogenes interactions with food contact surfaces.

76

CHAPTER 3

VARIATION IN BIOFILM FORMATION BY HEALTHY AND COLD-,
STARVE-, ACID-, AND CHLORINE-STRESSED LIS TERM

MONOC Y T OGENES

77

3.1 ABSTRACT

Presence of Listeria monocytogenes strains endemic to food processing
environments is presumably related to biofilm formation. Following exposure to various
environmental stresses, Listeria cells may be more prone to attach to surfaces. This study
quantified the degree of biofilm formation in a defined set of L. monocytogenes strains
when uninjured, cold-starved, cold-shocked, acid-shocked and chlorine-shocked.
Twenty-six L. monocytogenes strains (including clinical, food and dairy plant isolates)
were selected from a set of 196 strains previously characterized for biofilm formation. L.
monocytogenes (102 CFU/ml) was subjected to the previously mentioned stresses.
Uninjured cultures were used as controls. Biofilm formation by the uninjured and injured
bacteria was quantified in Modified Welshimer’s Broth (MWB) using 96-well untreated
polystyrene microtiter plates (3 wells/strain x 3 replicates). Following 4 days of
incubation at 22°C, the microtiter plate wells were emptied, rinsed and air-dried. After
staining cells that were fixed in 99% methanol with crystal violet, biofilm formation as
measured by optical density (OD) of the resolubilized dye was read
spectrophotometrically at 570 nm. Prior injury of L. monocytogenes by starvation
(28.5% injured) and cold (39.2% injured) lead to enhanced biofilm formation. Acid
injured (60.2% injured) and chlorine injured (55.1% injured) cultures showed a
diminished ability to form biofilms. Strains that comprised the extreme OD values in
biofilm formation remained consistent between all three treatments. Dairy plant isolates

did not predominate as strong biofilm formers.

78

3.2 INTRODUCTION

Prior to coming in contact with food processing surfaces, L. monocytogenes may
be exposed to various environmental stresses including nutrient deprivation, refrigeration
temperatures, low pH, limited available water, and sanitizers. These stresses can alter the
sensitivity of L. monocytogenes to other subsequent stresses, sometimes making it more
difficult to eradicate Listeria from the environment (Lou and Yousef, 1997;
Koutsoumanis et al., 2003; Koutsoumanis and Sofos, 2004; Gravesen et al., 2005;
Moorman et al., 2005).

Listeria is able to grow at refrigeration temperatures by altering its membrane
composition in order to maintain membrane fluidity and increase passive permeability.
This is achieved through changes in fatty acid composition (Neunlist et al., 2005). When
exposed to cold starvation conditions, L. monocytogenes undergoes shrinkage of the
cytoplasm, eventually resulting in holes in the cytoplasm (Dykes, 1999). The ability of L.
monocytogenes to respond to nutrient deprivation is a likely requirement for survival in a
biofilm. Mutants lacking the ability to mount a stringent response to amino acid
deprivation showed decreased attachment to surfaces, and were also avirulent in a mouse
model (Taylor et al., 2002).

Acid tolerance by L. monocytogenes has been studied by several researchers.
Listeria is able to better withstand lethal acid concentrations (pH 3.5) after habituation to
sublethal acid stress (pH 5-6), with maximum acid tolerance induced by habituation to
pH 5.5 (Koutsoumanis and Sofos, 2004; Koutsoumanis et al., 2005). Listeria
monocytogenes can survive without loss of viability for at least 20 h at pH 4.0, and also at

pH 3.5 in the presence of glucose (Shabala et al., 2002). In order to survive, L.

79

monocytogenes maintains a higher intracellular pH than the surrounding acidic
environment. While able to maintain an intracellular pH of 707.5 in glucose-containing
environments pH as low as 4.0, in the absence of glucose, L. monocytogenes is unable to
maintain a higher intracellular pH at pH 5.5 (Shabala et al., 2002). Other physiological
changes in response to acid stress include changes in protein synthesis and fatty acid
composition of the cell membrane (Koutsoumanis and Sofos, 2004). Changes to the cell
membrane result in increased surface hydrophobicity of acid adapted L. monocytogenes
(Lou and Yousef, 1997) with such changes having the potential to impact initial
attachment of Listeria to surfaces. For example, exposure to sublethal concentrations of
ethanol and isopropanol increased L. monocytogenes attachment at 10, 20 and 30°C
(Gravesen et al., 2005). Other oxidative stresses, such as chlorine and sanitizer stress
likely affect cellular proteins as evidenced by the fact that protein synthesis is essential
for subsequent cell repair and growth (Flanders et al., 1995).

Exposure to the aforementioned stresses can alter Listeria sensitivity to
quaternary ammonium sanitizers. After exposure to acid or starvation stress, Listeria
innocua was less sensitive to the quaternary ammonium sanitizer, cetrimide (Moorman et
al., 2005). This cross-protection did not occur after exposure to cold and heat stress,
which increased L. innocua sensitivity to cetrimide (Moorman et al., 2005). Listeria
monocytogenes strains that are resistant to quaternary ammonium compounds, other
sanitzers and antibiotics have been found to contain a gene (mer) that encodes an efflux
pump (Romanova et al., 2002). However, some resistant isolates do not appear to rely on
efflux pumps, and instead alter their cell membrane fatty acid profile in response to

sanitizer stress, which can prevent entry of foreign chemicals into the cytoplasm (To et

80

al., 2002). The same study also found that upon initial (1St 30 h) exposure to sublethal
levels of benzalkonium chloride, biofilm growth was favored (To et al., 2002).

Many types of bacteria, including L. monocytogenes, have the ability to attach and persist
on equipment over extended periods of time. Initial attachment occurs via electrostatic or
hydrophobic interactions between the bacterial surface and the contact surface (Arnold
and Bailey, 2000). It is thought that the rate of initial attachment is predictive of ultimate
biofilm forming ability—level and rate of L. monocytogenes attachment have been
correlated to the ability of strains to form biofilms and persist in the environment
(Lunden et al., 2000). Several studies have found that persistent strains of L.
monocytogenes are significantly more adherent than non-persistent strains (Lunden et al.,
2000; Norwood and Gilmour, 1999; Borucki et al., 2003). If biofilm-forming ability is
affected by hydrophobic or electrostatic interactions, then alterations to the cell
membrane from exposure to environmental stress suggest that biofilm formation should
be different from that seen in unstressed healthy cells. Hence, the objective of this study
was to determine whether exposure to common environmental stresses would alter
biofilm formation by a set of 26 L. monocytogenes isolates of varying biofilm-forming

abilities.

81

3.3 MATERIALS AND METHODS

3.3.1 Listeria monocytogenes strains.

A subset of 26 L. monocytogenes strains (Table 3. l) were selected from a larger
subset of 196 strains so that the distribution of their biofilm forming abilities as measured
by optical density reflected that of the entire set of 196 strains. The strains were assayed
for biofilm formation at 22 :t 2°C after being subjected to injury. All strains were
maintained at -80°C in trypticase soy broth containing 0.6% (w/v) yeast extract (TSB-
YE; Becton Dickinson, Sparks, MD ) and 10% (v/v) glycerol (Sigma Chemical

Company, St. Louis, MO).

3.3.2 Culture preparation.

All strains were subcultured in TSB—YE (Difco), incubated 18 h at 37°C, and
streaked to plates of trypticase soy agar containing 0.6% yeast extract (TSA-YE; Difco)
to obtain confluent growth after 18 h of incubation at 37°C. L. monocytogenes was
harvested from TSA-YE plates by flooding the agar surface with 10 ml of 0.1% sterile
peptone and suspending the cells using a sterile 10 pl inoculating loop (Becton
Dickinson). Cells were pipetted into a test tube and the concentration of the resuspended
culture was estimated from MacFarland Turbidity Standards (Acuff, 1992). The
resuspended culture was serially diluted to a final concentration of 102 CFU/ml in
Modified Welshimer’s Broth (MWB), which contained the following ingredients per
liter: KH2P04 (6.56 g), NazHPO4 - 7H20 (30.96 g), MgSO4 ° 7HzO (0.41 g), ferric citrate

(0.088g), glucose ( 10.0 g), L-leucine (0.1 g), L-isoleucine (0.1 g), L-valine (0.1 g), L-

82

methionine (0.1 g), L-arginine (0.1 g), L-cysteine (0.1 g), L-glutamine (0.6 g), riboflavin
(0.5 mg). thiamine (1.0 mg), biotin (0.5 mg) and thioctic acid (0.005 mg) (Premaratne et

al., 1991). All components of MWB were obtained from Sigma Chemical Company.

83

Table 3.1. L. monocytogenes strains and sources

 

 

Strain ID Source Serotype
CWD 1002a Pork Sausage 1/2c
CWD 1176 Ground Turkey 1/2b
CWD 1223 Pork Sausage 1/2b
CWD 1634 Ground Beef 1/2b
CWD 1734 Pork Sausage 3b
CWD 182 Unknown 4b
CWD 205 Unknown 4c
CWD 271 Dairy Plant Environment 4b
CWD 33 Unknown 4b
CWD 338 Dairy Plant Environment 1/2a
CWD 372 Dairy Plant Environment 1/2a
CWD 561 Dairy Plant Environment 1/2a
CWD 578 Dairy Plant Environment 4d
CWD 580 Dairy Plant Environment 1/2b
CWD 600 Dairy Plant Environment 1/2b
CWD 602 Dairy Plant Environment 1/2a
CWD 701 Cheese 1/2b
CWD 730 Dairy Plant Environment 1/23
CWD 764 Hotdog 1/2b
CWD 766 Hotdog 38
CWD 845 Dairy Plant Environment 1/2b
FSL J1-119b Human, epidemic, LA, 1985 4b
FSL J1-225 Human epidemic (Mass, 1983; Scott A) 4b
FSL N1-225 Human epidemic (US 1998—99) 4b
FSL R2-499 Human, epidemic (sliced turkey) (2000) 1/2a
FSL R2-501 Human, epidemic, North Carolina (2000) 4b

 

a CWD strains provided by Dr. Catherine Donnelly, University of Vermont

b F SL strains provided by Dr. Martin Wiedmann, Cornell University

84

3.3.3 Acid injured.

L. monocytogenes cells were harvested from TSA-YE into 10 ml of sterile TSB-
YE (pH 5.3) by flooding the agar surface with 10 ml of acidified TSB-YE and
suspending the culture using a sterile inoculating loop. After 4 h of incubation at 4°C the
cultures were serially diluted to contain 102 CFU/ml in MWB, as determined by
comparison to MacFarland Turbidity Standards.

3.3.4 Cold shocked.

L. monocytogenes cells were harvested from TSA-YE into 10 ml of sterile 0.1%
peptone broth and serially diluted to a final concentration of 102 CFU/ml (as determined
by comparison to MacFarland Turbidity Standards) in pre-chilled MWB. The cells were
then incubated for 2 h in a 4°C water bath.

3.3.5 Cold starved.

L. monocytogenes cells were harvested from TSA-YE into 10 ml of sterile
Butterfield’s Phosphate Buffer (BPB) and then centrifuged (Super T21, Sorvall Products,
Newtown, CT) at 9740 X g for 15 min at 4°C in sterile 50 ml polypropylene centrifuge
tubes (Corning Inc., Corning, NY). After resuspending the pellet in 10 ml of BPB,
starvation was achieved by holding the cells for 10 d at 4°C. Following incubation, the
cultures were serially diluted to a final concentration of 102 CFU/ml (as determined by
comparison to MacFarland Turbidity Standards) in MWB.

3.3.6 Chlorine injured.

L. monocytogenes cells were harvested from TSA-YE into 10 ml of sterile

phosphate buffered saline (PBS), then serially diluted into PBS containing 100 ppm

chlorine (Clorox; The Clorox Company, Oakland, CA) for 1 minute. Following

85

exposure, each strain was serially diluted into Neutralizing Buffer (Difco) to inactivate
the chlorine and then serially diluted in MWB to contain 102 CFU/ml, as determined by
comparison to MacFarland Turbidity Standards.

3.3.7 Quantification of injury.

After each of injury treatment, injured cultures were spread-plated on Tryptose
Phosphate Agar (TPA; Difco) and TPA with 4.5% NaCl (TPAN). Percent injury was
determined according to the following equation: % Injury = {(Count on non-selective
medium — count on selective medium) / (count on non-selective medium)} X 100
(Mathew and Ryser, 2002). Following the injury treatment, cultures were used in the
microtiter plate assay for biofilm formation.

3.3.8 Microtiter plate assay for biofilm formation.

A modification of the assay described by Stepanovic et al. (2000) was used to
assess biofilm formation by L. monocytogenes. After vortexing, 200 pl of the diluted
(102 CF U/ml) culture was pipetted into three wells of a 96-well untreated polystyrene
microtiter tissue culture plate (BD Falcon MicrotestTM Flat Bottom; Becton Dickinson
and Company, Franklin Lakes, NJ). Three wells per plate containing 200 pl of MWB
served as negative controls. Assays of injured cultures were carried out at 22 i 2°C for 4
d. At the end of incubation, the microtiter plate wells were emptied and rinsed three times
with physiological saline. The plates were gently shaken while rinsing to remove
unattached cells and were then allowed to air-dry. The remaining bacterial cells were
fixed to the well with 200 pl of 99% methanol (Fisher Chemicals, Fair Lawn, NJ). The
methanol was decanted 15 min later and the plates were allowed to air-dry. The

microtiter wells were stained with 200 pl of 2% crystal violet (Biochemical Sciences,

86

Inc., Swedesboro, NJ) for 5 min. After decanting the crystal violet, the wells were rinsed
five times with deionized water and were allowed to air-dry. The remaining dye was
resolubilized in 160 pl of 33% (v/v) glacial acetic acid (EM Science, Gibbstown, NJ) and
optical densities were read at 570 nm using a VmaxTM Kinetic Microplate Reader
(Molecular Devices, Menlo Park, CA).
3.3.9 Statistical Analysis.

All experiments were replicated three times. Statistical analysis was performed
using a general linear model procedure (SAS, Version 8, SAS Institute, Inc. Cary, NC).

Significance was determined at P < 0.05.

3.4 RESULTS

Overall, cold injury and cold starvation enhanced biofilm formation, while acid
injury and chlorine injury inhibited subsequent biofilm formation (Table 3.2). Cold
injured cells and cold starved cells were significantly better at forming biofilms than
uninjured cells (P < 0.05).

Within treatments, greater variation in biofilm formation was observed for
uninjured L. monocytogenes and strains subjected to cold injury and cold starvation
(Figures 3.1, 3.4, and 3.5), with a higher overall OD seen for cold injured (OD 0.12 —
3.73) and cold starved (OD 0.10 - 3.84) as opposed to uninjured bacteria (OD 0.14 —
2.61). Acid injured (OD 0.09 -— 1.27) and chlorine injured (OD 0.05 — 1.09) cultures of L.
monocytogenes exhibited less variability in biofilm formation by strain (Figures 3.2 and
3.3). Due to the criteria used to select isolates for this study (that the distribution of

OD570 at 22°C for the isolates follow the overall distribution for the complete collection

87

of 196 strains; Keskinen et al., 2006a), large standard deviations in mean OD570 were

observed for each treatment.

Table 3.2. Overall differences in injury and biofilm formation by 26 L.
monocytogenes strains following various treatments

 

Treatment ODm Injury (%)

 

Uninjured 0.83 :1: 0.66:11 0 1: 0a

Cold injured 1.28 :t 1.12b 39.2 :I: 15.3b
Cold starved 1.14 :l: 1.12b 28.5 :l: 14.0°
Acid injured 0.32 a 024° 60.2 a 2671'

Chlorine injured 0.26 :l: 0.25c 55.1 i 22.3d

 

n = 26
Means with different letters are significantly different (P < 0.05).

88

Figure 3.1. L. monocytogenes biofilm formation by uninjured cells

100 -i

i
s... ,
E l
E 60 ‘
¢ ;
2 40 l
g i
20 |

O mat-4w.— ~04. E. .4 .a m 3.. .2 .-L-.., _ L .1

0 0.5 1 1.5 2 2.5 3 3.5 4

00570

Figure 3.2 L. monocytogenes biofilm formation by acid-injured cells

 

100 1

E 80 ~ ’4'.
E. ’
5 60 — «39” . ’
E o
P 40 4
5
O. 20 ‘ O Q

l j o

0 —“ ‘b—TT ' _ _— _ T __fi” ”M_ _— - * w— _F— "T _—
0 05 1 15 2 25 3 35 4

89

Figure 3.3. L. monocytogenes biofilm formation by chlorine-injured cells

Percent Injury

 

100 ~-
I
80 el-I.
I
60 4.;
40 9"— '
I. I
20 ._
l I
0 i - .2
0 0.5

Figure 3.4. L. monocytogenes biofilm formation by cold-injured cells

Percent Injury

 

100“:
i
80*
60i , ‘
‘ A
404 fl ‘ ‘
201 “A
l A
o .- 44
0 0.5

90

Figure 3.5. L. monocytogenes biofilm formation by cold-starved cells

 

 

100 i
l
S 801
E 60 o
5 ’ .
g 404 . . .Q .
earns: . . . .
9. °
0 n" I- i» T.— —. w # fl 7 ’_k H T i""“ _— i _. _ _ T
0 0.5 1 15 2 2.5 3 35 4
00570

When the strains were ranked according to biofilm forming ability in relation to
each other (Table 3.3), the same strains consistently emerged as the strongest biofilm
formers, while different strains consistently predominated as the weakest biofilm formers.
Exposure to injury, even cold injury and cold starvation, did not significantly (P < 0.05)
enhance biofilm formation by the weakest biofilm forming strains in comparison to the
strong biofilm formers Additionally, no trends were observed in relative biofilm

formation by strains of the same serotype or strains from similar sources (clinical, food,

or environment; Table 3.1).

91

Table 3.3. Relative rankings of L. monocytogenes strains according to biofilm

forming ability (1 = strongest, 26 = weakest)

 

 

Strain ID Uninjured Acid- Chlorine- Cold- Cold-
injured injured injured starved
CWD 845 l l l 6 S
CWD 730 2 17 22 1 l
CWD 33 3 5 4 7 4
CWD 4 9 5 2 2
1734
CWD 338 5 7 8 5 6
CWD 764 6 3 3 3 3
CWD 600 7 19 13 10 11
CWD 766 8 6 2 4 7
CWD 580 9 26 19 12 10
CWD 10 11 6 9 8
1634
CWD 372 ll 12 14 l 1 l3
CWD 602 12 25 21 18 14
CWD 701 13 14 24 13 12
CWD 271 14 18 17 15 20
CWD 15 16 10 14 15
1176
CWD 16 23 20 8 9
1002
FSL R2- 17 4 7 17 17
501
CWD 18 21 23 21 22
1223
FSL J 1- 19 15 26 16 16
119
FSL N1- 20 2 1 1 19 18
225
CWD 561 21 13 16 23 19
FSL J l- 22 10 9 22 23
225
F SL R2- 23 8 12 20 21
499
CWD 578 24 22 25 24 24
CWD 182 25 24 15 25 25
CWD 205 26 29 18 26 26

92

3.5 DISCUSSION

Based on these results, environmental stresses to which L. monocytogenes may be
exposed in meat processing environments and delicatessens (cold, low nutrient
concentration) may lead to increased persistence and biofilm formation. However, this
only occurred after inoculation into MWB followed by incubation at ambient
temperature, which may explain why cold injury enhanced biofilm formation while
uninjured L. monocytogenes did not appear to form biofilms when incubated at 4°C
(Keskinen et al., 2006a).

Initial bacterial attachment to surfaces occurs via electrostatic or hydrophobic
interactions between the bacterial surface and the contact surface (Arnold and Bailey,
2000). Cell surface hydrophobicity can be affected by anything that will result in
changes to the cell surface. Therefore, changes in membrane fluidity that occur from
exposure to refiigeration temperatures are due to changes in the fatty acid profile of the
cell surface (N eunlist et al., 2005). Under these conditions, cell surface hydrophobicity
may have been sufficiently altered so that the hydrophobic interactions between injured
L. monocytogenes cells and the polystyrene surface were more favorable than for
uninjured cells. If initial attachment is more favorable, ultimate biofilm formation is
stronger compared to cells that exhibit poorer initial attachment (Lunden et al., 2000).
Acid and chlorine injury inhibited subsequent biofilm formation by L. monocytogenes.
Similar to cold injury, acid and sanitizer injury often decrease cell permeability by
altering the cell membrane fatty acid composition (Lou and Yousef, 1997; To et al.,
2002). Acid injury increases cell surface hydrophobicity (Lou and Yousef, 1997), which

affects initial attachment. Denaturation of proteins in the cell membrane by acid or

93

chlorine may also cause inhibition of biofilm formation. Cell surface proteins are
important for surface growth—the addition of 0.01% trypsin to growth media can reduce
adherent cell populations by 99.9%, as compared to control cultures without trypsin
(Smoot and Pierson, 1998). However, further research is required to determine whether
changes in surface hydrophobicity are indeed the cause for enhanced biofilm formation

by injured cells, or whether there are other factors that are more influential.

94

CHAPTER 4

IMPACT OF BIOFILM FORMING ABILITY ON TRANSFER OF SURFACE-

DRIED LIS T ERIA MONOC Y T OGENES FROM KNIFE BLADES TO ROAST

TURKEY BREAST

95

4.1 ABSTRACT

Listeria contamination of food contact surfaces can lead to cross-contamination of
ready-to-eat foods in delicatessens. In the present study, six previously identified strong
and weak biofilm-forming strains of L. monocytogenes were grown at 22°C for 48 h on
Trypticase soy agar containing 0.6% yeast extract and harvested in 0.1% peptone.
Thereafter, the strains were combined to obtain two 3-strain cocktails and resuspended in
turkey slurry to inoculate flame-sterilized grade 304 stainless steel knife blades at
concentrations of 108 and 106 CFU/blade. After incubation at ~78% relative humidity for
6 and 24 h, retail roast turkey breast was cut into 16 slices using the knives mounted on
an Instron Universal Testing Machine. In the evaluation of Listeria transfer from knife
blades to turkey breast, Listeria populations decreased 3-5 log CFU/slice after 16 slices.
Overall, total transfer to turkey was significantly greater for strong (4.4 log CFU total) as
opposed to weak biofilm formers (3.5 CFU total; P < 0.05). In addition, significantly
more listeriae were transferred at 6 h (4.6 log CFU total) than at 24 h (3.3 log CFU total;
P < 0.05). For both inoculum levels, transfer was observed out to the 16th slice. Greater
transfer was seen for the strong biofilm cocktail with increased survival of the strong
biofilm cocktail as observed via viability staining suggesting that these strains are better

adapted to survive stressful conditions than weak biofilm formers.

96

4.2 INTRODUCTION

The level and rate of Listeria monocytogenes attachment and biofilm formation
are useful predictors of persistence in the environment (Lunden et al., 2000; Norwood
and Gilmour, 1999; Borucki et al., 2003). In a recent study of L. monocytogenes transfer
from biofilms on stainless steel to a model food product, a transfer rate of 55% was
observed from pure culture biofilms with the presence of Kocuria varians (a Gram—
positive environmental isolate) increasing the L. monocytogenes transfer rate to 78%,
suggesting that this difference in transfer is related to differences in the adhesiveness of
L. monocytogenes in pure versus mixed culture (Midelet et al., 2006).

Food handling in home kitchens can lead to multiple routes of cross
contamination. Several studies have attempted to quantify transfer between food contact
surfaces and food in domestic kitchen-type scenarios. According to Kusumaningram et
al. (2003), 21-43% of the S. aureus, Campylobacterjejuni and Salmonella enteriditis
populations on inoculated (6.7 - 9.4 log CFU/sponge) wet sponges transferred to stainless
steel (AISI grade 304), with no significant differences seen between organisms in their
rate of transfer to stainless steel or to food products. Subsequently, 25-100% of the
available population transferred to roast chicken when applied to stainless steel for 10 s
with greater transfer observed when a 500-g weight was added to the chicken. Increasing
the product weight did not have the same effect on transfer to cucumber slices, which was
found to occur at 50-100% of the available bacterial population (Kusumaningram et al.,
2003). However, when compared to several other existing studies, a transfer rate of
100% is highly improbable with this overestimation of transfer likely due to inaccuracies

in estimating the surface inoculum. In the aforementioned study, an unorthodox contact

97

plate method was used to quantify the bacterial population on stainless steel with the agar
from the contact plate then suspended and homogenized in a peptone saline solution, and
diluted to a countable level before plating (Kusumaningram et al., 2003). In another
study, ground beef (75 - 100 g patties) with an average bacterial load of 6.7 log CFU/ g
transferred 2.5-3.0 log CF U/cm2 of E. coli OlS7:H7 to polyethylene and wood laminate
cutting boards after 30 min of contact, with no significant differences in transfer based on
cutting board material (Miller et al., 1996).

Given the lack of quantitative data for Listeria transfer in the existing literature, the
primary objective of this study was to determiner the transfer rate for L. monocytogenes
from knife blades to delicatessen turkey meat. The specific goals of the study were to (a)
determine whether differences exist in L. monocytogenes transfer based on biofilm
forming ability, particularly after desiccation on a stainless steel knife blade for extended
periods of time and (b) assess the viability of these same strains following desiccation on

stainless steel by viability staining.
4.3 MATERIALS AND METHODS

4.3.1 Listeria monocytogenes strains

Six strains of Listeria monocytogenes (obtained from Dr. Catherine W. Donnelly,
University of Vermont, Burlington, VT) were selected due to their ability to form weak
or strong biofilms in a microtiter plate assay (Keskinen, et al., 2003). Strong biofilm
forming strains included CWD 33 (unknown source, serotype 4b), CWD 730 (dairy plant
environmental isolate, serotype 1/2a), and CWD 845 (dairy plant environmental isolate,
serotype 1/2b); whereas the weak biofilm forming strains included CWD 182 (unknown

source, serotype 4b), CWD 205 (unknown source, serotype 4c), and CWD 578 (dairy

98

plant environmental isolate, serotype 4d). All strains were maintained at -80°C in
trypticase soy broth containing 0.6% yeast extract (TSB-YE; Becton Dickinson, Sparks,

MD) containing 10% (v/v) glycerol.

4.3.2 Preparation of turkey slurry

A turkey slurry was prepared for inoculation of knife blades by diluting 25 g of
retail restructured roast turkey breast (Gordon Food Stores, Lansing, MI)
1:10 in sterile deionized water and homogenizing in a model DIFP2 blender (General
Electric, Bridgeport, CT) at high speed for 1 min. The resulting slurry was filtered
through five layers of cheesecloth into sterile 50 ml conical polypropylene centrifuge
tubes (Corning, Corning, NY), heated in an 80°C water bath for 20 min, cooled, and

stored at -20°C. Before use, the turkey slurry was thawed overnight at 4°C.

4.3.3 Culture preparation

All frozen stock cultures were subcultured separately in TSB-YE (Becton
Dickinson) for 18 h at 37°C, and then streaked to plates of trypticase soy agar containing
0.6% yeast extract (TSA-YE; Becton Dickinson) to obtain confluent growth after 18 h of
incubation at 37°C. Listeria monocytogenes was harvested from the TSA-YE plates by
flooding and suspending the cells in 10 ml of 0.1% sterile peptone (Becton Dickinson).
Each Listeria suspension was then combined in equal volumes to produce two separate
cocktails containing three weak and three strong biofilm formers. The concentration of
each 3-strain cocktail was determined by optical density at 600 nm using a
spectrophotometer (GenesysZO Spectrophotometer, Therrno Electron Corp., Waltham,

MA) and by spiral plating (Autoplate® 4000 Spiral Plater, Spiral Biotech Inc., Norwood,

99

MA) on TSA-YE (Becton Dickinson) followed by 48 h of incubation at 35°C. Each
cocktail was then serially diluted in the turkey slurry to a concentration of 107 or 109

CFU/ml in turkey slurry for inoculation.

4.3.4 Knife blades

A set of six medium sharp electropolished grade 304 stainless steel knife blades
measuring 12 cm x 5 cm (product contact area of 60 cm2 on each side of the blade) with a
thickness of 1.4 mm were manufactured by ProAxis, Inc., (Lafayette, IN). Medium sharp
blades were machined to allow for a slightly dull blade by milling at a 45° angle 10 mm
from the end of the blade, and then machined with a blunt end 0.5 mm from the tip. The

end result was a blade meant to mimic a knife slightly dulled by routine usage.

4.3.5 Knife blade inoculation

After flame sterilizing in 95% ethanol, a set of 6 identical knife blades were
inoculated on one side with 100 pl of a 3-strain cocktail so as to contain 106 or 108 L.
monocytogenes CF U/blade. The inoculum was unifome spread over the 60 cm2 product
contact area with a 1 pl inoculating loop, allowed to dry on the blade for 5 min under
ambient conditions (~22°C and ~40% relative humidity), and then incubated at ambient
temperature (~22°C) at 78% relative humidity (R. H.; ASTM Standard Method E104) for
6 or 24 h before surface sampling or slicing. Relative humidity was monitored with a

hygrometer (Fisher Scientific; Hampton, NH).

100

4.3.6 Standardization of cutting force and speed

An Instron 5565 electromechanical compression analyzer (Instron; Canton, MA)
was used to standardize cutting force at a cutting speed of 8.3 mm/s. Each knife blade
was manufactured with a 1 cm x 2 cm flange at each end so that the blade could be
attached to a specially made support bracket and used with an Instron electromechanical
compression analyzer. A custom-made knife support bracket to which all knife blades
were attached was secured to the upper load cell (1124 lb) for cutting delicatessen turkey

meat (Figure 4.1).

Figure 4.1. Instron 5565 electromechanical compression analyzer with modified
upper load cell for knife blades

 

4.3.7 Restructured roast turkey breast

A retail brand of restructured roast turkey breast (2.5 to 2.9 kg each) was
purchased in chub-form from a local retailer (Gordon Food Service, Lansing, MI), stored
at 4°C, and used within 30 d of purchase. According to the package label, composition of

the roast turkey breast was as follows: turkey breast, turkey broth, < 2% each of salt,

101

dextrose and sodium phosphate. The restructured roast turkey breast averaged 78%

moisture, < 1% fat, and 19% protein (V orst et al., 2006).

4.3.8 Transfer of L. monocytogenes from inoculated grade 304 stainless steel knife

blades to uninoculated restructured roast turkey breast

Whole chubs of turkey were sliced using a knife blade inoculated at 108 or 106
CFU/blade to obtain sixteen slices. Each slice was diluted 1:5 (w/v) in either Phosphate
Buffered Saline (PBS; 108 CFU/blade) or University of Vermont Medium (UVM; Becton
Dickinson; 106 CF U/blade) for subsequent enrichment at 30°C, and homogenized in a
Stomacher (Seward, Norfolk, UK) for 1 minute. Samples obtained using blades
inoculated at 108 CFU/blade were spread-plated to Modified Oxford Agar (MOX; Becton
Dickinson). In contrast, samples sliced with blades inoculated at 106 CF U/blade were
pour-plated (5 ml into 25 ml of MOX) in duplicate in ISO-mm diameter disposable Petri
dishes (Fisher Scientific; Chicago, IL) and counted after 2 d of incubation at 35°C to
determine the number of listeriae transferred to each slice. When L. monocytogenes
could not be detected by direct plating, the UVM enriched samples were plated to MOX
after 2 d of incubation at 35°C to determine presence or absence of Listeria. Each

experiment was replicated three times.

4.3.9 Quantification of L. monocytogenes on used and unused knife blades

Two inoculated knife blades were surface sampled using the l-ply composite
tissue (CT) method developed by Vorst et al. (2004) after 6 and 24 h of incubation at
~22°C/78% R.H. as a positive control for each experiment. All knife blades were

sampled to determine numbers of L. monocytogenes remaining on the blade after 16

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slices. The CT was rehydrated with 10 ml of PBS in a Whirl-PakTM bag (N asco, Inc.,
Fort Atkinson, WI) and then used to swab the blade, after which the blade was dried
using a dry CT. After returning both CT to the original Whirl-PakTM bag, 40 ml of PBS
was added. The sample was then homogenized in a Stomacher for 1 min. Duplicate
samples were spread- or pour-plated, as previously described, and incubated at 35°C for 2

d before counting.

4.3.10 Cleaning and decontamination of knife blades

Knife blades were removed from the support bracket after use and soaked in an
activated 32% alkaline glutaraldehyde solution (CIDEX®; Advanced Sterilization
Products, Irvine, CA). Sanitized knife blades were washed in Tergizyme (Alconox, Inc.,
New York, NY), rinsed six times in tap water, followed by six rinses in deionized water.
The components were then dried using a CT. To prevent surface oxidation during
storage, the knife blades were coated with a thin layer of mineral oil. After storage, all
components were cleaned again with 70% ethanol and rinsed with sterile deionized water

immediately before use in order to remove the mineral oil film.

4.3.11 Evaluation of survival of L. monocytogenes on knife blades using confocal
scanning laser microscopy
A confocal scanning laser microscope (CSLM; Zeiss LSM 5 Pascal; Carl Zeiss,
Inc., Thomwood, NY) was used to evaluate the survival of L. monocytogenes on knife
blades after incubation for 1, 6 and 24 h at ~22°C/7 8% RH. Listeriae were grown on
TSA-YE, harvested into 5 m1 of 0. 1 % peptone and combined to form the two 3-strain

cocktails as previously described. The cocktails were then centrifuged (Super T21,

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Sorvall Products, Nevvtown, CT) at 9740 X g for 10 min at 4°C in sterile 50 ml
polypropylene centrifuge tubes. After decanting the supernatant, a 10 pl loop was used
to transfer the resulting pellet into 10 pl of turkey slurry on a flame-sterilized piece of
grade 304 electropolished stainless steel measuring 2.5 cm x 7.5 cm. Following 6 or 24 h
of incubation at ~22°C/78% R.H. in a humidity chamber, as previously described, the
bacteria were stained for viability using a LIVE/DEAD® BacLightTM Bacterial Viability
Kit (Molecular Probes, Inc., Eugene, OR) prepared as recommended by the manufacturer,
with propidium iodide and SYTO 9 mixed together for the staining stock solution.
Samples were stained by depositing 10 pl of stain on the sample followed by covering
with a glass coverslip. After 1 h incubation under ambient conditions, the stained cells
were observed using a CSLM equipped with a 100)( oil immersion objective (numerical
aperature = 1.3, Carl Zeiss, Inc., Thomwood, NY) and an argon-ion laser. Fluorescence
was detected using an excitation wavelength of 488 nm and emitted light was separated
through a 488 nm neutral density filter and a 545/635 nm secondary dichroic beam
splitter. Simultaneous dual-charmel imaging was used to create computer-generated
pseudocolor images of the live and dead bacterial cells. One channel was equipped with
a 505-530 nm band pass filter for detection of SYTO 9 stained cells (emission
wavelengths: 510-540 nm) whereas the second channel was equipped with a 560 nm long
pass filter for detection of propidium iodide stained cells (emission wavelengths: 620-650
nm). Five randomly chosen fields of view for each combination of time and cocktail (n =
45 micrographs) were printed and individual cells were counted by hand to determine the

percentage of live and dead bacteria were present under each set of conditions.

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4.3.12 Statistical analysis

All experiments were replicated three times, except for the microscopy
experiments, which were replicated until 5 micrographs with countable fields of view for
each treatment were obtained. The resulting data were analyzed using SAS (SAS
Version 8; SAS Institute, Cary, NC) software with a general linear mixed effects model
and analysis of variance (ANOVA) for least significant differences among the
combinations of treatments (P < 0.05). For analysis of transfer by biofilm forming
ability, time and inoculation level, results were analyzed based on the average transfer to

all 16 slices in a replicate.

4.4 RESULTS

4.4.1 Listeria transfer from knife blades to product

In the evaluation of Listeria transfer from knife blades to turkey breast, L.
monocytogenes populations decreased 3-5 log CFU/slice after 16 slices with a general
decrease observed in transfer to successive slices. (Figures 4.2 and 4.3). Significantly
greater overall transfer (P < 0.05) of Listeria was seen for strong (4.4 log CFU total) as
opposed to weak biofilm formers (3.5 log CFU) and after 6 (4.6 log CFU) as opposed to
24 h (3.3 log CFU) of desiccation on the blade. Greater total numbers of cells transferred
from knife blades inoculated at 8.9 log (6.4 log CFU transferred; Figure 4.2) than 6.9 log
CFU/blade (1.5 log CFU transferred; Figure 4.3) All two- and three-way interactions
(e.g., strong biofilm forming cocktail at 6 h vs. weak biofilm forming cocktail at 6 h)

were not significantly different for overall transfer.

105

Figure 4.2. Transfer of weak and strong biofilm forming cocktails of L.
monocytogenes from an inoculated knife blade (8 log CFU/blade; incubation = 6 and

24 h, 78 :l: 2% RH/22°C) to roast turkey breast (n = 3)

3 0 , 9 Strong, 6 h
. 0 Strong 24 h
3 .
mi A ’ o . AWeak,6h
8 6'0 6 . 9 ° . oWeak, 24h
= 50 - 9 A ’ A
("5 4.04 . C 3 A . . .
a 3.0 i . . O ' . x g .
3 20 ,I . . . . C
1.0 i
0.0 l,” ,3, .3 3 - _,_ 2 LL-.- r.-. ,.- ,_.,_.T,. 2.. -~~-. ___! >—-.
01 2 3 4 5 6 7 8 910111213141516

Slice number

Figure 4.3. Transfer of weak and strong biofilm forming cocktails of L.
monocytogenes from an inoculated knife blade (6 log CFU/blade; incubation = 6 and
24 h, 78 :l: 2% RI-I/22°C) to roast turkey breast (n = 3). Open symbols not
quantifiable by direct plating, but were positive by enrichment.

 

6'01; OStrong,6h
‘ IStrong,24h
5'01 AWeak,6h
i ° oWeak,24h
8 4.0 1 I ‘
g A 9 ° X x o
0 o A A o I I -
3’ I I 0 3 I A
..l 2.0 l
..oi
l
0.0 i ee-e—e ”O c} , _._ oeC-Q-H-e-Hovoz
012 3 4 5 6 7 8 91011121314151617

Slice Number

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Table 4.1. Number of direct counts and positive enrichments for roast turkey breast
sliced with L. monocytogenes-contaminated knife blades after 6 and 24 h

10° CFU/Blade 10° CFU/Blade

 

Strong cocktail Weak cocktail Strong cocktail Weak cocktail

 

Slice 6h 24h 6h 24h 6h 24h 6h 24h

 

1 3/3a 3/3 3/3 1/3 3/3 3/3 3/3 3/3
2 3/3 2/3 3/3 2/3 3/3 3/3 3/3 3/3
3 3/3 2/3 2/3 0/3 3/3 3/3 3/3 3/3
4 3/3 3/3 3/3 0/3 3/3 3/3 3/3 3/3
5 3/3 1/3 3/3 1/3 3/3 2/3 3/3 2/3
6 2/3 1/3 2/3 1/3 3/3 2/3 3/3 1/3
7 2/3 2/3 2/3 1/3 3/3 2/3 3/3 1/3
8 2/3 2/3 1/3 0/3 3/3 2/3 3/3 0/3
9 1/3 1/3 1/3 0/3 3/3 1/3 3/3 1/3

10 2/3 1/3 0/3 0/3 3/3 1/3 3/3 2/3
11 NTb NT 071° 0/1° 3/3 1/3 3/3 2/3
12 2/3 0/3 3/3 1/3 3/3 1/3 3/3 2/2
13 NT NT OH" OH" 2/3 1/3 3/3 2/3
14 1/3 1/3 1/3 0/3 3/3 1/2 3/3 2/3
15 NT NT 1/1° 0/10 3/3 2/3 3/3 2/3
16 0/3 1/3 1/3 0/3 3/3 1/2 3/3 2/2
aDirect counts / enrichment results for 3 replicates

bNT = Not tested
°Direct counts / enrichment result for 1 plated replicate

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4.4.2 Survival of L. monocytogenes on knife blades over time

Strong biofilm formers survived drying on knife blades in significantly greater
numbers (P < 0.05) than weak biofilm formers, with survival rates of 51.4% i 23.2 and
38.7% i 24.9, respectively. Listeria viability was significantly greater after 1 h (53.0% i
17.5; P < 0.05) as opposed to 6 (38.9% i 28.8) or 24 h (43.9% i: 25.1) of incubation on

stainless steel.

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4.5 DISCUSSION

In a related Listeria transfer study by Vorst (2005) using knife blades inoculated
with L. monocytogenes at 8 log CF U/blade a 2 log CFU decrease in transfer was seen
over 12 slices of roast turkey breast with the pathogen quantifiable out to 30 slices.
However, when the inoculum level was decreased to 5 log CFU/blade, transfer was only
quantifiable for the first 20 slices with slices 26 through 30 negative by enrichment. In
this study, transfer was only quantifiable to 16 slices of turkey using a similar a knife
blade initially inoculated at 6 log CF U/blade. However, given the extended incubation
times of 6 and 24 h on the knife blade as opposed to l h in the Vorst (2005) study, the
surviving population on the blade after surface drying was likely ~5 log CF U/blade,
making the initial inoculum levels roughly similar. This reduction in number of L.
monocytogenes was confirmed by viability staining (Table 4.2), which showed that
significantly lower numbers of Listeria survived 6 and 24 h of incubation, as opposed to
1 h.

According to Vorst (2005), greater transfer was seen using AISI grade 304 than
grade 316 stainless steel knife blades reinforcing increased cleanability of the latter
(Arnold and Bailey, 2000; Leclercq-Pelat and Lalande, 1994). Therefore, in this study,
AISI grade 304 stainless steel blades were used in order to mimic the worst-case scenario
for harboring Listeria during extended incubation times on a surface in the absence of
water.

The two sets of L. monocytogenes strains used in this study were among the
strongest and weakest biofilm formers from a set of 122 strains that was previously

characterized for biofilm formation using a microtiter plate assay. According to Borucki

109

et al. (2003), persistent strains were better able to form biofilms, as measured by a

microtiter plate assay for biofilm formation. Lunden et al. (2000) reported that a
persistent strain of L. monocytogenes was transferred to three different processing plants
via a dicing machine and was significantly more adherent than non-persistent strains, a
trend which also has been previously observed (Lunden et al., 2000; Norwood and
Gilmour, 1999; Borucki et al., 2003). Kalmokoff et al. (2001) reported variation in the
ability of L. monocytogenes strains to adhere to stainless steel with the extent of
subsequent biofilm formation also varying. Therefore, the relationship between assumed
persistence of L. monocytogenes according to biofilm formation and extent of transfer
from stainless steel to roast turkey breast was also assessed.

Significantly higher total transfer was observed by strong (and assumedly
persistent) biofilm forming strains of L. monocytogenes as opposed to weak biofilm
formers. This would run counter to some of the assumptions one might make regarding
persistence—if persistence predicts strength of adhesion to a surface, one would predict
greater transfer of the less persistent strains. Since the opposite proved to be the case, it
was decided to examine survival by strong and weak biofilm forming strains dried on the
stainless steel. Although viability staining showed that strong biofilm formers survived
in greater numbers than weak biofilm formers, the ability of some L. monocytogenes
strains to form stronger biofilms may enhance survival to various environmental stresses
including lack of moisture and sanitizer exposure. Thus, greater transfer of strong

biofilm formers to turkey during slicing may be at least partially due to their increased

survival as compared to the weak biofilm forming strains.

110

Greater transfer of strong biofilm formers may also be related to differences in
attachment to stainless steel due to cell surface hydrophobicity. In a previous study
(Keskinen et al., 2003), same weak biofilm forming strains used here were significantly
more hydrophobic than the strong biofilm forming strains, however the literature is
contradictory as to whether hydrophobic or hydrophilic cells will attach more strongly to
stainless steel. While hydrophilic and negatively charged L. monocytogenes cells
adhered better to stainless steel, than to polytetrafluoroethylene (Chavant et al., 2002),
Briandet et al. (1999) observed better adherence to stainless steel among L.
monocytogenes strains that were slightly more hydrophobic. Finally, Midelet and
Carpentier (2002) reported stronger attachment of L. monocytogenes biofilms to
polyvinyl chloride and polyurethane than to stainless steel, and also noted that all of these
surfaces were hydrophobic, with these same surfaces becoming hydrophilic after
exposure to meat exudate based on contact angle measurement. Hence, further research
into the surface characteristics of both L. monocytogenes and common food contact
surfaces is required to more fully determine whether attachment influences persistence

and transfer of L. monocytogenes during slicing of delicatessen meats

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CHAPTER 5

IMPACT OF BACTERIAL STRESS AND BIOFILM FORMING ABILITY ON

TRANSFER OF SURFACE-DRIED LIS T ERIA MONOC Y T OGENES DURING

SLICING OF DELICATESSEN MEATS

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5.1 ABSTRACT

Listeria contamination of delicatessen slicer blades can lead to cross-
contamination of luncheon meats. In the present study, six previously identified strong
and weak biofilm-forming strains of L. monocytogenes were grown at 37°C/18-24 h on
trypticase soy agar containing 0.6% yeast extract, harvested in 0.1% peptone and then
combined to obtain two 3-strain cocktails. The cocktails were resuspended in turkey
slurry with or without prior cold-shock at 4°C/2h and then used to inoculate flame-
sterilized stainless steel delicatessen slicer blades at a concentration of 106 CFU/blade.
After incubation at 22°C/78 i 2% relative humidity for 6 and 24 h, the inoculated blades
were attached to a gravity-fed delicatessen slicer and used to generate 30 slices from
retail chubs of roast turkey breast or Genoa salami. Slices (~25 g) were diluted 1:5 in
phosphate buffered saline or University of Vermont Medium and then pour-plated (5 ml)
into ISO-mm dia. Petri plates using 20 ml of Tryptose Phosphate Agar containing esculin
and ferric ammonium citrate with the transfer results reported as the average of 30 slices.
Overall, more strong biofilm-formers transferred (3.62 log CFU) than weak biofilm-
forrners (3.12 log CFU), cumulatively. Significantly greater transfer to turkey (3.61 log
CF U) than to salami (3.12 log CF U) was observed. Previous cold-shock significantly
increased subsequent Listeria transfer (3.69 log CFU) compared to healthy (3.30 log
CPU) and chlorine-injured cells (3.12 log CFU). Length of desiccation on the blade also
significantly affected overall transfer, with greater transfer after 6 h of desiccation. These
results are likely due to differences in both product composition and survival of L.

monocytogenes that were observed via viability staining.

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5.2 INTRODUCTION

In retail food handling environments, bacterial contaminants including Listeria are
most often found in difficult to clean areas that contain food particulates and adequate
moisture. Bacteria within these harborage sites are typically exposed to stressful
conditions including sanitizers, dehydration, starvation, and extremes in both temperature
and pH. Under these extreme conditions, L. monocytogenes can become sublethally
injured with the pathogen then unable to grow on many commonly used selective plating
media. Even under these unfavorable environmental conditions, bacterial foodbome
pathogens can remain viable on common food contact surfaces for days or weeks and
cross-contaminate other products. In one early report, 469 cases of typhoid fever were
traced to a single can of delicatessen-sliced corned beef with Salmonella Typhi
transferred from the delicatessen slicer to other deli meats that were subsequently sold
and consumed (Howie, 1968). The greater prevalence of L. monocytogenes in
delicatessen- as opposed to manufacturer-sliced meat is at least partly due to cross-
contamination in the delicatessen with one of the most obvious contact points being the
delicatessen slicer (Gombas et al., 2003).

In recent bacterial transfer work with mechanical delicatessen slicers, L.
monocytogenes was shown readily transfer both to and from slicer blades and deli meats.
Based on the work of Vorst et al., (2006), L. monocytogenes transferred from a blade
inoculated at 8 log CFU/blade to 30 successive slices of roast turkey breast with transfer
decreasing logarithmically to 2 log CF U/slice by the 30th slice. At lower inoculums (5
log and 3 log CFU/blade), transfer was not quantifiable beyond the 5th slice, with

negative enrichments after 27 and 15 slices, respectively (Vorst et al., 2006). In the same

114

study, transfer to salami was more continuous throughout the 30 slices than to turkey or
bologna, both of which were higher in moisture and lower in fat than salami. The
difference in transfer between the products was attributed to the layer of fat that
accumulated on the slicer blade during slicing of salami, which was not seen for the other
two products (Vorst et al., 2006).

Lin et al. (2006) conducted a study in which the blade of a commercial-scale meat
slicer used to slice roast turkey breast, salami and bologna was inoculated to contain L.
monocytogenes at levels of l, 2, or 3 log CFU/blade (1 and 2 log CFU/blade inoculum
used with turkey only). More slices tested positive by enrichment using 3 log CFU/blade
than at l or 2 log CFU/blade (Lin et al., 2006). Additionally, Lin et al. (2006) found that
more equipment samples were positive for L. monocytogenes after slicing salami (8
samples) than turkey (3 samples) or bologna (1 sample), which supports a longer
residence time for L. monocytogenes on fat-coated slicers as suggested by Vorst et al.
(2006)

In a study of L. monocytogenes transfer from meat industry biofilms, transfer to a
trypticase soy agar cylinder used as a model food product was quantified and a transfer
rate of 55% was observed fi'om pure culture biofilms, while the presence of Kocuria
varians (a Gram-positive environmental isolate) increased the L. monocytogenes transfer
rate to 78% (Midelet et al., 2006). Exposure to chlorine shock increased the adhesiveness
of L. monocytogenes to the stainless steel surface, resulting in less transfer (Midelet et al.,
2006)

Given these previous findings, the specific goal of this study was to determine

whether differences exist in the transfer of L. monocytogenes strains based on their ability

115

to persist in the environment (as determined by biofilm forming ability), particularly after
desiccation on a stainless steel slicer blade for extended periods of time. Strong and
weak biofilm forming cocktails of L. monocytogenes were also subjected to sublethal
cold- and chlorine-injury before desiccation on slicer blades, to assess the impact of
injury on subsequent transfer while slicing turkey or salami. These same cocktails were
also compared in regards to their ability to survive desiccation, with and without prior

sublethal cold- and chlorine-injury, as measured by viability staining.

5.3 MATERIALS AND METHODS

5.3.1 Listeria monocytogenes strains

Six strains of Listeria monocytogenes (obtained from Dr. Catherine W. Donnelly,
University of Vermont, Burlington, VT) were selected due to their ability to form weak
or strong biofilms in a microtiter plate assay (Keskinen et al., 2003). Strong biofilm
forming strains included CWD 33 (unknown source, serotype 4b), CWD 730 (dairy plant
environmental isolate, serotype U28), and CWD 845 (dairy plant environmental isolate,
serotype 1/2b); whereas the weak biofilm forming strains included CWD 182 (unknown
source, serotype 4b), CWD 205 (unknown source, serotype 4c), and CWD 578 (dairy
plant environmental isolate, serotype 4d). All strains were maintained at -80°C in
trypticase soy broth containing 0.6% yeast extract (TSB-YE; Becton Dickinson, Sparks,

MD) containing 10% (v/v) glycerol.

116

5.3.2 Preparation of turkey slurry

A turkey slurry was prepared for inoculation of delicatessen slicer blades by
diluting 25 g of retail restructured roast turkey breast (Gordon Food Stores, Lansing, MI)
1 :10 in sterile deionized water and homogenizing in a model DIFP2 blender (General
Electric, Bridgeport, CT) at high speed for 1 min. The resulting slurry was filtered
through five layers of cheesecloth into sterile 50 m1 conical polypropylene centrifuge
tubes (Corning, Corning, NY), heated in an 80°C water bath for 20 min, cooled, and

stored at -20°C. Prior to use, the turkey slurry was thawed overnight at 4°C.

5.3.3 Culture preparation, uninjured cocktails

All frozen stock cultures were subcultured separately in TSB-YE (Becton
Dickinson) for 18 h at 37°C, and then streaked to plates of trypticase soy agar containing
0.6% yeast extract (TSA-YE; Becton Dickinson) to obtain confluent growth after 18 h of
incubation at 37°C. Listeria monocytogenes was harvested from TSA-YE by flooding
the plates and suspending the cells in 10 ml of 0.1% sterile peptone (Becton Dickinson).
Each individual Listeria suspension was then combined in equal volumes to form two 3-
strain cocktails consisting of weak and strong biofilm formers. The concentration of each
3-strain cocktail was determined by optical density at 600 nm using a spectrophotometer
(Genesys20 Spectrophotometer, Therrno Electron Corp., Waltham, MA) and by spiral
plating (Autoplate® 4000 Spiral Plater, Spiral Biotech Inc., Norwood, MA) on TSA-YE
followed by 48 h of incubation at 35°C. The cocktail was then serially diluted to a

concentration of 107 CFU/ml in turkey slurry for inoculation.

117

5.3.4 Culture preparation, cold-injured cocktail

Listeria monocytogenes strains were grown as previously described, individually
harvested from TSA-YE plates by flooding the surface with 10 ml of Butterfield’s
Phosphate Buffer, and then combined in equal volumes to form two 3-strain cocktails of
weak and strong biofilm formers. After determining the cell concentration by optical
density at 600 run each cocktail was incubated for 2 h in an ice water bath and then
serially diluted to a concentration of 107 CFU/ml in turkey slurry. Injury was quantified
by spiral plating (Spiral Biotech Inc.) to tryptose phosphate agar (TPA; Becton
Dickinson) and tryptose phosphate agar containing 4.5% sodium chloride (TPAN; Becton
Dickinson) followed by 48 h of incubation at 35°C. Percent injury was determined using

the following equation:

Percent injury = [(TPA count — TPAN count)/T PA count] * 100

5.3.5 Culture preparation, chlorine-injured cocktail

Listeria monocytogenes strains were grown as previously described, individually
harvested from TSA-YE plates by flooding the surface with 10 ml of Phosphate Buffered
Saline, and then combined in equal volumes to form two 3-strain cocktails of weak and
strong biofilm formers. After determining the cell concentration by optical density at 600
nm, each cocktail was then injured by exposure to 100 ppm chlorine (Clorox; The Clorox
Company, Oakland, CA) for 1 minute. Following exposure, each cocktail was serially

diluted in Neutralizing Buffer (Becton Dickinson) to inactivate the chlorine and then

118

serially diluted to a concentration of 107 CFU/n11 in turkey slurry. Injury was quantified

by plating on TPA and TPAN as previously described.

5.3.6 Delicatessen slicer inoculation

A corrrmercial gravity-fed delicatessen slicer (Model 220F, Omcan
Manufacturing; Niagara Falls, NY) was used for slicing with eight additional
electropolished grade 304 stainless steel slicer blades also obtained from the same
manufacturer. None of the remaining slicer components were electropolished. After
flame sterilizing in 95% ethanol, the product contact surface of four slicer blades, as
determined using Glo-GerrnTM powder (Vorst et al., 2005), were inoculated with 300 pl
of turkey slurry so as to contain 106 CFU/blade and then incubated for 6 or 24 h at
ambient temperature (~22°C) and 78% R. H. (ASTM standard method E104) before
surface sampling or slicing.

Figure 5.1. Contact areas of gravity fed delicatessen slicer

 

(T) = table, (BP) = back plate, (B) = blade, (G) = guard, (C) = collection area

119

5.3.7 Delicatessen meats

One retail brand each of restructured roast turkey breast and Genoa hard salami
(5.5 to 6.5 lbs each) was purchased in chub-forrn from a local retailer (Gordon Food
Service, Lansing, MI), stored at 4°C, and used within 30 d of purchase. According to the
package label, composition of the roast turkey breast was as follows: turkey breast,
turkey broth, < 2% each of salt, dextrose and sodium phosphate. The stated product
composition of the Genoa salami was as follows: pork, beef, salt < 2% each of dextrose,
water, natural spices, sodium ascorbate, lactic acid starter culture, garlic powder, sodium
nitrite, BHA, BHT, and citric acid. The restructured roast turkey breast averaged 78%
moisture, < 1% fat, and 19% protein, while the Genoa hard salami contained 43%

moisture, 36% fat, and 17% protein (V orst et al., 2005).

5.3.8 L. monocytogenes transfer from an inoculated delicatessen slicer blade to

uninoculated product

Whole chubs of turkey and salami were sliced using an inoculated slicer blade to
obtain 30 2- to 3- mm thick slices weighing approximately 25 g each. Each slice was
diluted 1:5 (w/v) in University of Vermont Medium (UVM; Becton Dickinson),
homogenized in a Stomacher (Seward; Norfolk, UK) for 1 minute, and pour-plated (5 ml
into 25 ml of agar) in duplicate in 150 mm diameter disposable Petri dishes (Fisher
Scientific; Chicago, IL) in modified TPA (mTPA) and TPAN (mTPAN) containing ferric
ammonium citrate (0.5 g/L), esculin (1 g/L), and lithium chloride (3.75 g/L). Ferric
ammonium citrate and esculin allow for the differentiation of L. monocytogenes from
background microflora. Lithium chloride was added at one-quarter of the strength that is

found in Modified Oxford Agar, which was found to be adequate to select against the

120

lactic acid bacteria found in salami which can also react with ferric ammonium citrate
and esculin (data not shown). After 4 d of incubation at ambient temperature (~22°C),
the plates were counted to determine the number of listeriae and percent injury per slice,

with percent injury determined as follows:

Percent injury = [(TPA count — TPAN count)/T PA count] * 100

When L. monocytogenes could not be detected by direct plating, the UVM enriched
samples were plated to Modified Oxford Agar (MOX; Becton Dickinson) after 4 d of
incubation at ambient temperature (~22°C) to determine presence or absence of Listeria.

Each experiment was replicated three times.

5.3.9 Quantification of L. monocytogenes on used and unused slicer blades

An inoculated slicer blade was surface sampled using the l-ply composite tissue
surface sampling (CT) method developed by Vorst et al. (2004) after 6 and 24 h of
incubation at ambient temperature (~22°C) and 78% RH. as a positive control for each
experiment. All slicer blades were also similarly sampled to determine numbers of L.
monocytogenes remaining on the blade after 30 slices. The CT was rehydrated with 10
ml of PBS in a Whirl-PakTM bag (N asco, Inc., Fort Atkinson, WI) and then used to swab
the blade, after which the blade was dried using a CT. After returning both CT to the
original Whirl-PakTM bag, 40 ml of UVM was added. The sample was then homogenized
in a Stomacher for 1 min. Duplicate samples were pour-plated using mTPA and mTPAN
and incubated at ambient temperature (~22°C) for 4 d, as previously described. Percent

injury was calculated as previously described.

121

5.3.10 Cleaning and decontaminating the slicer

After use and disassembly, the slicer table, guard and blade (Figure 5.1) were
wiped with a CT and soaked for 30 min in a pan containing an activated 32% alkaline
glutaraldehyde solution (CIDEX®; Advanced Sterilization Products, Irvine, CA). Non-
removable components of the slicer were wiped with a CT, disinfected with a 32%
alkaline glutaraldehyde solution, and then air-dried for 30 min. After disinfection, non-
removable components were wiped with a CT soaked in 70% ethanol (v/v), followed by a
CT soaked in deionized water, and dried using a CT. Sanitized removable slicer
components were washed in Tergizyme (Alconox, Inc., New York, NY), rinsed six times
in tap water, followed by six rinses in deionized water. The components were then dried
using a CT. After storage, all components were cleaned again with 70% ethanol and

rinsed with sterile deionized water immediately before use.

5.3.11 Evaluation of survival of L. monocytogenes on knife blades using confocal

scanning laser microscopy

A confocal scanning laser microscope (CSLM; Zeiss LSM 5 Pascal; Carl Zeiss,
Inc., Thomwood, NY) was used to evaluate the survival of L. monocytogenes on knife
blades after 1, 6 and 24 h of incubation at ~22°C/78% R.H. Listeriae were grown on
TSA-YE, harvested into 5 ml of 0.1% peptone and combined to form the two 3-strain
cocktails, as previously described. The cocktails were then centrifuged (Super T21,
Sorvall Products, Newtown, CT) at 9740 x g for 10 min at 4°C in sterile 50 ml
polypropylene centrifuge tubes (Corning Inc., Corning, NY). After decanting the
supernatant, a 10 pl loop was used to transfer the resulting pellet into 10 pl of turkey

slurry on a flame-sterilized piece of grade 304 electropolished stainless steel measuring

122

2.5 cm x 7.5 cm. Following 6 or 24 h of incubation at ~22°C/78% R.H. in a humidity
chamber, as previously described, the bacteria were stained for viability using a
LIVE/DEAD® BacLightTM Bacterial Viability Kit (Molecular Probes, Inc., Eugene, OR)
prepared as recommended by the manufacturer, with propidium iodide and SYTO 9
combined to obtain the staining stock solution. Samples were stained by depositing 10 pl
of the stock solution of on the sample followed by a glass coverslip. After 1 h of
incubation under ambient conditions, the stained bacteria were observed using a CSLM
equipped with a 100x oil immersion objective (numerical aperature = 1.3, Carl Zeiss,
Inc., Thomwood, NY) and an argon-ion laser. Fluorescence was detected using an
excitation wavelength of 488 nm with transmitted light separated through a 488 nm
neutral density filter, a 545/635 nm secondary dichroic beam splitter. Simultaneous dual-
channel imaging was used to create computer-generated pseudocolor images. One
channel was equipped with a 505-530 nm band pass filter for detection of SYTO 9
stained cells (emission wavelengths: 510-540 nm) and the second channel was equipped
with a 560 nm long pass filter for detection of propidium iodide stained cells (emission
wavelengths: 620-650 nm). Five randomly chosen fields of view for each combination of
time and cocktail (n = 45 micrographs) were printed and individual cells were counted by
hand to determine the percentage of live and dead bacteria were present under each set of

conditions.

5.3.12 Statistical analysis

All experiments were replicated three times, except for the microscopy
experiments, which were replicated until 5 micrographs with countable fields of view for

each treatment were obtained. The resulting data were analyzed using SAS (SAS

123

Version 8; SAS Institute, Cary, NC) software with a general linear mixed effects model
and analysis of variance (ANOVA) for least significant differences among the
combinations of treatments (P < 0.05). For analysis of transfer by biofilm forming
ability, injury, time and inoculation level, results were analyzed based on the average

transfer to all 30 slices in a replicate.

5.4 RESULTS

5.4.1 Transfer of surface-dried L. monocytogenes from an inoculated delicatessen

slicer blade to uninoculated product

Listeria monocytogenes transfer from an inoculated slicer blade containing 106
CF U/blade to Genoa salami and roast turkey breast differed depending on the biofilm-
forrning ability of the inoculum and the injury to which the inoculum was exposed prior
to inoculation on the slicer blade. While there was an overall decrease in the amount of
transfer to each successive slice (Figures 5.2 — 5.13), transfer was not generally linear (R2

< 0.70) or logarithmic (R2 < 0.70).

124

Figure 5.2. Transfer of healthy, strong biofilm forming L. monocytogenes from an
inoculated slicer blade (6 log CFU/blade; 6 and 24 h/ 78 :l: 2% RHI22°C) to
uninoculated turkey (n = 3). Open symbols not quantifiable by direct plating,
positive by enrichment.

3.50 7 O
3.00 l . o 9

i
2501 . ’0 .
O O oStrong, 6 h
2th I o

i ’ 9. ”3.0 . a Strong, 24h
1.50 ]0 II

(I

 

Log CF Ulslice

1.00
0.501 . .I. u. . _ u

0.00 m" -, - -~. flour—cr—ee. ,
0 5 10 15 20 25 30

Slice number

 

Figure 5.3. Transfer of healthy, weak biofilm forming L. monocytogenes from an
inoculated slicer blade (6 log CFU/blade; 6 and 24 h/ 78 :l: 2% RH/22°C) to
uninoculated turkey (n = 3). Open symbols not quantifiable by direct plating,
positive by enrichment.

O

.2

E

a A Weak, 6 h
o 0 Week, 24 h

0.50 - O O I MAA

 

0.00 e ~~-. ""‘OO—r’ro—‘HT’ e —+0--—AQAAAAA
0 5 10 15 20 25 30

Slice number

125

Figure 5.4. Transfer of healthy, strong biofilm forming L. monocytogenes from an
inoculated slicer blade (6 log CFU/blade; 6 and 24 h/78 :l: 2% RH/22°C) to
uninoculated salami (n = 3). Open symbols not quantifiable by direct plating,

positive by enrichment.

4.00 e,

O O OStrong, 6 h
. O I Strong, 24 h

Log CFU/slice
N
. . . . .0 . .
O
_ - -.L _.. fizu.._
I
I
O
0

Slice number

Figure 5.5. Transfer of healthy, weak biofilm forming L. monocytogenes from an
inoculated slicer blade (6 log CFU/blade; 6 and 24 h/ 78 :l: 2% RHI22°C) to
uninoculated salami (n = 3). Open symbols not quantifiable by direct plating,
positive by enrichment.

4.00 ‘1

3.50 '

O
3.00 I . A Weak. 6 h
l O 0.00:.
2-50 1 0 Weak, 24 h

A
2.00 -« A AAAA‘

0 CF Ulsllce

 

 

Slice number

126

Figure 5.6. Transfer of chlorine-injured, strong biofilm forming L. monocytogenes
from an inoculated slicer blade (6 log CFU/blade; 6 and 24 h/ 78 :l: 2% RHI22°C) to
uninoculated turkey (n = 3). Open symbols not quantifiable by direct plating,

positive by enrichment.

4.00 - O
3 0 i ..o OStrong,6h
'5 i . 090,. IStrong,24h
3001 9’9
d) ' I .
.2 » 99
7’ 2.50 i I .
5 7| 9 e
5’ 1.50 i . I . . .
3 l I .9
1.00 l I.
l I I II I I
0.00 -L- ._ - —- Elw- r-DCI- w—tElU—u-DDUDDU
0 5 10 15 20 25 30

Slice number

Figure 5.7. Transfer of chlorine-injured, weak biofilm forming L. monocytogenes
from an inoculated slicer blade (6 log CFU/blade; 6 and 24 h/ 78 i 2% RH/22°C) to
uninoculated turkey (n = 3). Open symbols not quantifiable by direct plating,

positive by enrichment.

400,
$ A A Weak, 6 h
3'50 ‘ 0 Weak, 24 n
3001
3 ' ‘
g Zfl)j
‘ l A
E 2m11 ' g .
o i ' ‘AA 0
1001 0 A 3A
A o A AAA AA
050i .‘ “ ‘.
0.00 1 ——~ -. e — e-4;Ho_p-o_.m -_____-O
0 5 10 15 20 25 30

Slice number

127

Figure 5.8. Transfer of chlorine-injured, strong biofilm forming L. monocytogenes
from an inoculated slicer blade (6 log CFU/blade; 6 and 24 h/ 78 :l: 2% RH/22°C) to
uninoculated salami (n = 3). Open symbols not quantifiable by direct plating,
positive by enrichment.

4.00 1‘

3.50 4‘ OStrong, 6 h
300 % IStrong.24h
2.50 -

2.00 ~
0

'0
1.50i . . . O . O O... ...
° 9 o 90 , ,
o

o o O o
0'50“ I .00... I II
0.00 .-_- — e ee- 0- C——-o-C—o—ee

0 5 1o 15 20 25 30

Slice number

Log CFU/slice

1.00 *

 

Figure 5.9. Transfer of chlorine-injured, weak biofilm forming L. monocytogenes
from an inoculated slicer blade (6 log CFU/blade; 6 and 24 h/ 78 :l: 2% RH/22°C) to
uninoculated salami (n = 3). Open symbols not quantifiable by direct plating,
positive by enrichment.

4.00 1
l A Weak, 6 h

3.50 -1
300 fi lWeak, 24 h

2.50 4 A I
2.00 a A
1.50 i A

Log CFU/slice

1.00 - A I
I. ‘ I A
0.50 ’1 A
II I IA I'l ' I
0.00 all; . AA A—-—ADAAA*ADDCIAAAACIA~ 13A
0 5 10 15 20 25 30

Slice number

 

 

128

Figure 5.10. Transfer of cold-injured, strong biofilm forming L. monocytogenes
from an inoculated slicer blade (6 log CFU/blade; 6 and 24 h/ 78 :l: 2% RH/22°C) to

uninoculated turkey (n = 3).

 

5.00 -
4,50 _. . OStrong, 6 h
4.00 ~ I’. I Strong, 24 h
,8 3.50 4 I o . .
g 3.00 a -M 0 0 o.
=5 3:33 i "-3’ 538:“ W
3’ . I .‘II I I
1.00 9 I ' I
0.50 I
0.00 T W T l— — '— _T—__T_‘—“

Slice number

Figure 5.11. Transfer of cold-injured, weak biofilm forming L. monocytogenes from
an inoculated slicer blade (6 log CFU/blade; 6 and 24 h/ 78 :l: 2% RHI22°C) to
uninoculated turkey (n = 3). Open symbols not quantifiable by direct plating,

positive by enrichment.

5.00 —
4.50 - AWeak, 6 h

4.00 a A OWeak, 24 h
3.50l .‘A

3.00 1 O M‘

2.50 g ‘A ‘AA

2.00 i A “M ‘A

- 9 A A

A O

1.00 3 . C 0 . “‘ .
0.50 i O. C. O. O .. g
0.00 "h__—fi'l“""‘**l—O“——FO-O_T‘OO*’"O~‘_OAU

0 5 10 15 20 25 30

Slice number

Log CFU/slice

 

129

Figure 5.12. Transfer of cold-injured, strong biofilm forming L. monocytogenes
from an inoculated slicer blade (6 log CFU/blade; 6 and 24 h/ 78 :l: 2% RH/22°C) to

uninoculated salami (n = 3).

4.00 .
3.50 —
3.00 ~ I
2.50 ~
2.00 ~ I ” g

OStrong, 6 h
I Strong, 24 h

o CFU/slice

L9
A—l
our
0::

O
I

 

 

Slice number

Figure 5.13. Transfer of cold-injured, weak biofilm forming L. monocytogenes from
an inoculated slicer blade (6 log CFU/blade; 6 and 24 bl 78 :l: 2% RHI22°C) to
uninoculated salami (n = 3). Open symbols not quantifiable by direct plating,
positive by enrichment.

4.00 w.

350 4 AWeak,6h
8 3.00 ~ A OWeak,24h
= H A
3 200 a A‘ ‘ h“
8 . ' e e 0 ml“ ‘
" 1.00 e 0 0'0.“ 0 00 e 0

WA C . . . . g
0.50 . . . .
0.00 +~ ~~ . -._s__n__ cw #0 “mm

 

 

0 5 10 15 20 25 3O
Slice number

130

Overall, less transfer was observed to the first slice for each combination of
conditions. This is likely due to Listeria drying and adhering to the blade since once
moisture and friction are introduced to the surface, a larger number of cells subsequently
transfer from the blade to the product. Transfer was observed out to 30 slices for most
cocktail/injury/incubation time/product combinations. However, direct counts could not
be obtained for every slice, with some slices negative for L. monocytogenes in all three

replicates for specific treatment combinations (Tables 5.1 and 5.2).

131

Table 5.1. Number of direct counts and positive enrichments for salami sliced with
slicer blades contaminated with healthy, cold- or chlorine-injured L. monocytogenes
(6 log CFU/blade)

Slice Strong Biofilm Former Weak Biofilm Former
Healthy Cold Chlorine Healthy Cold Chlorine
6h 24h 6h 24h 6h 24h 6h 24h 6h 24h 6h 24h

 

 

3/3a 3/3 2l3 3/3 1/1 0/0 3/3 3/3 1/3 0/0 1/1 0/1
3/3 3l3 3/3 3l3 3/3 2/2 3/3 3/3 3/3 2/3 2/3 1/1
3/3 3/3 3/3 2/2 3/3 1/1 3/3 3/3 3/3 3/3 3/3 1l2
3/3 3/3 3/3 3/3 2/2 0/0 3/3 3/3 3/3 2/3 2/3 1/1
3/3 3/3 3/3 3l3 2/2 1l1 3/3 3/3 3/3 2/2 1/2 2/2
3/3 3/3 3/3 3/3 2/2 2/2 3/3 3/3 3/3 2/3 1l2 1l1
3/3 2/3 3/3 3/3 3I3 2/2 2/2 3/3 3/3 1/2 2/3 0/0
3l3 3/3 3/3 2/3 3/3 1/1 3/3 3/3 3I3 2/3 0/2 1l1
3/3 2/2 3/3 3/3 3/3 3/3 3/3 3/3 3/3 2/2 0/2 0/0
3/3 2/3 3/3 3/3 2/2 0/0 3/3 3/3 3/3 2/2 0/3 1/1
3/3 2/3 3/3 3/3 1/1 1/1 2I3 0/1 3/3 2/2 2/3 2/2
3/3 2/3 3/3 2/3 3/3 0/1 2/3 1l1 3/3 2/3 0l2 1/1
3/3 2/3 3/3 3/3 2/2 1/1 2/3 0/0 3/3 2/2 1/2 0/1
3/3 2l3 3/3 3/3 3l3 1/1 1/3 0/1 3I3 2/2 0/1 0/0
3/3 1/3 3/3 2/3 1/1 0/0 1/3 0/0 3/3 2/2 0/2 1/1
3/3 3/3 3/3 3/3 1/3 1/1 1/3 1/2 3/3 2/3 0/1 1/2
3/3 1I3 3/3 3/3 2/2 0/1 2/2 0/0 3/3 2/3 1/2 1/1
3I3 2/2 3/3 3/3 2/2 1/1 1/3 0/0 3/3 2/2 0/2 0l0
3/3 2/2 3/3 2/3 3/3 0/0 2/3 1l1 3/3 2l3 1l3 0l1
3/3 0/2 3/3 3/3 2/2 1 /1 1/3 0/0 3/3 1/2 0/2 0/1

@GNOO’ItfiUN-fi

”aaaaaaaaaa
OGQNOUI-th-IO

21 3/3 0/3 3/3 3/3 3/3 0I0 2/3 1/1 3/3 1l2 0/2 0/1
22 3/3 0l2 3/3 3/3 3/3 0/0 1/3 1/1 3/3 2l2 0/1 3/3
23 3/3 1/2 3l3 2/3 2l2 0/0 1/2 1l1 3/3 1/2 0/2 0/0
24 2/3 113 3/3 3/3 2/2 0/0 2/3 1/1 3/3 1l2 0/2 0/0
25 3/3 1/2 3l3 2/3 3/3 1l1 1/3 0/2 3/3 1/2 0/1 1/2
26 3/3 0/3 3/3 2/2 2/2 1/2 1/3 0/1 3/3 1l2 0/2 0/1
27 3/3 1l2 3/3 3/3 2/3 0/0 1/1 1/1 3/3 1l2 0/2 0/0
28 3/3 1/2 3/3 3/3 3/3 1/1 0/0 0/0 3/3 1/2 1/1 1l1
29 3/3 0/2 3/3 2/3 2/2 111 2/3 1/1 2/3 2/2 0/1 0/2
30 3/3 0l2 3/3 1/3 1/2 0/0 2/3 0/2 2/3 1/2 0/0 0/0

aDirect counts / enrichment results for 3 replicates

132

Table 5.2. Number of direct counts and positive enrichments for roast turkey breast
sliced with slicer blades contaminated with healthy, cold- or chlorine-injured L.
monocytogenes (6 log CFU/blade)

Slice Strong Biofilm Former Weak Biofilm Former
Healthy Cold Chlorine Healthy Cold Chlorine
6h 24h 6h 24h 6h 24h 6h 24h 6h 24h 6h 24h

 

 

 

1 3/32‘ 1/1 1/1 1/3 3/3 1/3 2/2 3/3 3/3 1l2 2/2 0/0
2 3/3 2/2 3/3 3B 3B 3/3 3l3 2/2 3/3 3/3 3/3 3I3
3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/ 3 3/3 3/3 3l3
4 3/3 2/2 3/3 3/3 3/3 3/3 3/3 1/1 3/3 3/3 3/3 2/3
5 3/3 2/2 3/3 3/3 3/3 3/3 3/3 0/0 3/ 3 2/3 3/3 2/3
6 3/3 2/2 3/3 3/3 3/3 3I3 2/2 1/1 3/3 3I3 3/3 2/3
7 2/2 2/2 3/3 3/3 3/3 2/2 3/3 0/0 3/ 3 2/3 3/3 2/3
8 2/2 2/2 3/3 3/3 3/3 3/3 2/2 0/1 3/3 2/3 3I3 2/3
9 2/2 1/ 1 3/3 3/3 3/3 2/2 3/3 0/1 3/3 2/3 2/2 2/2
10 2/2 1/1 3/3 3/3 3/3 1/3 2/2 1/1 3/3 1l3 2/2 2/3
11 2/3 1/1 3/3 3/3 3/3 1/2 1/2 0/0 3/3 1/3 2/2 2I3
1 2 2/2 0/1 3/3 3/3 3/3 0/2 2l2 0/1 3/3 0/3 3/3 1/1
13 1l2 1/1 3/3 3/3 3/3 1/2 1l2 0/0 3/3 1/3 2/2 1/1
14 1/2 0/1 3/3 3/3 3/3 2/3 2/3 0/0 3/3 1I3 2/2 112
15 2/2 0/0 3/3 2/3 3/3 1l2 1/2 1/1 3/3 1l2 1l2 0/2
16 2/3 1l1 3/3 2/3 3/3 0/2 1/2 0/0 3/3 1/1 2/2 0/1
17 2/2 1/1 3/3 3/3 3/3 0/2 112 1/2 3/3 1/3 2/2 1/2
18 2/2 0/1 3/3 2/3 3/3 2/3 1/1 1l1 3/3 0/2 2/3 0/1
19 2/2 0/1 3/3 2/3 3/3 1/3 0/1 1/1 2/3 1/2 2/2 1/1
20 2/2 0/0 3/3 2/3 3/3 0/2 1 I 1 1/1 2/2 1l1 2/2 0/0
21 1/2 0/0 3/3 2/3 3/3 0/2 1/1 0/1 2/3 0/2 0/0 0/0
22 2/3 0/1 3/3 3/3 3/3 0/2 1/1 0/0 2/2 0/3 1l1 0/1
23 2/2 1/1 3/3 3/3 3/3 1/2 2/2 0/0 2/3 1/2 1 I2 0/0
24 1/2 0/0 3/3 3/3 3/3 1/2 0/1 0/0 2/3 1/2 1 I 1 0/1
25 1/2 2/2 3/3 2/3 3/3 0/2 OH 0/1 2/3 0/1 1/1 0/1
26 2/2 0/0 3/3 3/3 3/3 0/2 0/2 0l0 2/ 3 1/2 1/1 0/0
27 2/2 0/0 3/3 3/3 3/3 0/2 0/1 0/0 1/3 1/3 0/0 0/0
28 2/2 0/0 3/3 3/3 3/3 0/2 0/2 0/0 2/ 3 0/3 1l1 0/0
29 2/2 0/1 3/3 3/3 2/3 0/2 0/1 0/0 2/3 1/2 1 I 1 1/1
30 1/2 0/0 3/3 2I3 2/ 3 0/2 0/1 0l0 2/ 3 0/0 2/3 0/1

aDirect counts / enrichment results for 3 replicates

133

5.4.2 Affect of biofilm forming ability, injury, incubation time and product on

transfer of L. monocytogenes

When the results for all 30 slices are summed for each treatment, significantly
greater cumulative transfer (P < 0.05) can be seen to turkey (3.61 d: 0.89 CFU) than to
salami 3.12 :l: 0.64 log CFU Furthermore, significantly greater cumulative transfer was
seen for cold-injured as opposed to chlorine-injured or healthy L. monocytogenes cells.
(Table 5.3). More Listeria were transferred after being dried on the slicer blade for 6 h
(3.72 i 0.69 log CFU) than 24 h (3.01 i 0.78 log CF U), however, no significant
difference in percent injury of the transferred bacteria was seen between the two
desiccation times. Strong biofilm formers transferred to meat in significantly higher (P <
0.05) numbers (3.62 :t 0.79 log CFU) than weak biofilm formers (3.12 :t 0.76 log CFU.)
with the biofilm formers (73.2 :t 23.1% injury) being significantly more injured at the
time of transfer than strong biofilm formers(54.3 i 34.2% injury). Significant differences
were also observed for overall transfer (Figures 5.14 — 5.16) and injury at the time of

transfer (Figure 5.17) based on combined affects of product, time, cocktail and injury.

Table 5.3. Cumulative log transfer of previously injured and uninjured L.
monocytogenes to delicatessen meat and percent injury at the time of transfer.

 

Cumulative Log CFU Injury (% of total CFU

 

transferred)
Healthy 3.30 :1: 0.713 50.7 :1: 27.13
Cold 3.69 i 0.79b 47.8 :1: 26.23
Chlorine 3.12 :l: 0.853 92.8 :l: 11.0b

 

Means with different superscripts are significantly different (P < 0.05)

134

Figure 5.14. Cumulative log transfer of L. monocytogenes to delicatessen meat by
product and incubation time (n = 18). Means with different superscripts are
significantly different for total transfer (P < 0.05).

Log CFU

5
4.5 ‘
4 .

0.5
o .

    
       
         
 

          

Q
Q

’V'I"

at.
s“
ssssssv

\\\\\\
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\

z
$§§§§§§“
at

s
\\
§§

\\
RR

l
Ill!
95%
lllllllll
lllllllll
lllllllll
lllllllll
lllllllll
llllllll
lllllll
was
llllllll
I

ll!!! lg

\\\\\

 
       

E Salami, 6 h
E?! Salami, 24 h
Turkey, 6 h
a Turkey, 24 h

Figure 5.15. Cumulative log transfer of L. monocytogenes to delicatessen meat by
product and injury treatment (n = 12). Means with different superscripts are
significantly different for total transfer (P < 0.05).

Log CFU

 

‘wy
\\§
‘

§sssssss . :

W

VVV
\V e\\

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135

E Salami, Chlorine
E1 Salami, Cold
Salami, Healthy
5 Turkey, Chlorine
l Turkey, Cold

El Turkey, Healthy

Figure 5.16. Cumulative log transfer of L. monocytogenes to delicatessen meat by
product and cocktail (n = 18; Strong = Strong biofilm-former cocktail; Weak =
Weak biofilm-former cocktail). Means with different superscripts are significantly
different for total transfer (P < 0.05).

 

5
4.5
4
D 3.: E Salami, Strong
tu-5 2 5 El Salami, Weak
E; '2 Turkey, Strong
1.5 a Turkey, Weak
1
0.5
0

Figure 5.17. Percent injury of L. monocytogenes at the time of transfer to
delicatessen meat by previous injury treatment and cocktail (n = 12; Strong =
Strong biofilm-former cocktail; Weak = Weak biofilm-former cocktail). Means
with different superscripts are significantly different from percent injury (P < 0.05).

 

... 100

i 90

E 80 a Chlorine, Strong
3 70 ‘ EChlorine, Weak
g 60

g 50 1 Cold, Strong
3 4o ‘ ECold,Weak
£3 30 ; %7/ 9. ElHealthy, Strong
8 20 ,, %% .3. Healthy, Weak
“ // ”53$

3- 10 t We???

E 0

136

5.4.3 Survival of L. monocytogenes on slicer blades over time

Strong biofilm formers survived desiccation on slicer blades in significantly
greater numbers than weak biofilm formers, with survival rates of 51.4% d: 23.2 and
38.7% i 24.9, respectively. Listeriae that were cold-injured before desiccation on
stainless steel exhibited significantly greater survival (55.8% i 20.9; P < 0.05) compared
to uninjured (40.1% :1: 28.2) and chlorine-injured cells (39.3% at 21.7). Listeria viability
was significantly greater after 1 h (53.0% i 17.5; P < 0.05) as opposed to 6 (38.9% 3c

28.8) or 24 h (43.9% :t 25.1) of incubation on stainless steel.

137

5.5 DISCUSSION

While there was an overall decrease in the amount of transfer to each successive
slice, transfer was not generally linear (R2 < 0.70) or logarithmic (R2 < 0.70). In this
respect, the transfer observed afier 6 and 24 h of desiccation on the slicer blade resembled
the transfer from slicer blades inoculated at 103 CFU/blade previously described by Vorst
et al. (2006) Using an initial inoculum of 6 log CFU/blade, L. monocytogenes transfer
was quantifiable to all 30 slices, particularly for both cold-injured cocktails. However,
Vorst et al. (2006) were unable to quantify transfer beyond the 5th slice using inoculum
levels of 5 and 3 log CFU/blade. Greater overall transfer of cold-injured as opposed to
healthy and chlorine-injured cells of L. monocytogenes may be related to the higher
overall survival of the former as determined by viability staining. Healthy and chlorine-
injured cells showed no difference in overall transfer. However, the chlorine-injured
cells that transferred exhibited significantly greater injury at the time of transfer than the
uninjured and cold-injured cells, indicating that refrigeration extended the persistence of
L. monocytogenes in the environment for as long as 24 h following exposure.

To the contrary, exposure to chlorine at sublethal levels will not significantly
impair cell survival and transfer as compared to their uninjured counterparts.
Furthermore, in contrast to the findings by Midelet et a1 (2006), in which chlorine
increased the adherence of L. monocytogenes to stainless steel, under the conditions used
in this study, no significant difference was seen in the ability of uninjured and chlorine-
injured cells to be transferred from stainless steel. However, in this study, exposure to
chlorine occurred before Listeria was inoculated on the stainless steel surface, suggesting

that a greater reduction in transfer and a corresponding increase in adhesiveness to the

138

surface may have resulted if Listeria had been exposed to chlorine afier inoculation onto
stainless steel.

Strong biofilm forming strains of L. monocytogenes transferred to delicatessen
meats in greater numbers than weak biofilm formers. The strains chosen for this study
consisted of L. monocytogenes that formed either the strongest biofilms (as determined
by a microtiter plate assay for biofilm formation), or the weakest biofilms out of 122
strains that were previously tested for biofihn-forming ability (Chapter 2). According to
Borucki et al. (2003), persistent strains were better able to form biofilms, as measured by
a microtiter plate assay for biofilm formation with other studies showing that persistent
strains are significantly more adherent to surfaces (Lunden et al., 2000; Norwood and
Gilmour, 1999). Therefore, it was decided to see whether this assumed persistence of L.
monocytogenes according to biofilm formation would impact transfer from delicatessen
slicers to delicatessen meats.

Significantly higher total transfer was observed for strong biofilm forming strains
of L. monocytogenes as opposed to weak biofilm formers. Since this was the opposite of
what would be expected if biofilm formation is a predictor of persistence, survival of
strong and weak biofilm forming strains was examined during desiccation. Viability
staining showed that strong biofilm formers survived in greater numbers under desiccated
conditions than weak biofilm formers, suggesting that the ability to form thick biofilms is
advantageous for survival of Listeria in stressful environments. Greater overall transfer
of the strong biofihn-forming strains to turkey may be partially due to their increased
ability to survive desiccation. In a previous study (Chapter 2), the weak biofilm forming

strains used here were significantly more hydrophobic than the strong biofilm forming

139

strains, however the literature is contradictory as to whether hydrophobic cells or
hydrophilic cells will attach more strongly to stainless steel (Chavant et al., 2002;
Briandet et al., 1999; Midelet and Carpentier, 2002). Hence, further research into the
surface characteristics of both L. monocytogenes and common food contact surfaces is
needed to better assess the impact of attachment and persistence on L. monocytogenes
transfer.

Significantly greater transfer was seen after 6 h as opposed to 24 h, however, this
cannot be explained by survival differences between the two time points since no
significant difference in survival was seen by viability staining. In the aforementioned
Vorst et a]. (2006) study, transfer to salami was more continuous throughout the 30 slices
than to turkey or bologna, both of which were higher in moisture and lower in fat than
salami. Similar trends were observed in this study, which showed that L. monocytogenes
had lower overall cumulative transfer to salami than to turkey. This fiirther reinforces the
conclusions drawn by Lin et al (2006) and Vorst et al. (2006) that the fat deposited by the
salami may help L. monocytogenes remain on equipment longer, and therefore result in
continual transfer of low numbers.

Various combinations of factors including time and product, injury and product, and
biofilm forming ability and product all significantly impacted Listeria transfer during
slicing. The greatest overall transfer was generally observed to turkey under conditions
that allowed significantly greater survival, whether by strong biofilm formers, or cold-
injured cells. Hence, survival appears to play a large role in transfer of L. monocytogenes

over extended periods of time, thereby reinforcing the importance of proper cleaning and

140

sanitizing of food contact surfaces to prevent the establishment of persistent L.

monocytogenes strains in niches within food processing and retail environments.

141

CHAPTER 6

VALIDATION OF A PREDICTIVE MODEL FOR LIS T ERIA

MONOC Y T OGENES TRANSFER DURING SLICING OF DELICATESSEN

MEATS

142

6.1 ABSTRACT

Despite careful attention to cleaning and sanitizing, Listeria monocytogenes can
persist for weeks or months on difiicult-to-clean stainless steel surfaces including
delicatessen slicer blades. Consequently, improperly cleaned slicers can contain small
numbers of cells that can potentially be transferred to deli meats during slicing. In
response to these concerns, transfer of healthy, chlorine-injured and cold-shocked L.
monocytogenes cells of weak and strong biofilm-forming ability was assessed after 6 and
24 hours of drying on delicatessen slicer blades. These data were then used to test a
previously developed predictive model for Listeria transfer to and from mechanical
delicatessen slicers and kitchen knives.

The model and subsequent computer program in GWBasic are based on the
following two assumptions: 1) the number of Listeria cells transferred from the blade to
the meat during slicing is a fraction of the number of Listeria cells on the blade just
before each sequential slice, and 2) the number of Listeria cells transferred to
surrounding areas is a different fraction of the number of cells on the blade just before
each sequential slice. The model predicts an exponential decay in the number of cells
versus slice number. Observed and predicted values were similar for the transfer of
healthy cells to salami and roast turkey breast (R2 = 0.88). However, the model was least
accurate when used to predict transfer of previously cold-injured cells. Greater variance
was seen between the observed values and the predicted values (R2 = 0.65). For all other
scenarios, which included transfer fi'om strong and weak-biofilm forming cells to salami
and turkey, and transfer after 6 and 24 h of desiccation on a slicer blade, the model was

an accurate predictor of transfer (0.77 S R2 S 0.94).

143

6.2 INTRODUCTION

In the federal Listeria Risk Assessment (FDA/USDA/CDC, 2003), delicatessen
meats were ranked as posing the highest relative risk of exposing the public to L.
monocytogenes out of 23 categories of ready-to-eat (RTE) foods. In the same risk
assessment, quantitative transfer to and from commercial meat slicers, knives and cutting
boards in delicatessens was identified as a key informational gap for assessing exposure
(FDA/USDA/CDC, 2003). In a survey of L. monocytogenes in RTE foods, luncheon
meats that were store-packaged were more frequently contaminated with L.
monocytogenes (6.8 times as likely to be contaminated) than manufacturer-packaged
meats (Gombas et al., 2003). However, the samples contaminated at levels higher than
102 CFU/ g were more likely to be manufacturer-packaged, with most positive samples
containing less than 1 CFU/g (Gombas etal., 2003). The higher prevalence of L.
monocytogenes in delicatessen meat sliced at retail strongly suggests that the delicatessen
slicer is an important vehicle for cross-contamination of products.

Within the last decade, risk assessments have necessitated the development of
dynamic models that provide estimates of bacterial survival, growth, and distribution
throughout food processing and storage. Microbiological risk assessments depend upon
exposure assessments, however, these exposure assessments rely on existing data for
presence of bacteria in foods, the accuracy of which may be limited by sample size and
different test methods (Gardner, 2004).

Predictive modeling can be used to estimate microbial contamination levels,
distributions and rates of transfer in the environment. Schaffner (2004) has described the

basic mathematical framework for modeling L. monocytogenes cross-contamination in

144

food processing facilities. Using the resultant models can help a processor determine
whether an overall greater reduction in L. monocytogenes prevalence in a production
facility could be achieved by requiring better raw material quality or by improved
sanitation efforts.

A model with similar benefits, in terms of determining the best testing sites to
minimize contamination of ground beef produced using a commercial grinder, was
developed by Flores and Stewart (2004). According to their model, rather than random
sampling of a ground beef lot to determine E. coli 01 57:H7 contamination, testing the
collar that fixes the grinder die and blade to the meat grinder was shown to be a more
accurate predictor of contamination.

In models that have been specifically developed to assess transfer of L.
monocytogenes, one assessed the risk of L. monocytogenes transfer and subsequent
grth due to contact with bare or gloved hands (Perez-Rodriguez et al., 2006). This
model predicted that the highest risk of contamination comes from handling raw and
ready-to-eat meats with the same gloves. This risk was higher than the risk of cross-
contamination from bare, washed hands (Perez-Rodriguez et al., 2006).

Thus far, Vorst (2005b) has developed the only model to predict L.
monocytogenes transfer during slicing of RTE delicatessen meats on a commercial
delicatessen slicer. Their exponential decay for direct CFU model predicts the number of
CF U transferred to any given slice, as well as the number CFU lost to the environment
through aerosols and bacterial death. Under the conditions tested by Vorst (2005b), this

model had a correlation coefficient varying from R2 = 0.40 when slicing salami, to over

145

0.90 when slicing turkey or bologna with a slicer blade inoculated at 8 log CFU/blade
(Vorst, 2005b).

The objective of this study was to verify the predictive model developed by Vorst,
2005b) for Listeria transfer based on quantitative data obtained from slicing of turkey and
salami with an inoculated delicatessen slicer blade with additional variables including the
physiological state (healthy vs. injured), and biofilm-forming ability (strong vs. weak) of

L. monocytogenes and length of desiccation on the blade before slicing (6 vs. 24 h).

6.3 MATERIALS AND METHODS
6.3.1 Transfer coefficients for surface-dried, uninjured and injured L.
monocytogenes during slicing of turkey and salami
Transfer data were obtained from slicing roast turkey breast and salami with a
delicatessen slicer that was inoculated with an uninjured, cold-injured or chlorine-injured
cocktail of L. monocytogenes and then held for 6 or 24 h at 78% RH before slicing
(Chapter 5). The previously developed model of Vorst (2005) was used to determine
transfer coefficients for L. monocytogenes from contaminated knife and slicer blades to

uncontaminated product.

6.3.2 Predictive modeling of L. monocytogenes transfer during slicing of roast

turkey breast and salami

A model based on the following three assumptions was developed to predict the
previously calculated transfer coefficients by Vorst (2005b): a) the number of Listeria
cells transferred from the blade to the meat during slicing is a fraction (f.) of the number

of Listeria cells on the blade just before each sequential slice, b) the number of Listeria

146

cells transferred to surrounding areas is a different fraction (f2) of the number of cells on
the blade just before each sequential slice, and c) the CFU on the blade before any slicing
begins is No.

The consequences of these assumptions are as follows (V orst, 2005b):

lSt Slice

CFU on Meat = f,N0 (1a)
CF U to Surroundings = sz0 (1b)
CFU left on Blade = N0 -f,N0 —f2N0 =(1—fl —f2)N0 (10)
2nd Slice

CFU on Meat = f,(1—fl —f2)N0 (2a)
CFU to Surroundings = f2(1—fl — f2)N0 (2b)

CFU lefi on Blade: (l—f, —f,)N0 —f,(1—f, —f,)N,, —f,(1—f, —f,)N0

=(1—f. wow. (2.)
3rd Slice
CFU on Meat = f,(1—fl - f,)2N0 (3a)
CFU to Surroundings = f,(1—f, — f2)2N0 (3b)

CFU left on Blade= (l—f, —f,)2N0 —f,(1—f, —f2)2N0 -f2(l—f, —f,)21v0

147

=(1—fl—f2YN. (3c)

Xth Slice
CFU on Meat = f,(1— f} —f2)’("‘N0 (4a)
CFU to Surroundings = f2(1- fl — f2)X"N0 (4b)

CFUlefion Blade=(1—fl —f2)X_lN0 -fl(1-fr ‘fzy‘HNo rfzfl-f. —f2)X—IN0

=(1—f.—f2)XNo <4c)

6.3.3 Predicting CFU on meat as a function of slice number (X)

The model predicts that the number of CFU transferred to slice X is:
CFU (X>= f.(1—f. ~13)‘“'No (5a)

This can be arranged as:

CFUtX)=i—_-f}—’Z_°—f— (l—f.—f.)" (5b)

This can be rewritten as:

CFU (X) = Ira" (5c)

Where “k” and “a” are constants related to the model parameters fl, f2, and No.

6.3.4 Fitting the equation to experimental data (finding “k” and “a”)

Taking the natural log of the predictive equation gives the general equation for a straight

line.

148

This equation can then be fitted to the data to find the SIOpe (m) and intercept (b), where:

y = ln(CF U )
b = ln(k)
m = ln(a)

x = slice number

It then follows from equations (5b) and (5c) that:
a=1—f,—f2=e"’ (6b)

k=./INO(1_fl—f2)=eb (66)

6.3.5 Interpretation of fit results

Given that m and b are known from the straight line fit to y = In (CF U) vs. x = slice
number, equations (6b) and (6c) can be used to find f1 and f; if the original inoculum

level, No, is known. The parameter “a” is the fraction of CF U remaining on the blade

after any slice. The slope “m” from the fit will always be negative, so (1 — fl — f2) <1.

The number of CFU transferred from the blade to the first slice is fiNo. From the

relationships between the fit parameters “m” and “b” and the model parameters “ fl ”,

“f'z 99 and cc N0 99 it follows that:

Fraction remaining on blade = e’"

CFU transferred to 1St slice = e”””

149

99 66

Given the inoculation level or original number of CF U on the blade, “ fl , f2 ” and

“ N0 ” can be found as follows using these previous equations:

 

f. = 8N0 (7a)
f2 =1— f. — e’" (7b)
6.4 RESULTS

Listeria cumulative transfer amounts based on the experimental data for each
mechanical slicer scenario are presented in Figures 6.1 — 6.4. A similar trend was seen
for all scenarios, with 99% of the total Listeria transfer occurring within the first 10
slices. Significantly more transfer to turkey than to salami had been observed previously
(Chapter 5). This resulted in smaller transfer coefficients f1 and f2 for salami than for
turkey. This is reflected in the fact that cumulative transfer for salami does not reach a
plateau, with small numbers of listeriae continually transferred over more slices than
what is observed for transfer to turkey. For the scenarios studied—biofilm forming
ability, time, and injury—a plateau was reached in the transfer of each, with 99% of the

total transfer generally occurring within the first 10 slices.

150

Figure 6.1. Cumulative L. monocytogenes transfer from an inoculated slicer blade
(6 log CFU/blade) to turkey and salami

N

_x
01

’r AAAAAAAAAAAAAMMMA
- AAA
AAA

A A Turkey
El Salami

.0
01
l>

 

Cumulative transfer (%)

0
"it.
1
l
i
i

 

O 5 1O 15 20 25 3O
Slice number

Figure 6.2. Cumulative transfer by strong and weak biofilm forming L.
monocytogenes from an inoculated slicer blade (6 log CFU/blade) to turkey and
salami

 

 

 

$3 2 l

‘5' I

“g 1 5 : AAAA

.g A El Weak
‘5 .

- 0.5 A

3 IllllHlTllllllllllllll

g DWI I I

o O *fl’rrw—r-W '"""—' ##4”—"”"l " ' l ' tarry“ l

 

5 1O 15 20 25 3O
Slice number

0

151

Figure 6.3. Cumulative L. monocytogenes transfer from an inoculated slicer blade
(6 log CFU/blade; 6 and 24 h incubation, 22°C/78% RH) to turkey and salami

N

 

 

§ 1

t l

gloj A ,,,,,,,QMM

é , A 024 h
m

- 0.5 1 A

3 FfllflfllIlLflLJfllllfllflfllllflfllfl]

E Eflfiljfl

3 0 » gfiflflfl “fig”. _— T“ 'TT'"T".””‘_fil’ _“ *‘l

0 5 10 15 20 25 30

Slice number

Figure 6.4. Cumulative transfer by uninjured and cold-injured L. monocytogenes
from an inoculated slicer blade (6 log CFU/blade) to turkey and salami

0.5 I ”W

10 20 25 30
Slice number

I
i
l

Cumulatlve transfer (%)
'01
D
0
0.
O.

O
(’1
_x
01

152

6.4.1 Predictive model for L. monocytogenes transfer during slicing of turkey and

salami using a mechanical slicer

Using the aforementioned predictive model (Sections 6.3.2 — 6.3.3), a program
was developed using GWBasic to find the transfer coefficients f1 and f2 for the different
Slicer scenarios, including experimental variables; product (turkey and salami),
incubation time on the stainless steel blade (6 and 24 h), bacterial injury (healthy and
cold-injured), and biofilm-forming ability (strong and weak) which had been identified in
previous studies as being significantly different in total cumulative transfer (Chapter 5).
One inoculum level was used for all scenarios (N0 = 106 CFU/blade). All data replicates
were averaged with regards to the aforementioned scenarios, resulting in a minimum of
24 (healthy vs. cold-injured) and a maximum of 36 (all other scenarios) averaged
replicates for use in the GWBasic model.

The first slice for each replicate was not modeled. Due to the length of incubation
on the blade, the first Slice generally had less transfer than the second slice. This is an
artifact of the experiment, it is unlikely that L. monocytogenes would completely dry on a
slicer blade for 6 or 24 h without disturbance. Therefore, modeling began with the
second slice, which followed the output of the program shown in Figure 6.5.

Transfer of weak biofilm-forming L. monocytogenes strains to turkey and salami
resulted in the lowest variance (R2 = 0.94) for observed vs. predicted values for all
models tested (Figures 6.6 and 6.13). Transfer of cold-injured L. monocytogenes to
turkey and salami showed the greatest deviation from the predicted values (R2 = 0.65;
Figure 6.11). Since the transfer to the first slice was typically less than 100 CFU, it was

not necessary to modify the No used in the program—the starting number of bacteria

153

available to transfer to the second slice was still approximately 106 CFU. Likewise, when
percent survival (~40 — 60% of initial inoculum, Chapter 5) at the time of transfer was
input into the model, it simply resulted in a 0.009 - 0.01 decrease for the f; calculated by
the model, with no affect on the predicted transfer to each slice or the correlation
coefficient (data not shown). Again, this is due to the fact that ~40 — 60% of an initial
inoculum of 106 CFU is still approximately 106 CFU. In all possible combinations of
variables, the fraction transferred to the surroundings (f2) always exceeded the fraction
transferred to each Slice of delicatessen meat (f1). This is shown in Table 6.1, which
summarizes the model results for all of the scenarios tested.
Figure 6.5. Example: GWBasic output for turkey and salami sliced using a knife
blade inoculated with weak biofilm forming L. monocytogenes (l06 CFU/blade).

Fraction left on blade during each slice = .8541745

CFUs transferred to 1’t slice = 403.5263

Above results are independent of No

If initial CFUs on the blade = 1E+06, then

Fraction transferred to the product during each slice = 4.035263E-04

Fraction transferred to surroundings during each slice = .145422

Fitted equations (all equivalent) are:

1) 1n CFU(S) = -.1576198 *3 + 6.157861

2) CFU(S) = 472.4167 * .8541745As

3) CFU(S) = 472.4167 * e"(-.1576198 *s)

4) CFU(S) = 472.4167 * 10"(-6.845341E-02 *3)

Correlation coefficient for fit is R2 = .9410404

154

Table 6.1. Model predicted fraction of transfer of Listeria monocytogenes from
delicatessen slicers to delicatessen meat (fl) and environment (f2) by product, biofilm
forming ability, injury, and incubation time on stainless steel blade

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Product Biofilm Type of Time (h) f, f; R2
forming Injury
ability
Turkey Weak Cold 6 2.51 x 10'3 0.18 0.93
24 1.42 x 103‘ 0.15 0.70
Chlorine 6 4.45 x 10“ 0.21 0.74
24 1.85 x 104 0.24 0.74
Uninjured 6 1.68 x 10“ 0.15 0.83
24 3.63 x 10'6 0.16 0.47
Strong Cold 6 2.37 x 10'3 0.07 0.47
24 1.66 x 10'3 0.15 0.85
Chlorine 6 9.43 x 10'3 0.19 0.95
24 7.49 x 10'5 0.11 0.66
Uninjured 6 1.57 x 10‘3 0.15 0.83
24 2.68 x 10'4 0.28 0.84
Salami Weak Cold 6 4.78 x 10‘4 0.08 0.90
24 2.67 x 10‘5 0.04 0.53
Chlorine 6 2.11 x 10'5 0.08 0.47
24 3.24 x 10'6 0.09 0.26
Uninjured 6 1.92 x 101 0.14 0.65
24 3.00 x 10" 0.24 0.58
Strong Cold 6 2.37 x 10“ 0.05 0.69
24 3.07 x 10'5 0.02 0.11
Chlorine 6 4.60 x 10'5 0.04 0.43
24 1.48 x 10'5 0.18 0.49
Uninjured 6 5.34 x 10“ 0.12 0.87
24 1.71 x 10“ 0.13 0.82

 

 

 

 

 

 

 

155

 

Figure 6.6. Plotted output using GWBasic for assessing L. monocytogenes transfer
from an inoculated slicer blade (6 log CFU/blade) to salami

4 ,, —o—Obselved
3.5 l ------- Predicted
.3 1
§ , - f1 = 2.0x10'4
£3" ' R2 = 0.85

 

O “'1 T‘ ’1 —'—‘ "l T‘. _*_' 7’ ' 'F T" ____1
0 5 10 15 20 25 30

Slice number

Figure 6.7. Plotted output using GWBasic for assessing L. monocytogenes transfer
from an inoculated slicer blade (6 log CFU/blade) to turkey

  
  

——+— Observed
------- Predicted

 

0

g

a 2.5

(a) 2 -‘ f1=1.0x10'3

gl 1? "* f2=0.11

_l i 2 =
0.51 R 0.77

0 ‘l ”—_ T— T 1 l_— — _— —l

O 5 10 15 20 25 30
Slice number

156

Figure 6.8. Plotted output using GWBasic for assessing transfer of strong biofilm-
forming L. monocytogenes from an inoculated slicer blade (6 log CFU/blade) to
turkey and salami

 

 

44

3.5 ~ —+—Observed
g 3 1 ------- Predicted
£ 2.5 -
(35 2 f1= 9.0x10“4
8) 1f "'1 f2: 0.10
_l 2 _

05 j R —0.77

O i W“— " l "A" g 1' I!" W" ”H "7-. 'F- ’#l - “"1

0 5 10 15 20 25 30
Slicenumber

Figure 6.9. Plotted output using GWBasic for assessing transfer of weak biofilm-
forming L. monocytogenes from an inoculated slicer blade (6 log CFU/blade) to
turkey and salami

4 1 _+_ Observed
------- Predicted
0
g
g f1 = 4.0x10"
5 f2 = 0.15
8 R2=094
..l

 

 

 

oaw—fiWMfimmfimmm a i

0 5 10 15 20 25 30

Slice number

157

Figure 6.10. Plotted output using GWBasic for assessing transfer of uninjured L.
monocytogenes from an inoculated slicer blade (6 log CFU/blade) to turkey and
salami

 

4 3,. —+—Observed
m 3.5 i ------- Predicted
.2 3
g 2.5 i
3 2 i f1= 5.0x10“
c» 1'5 1 f2 =0.15
3 1 i ‘ - - 2

0'5l - R =0.88
0 7‘ - L L _ _ _ __ “L, ._ L- ._

0 51015202530

Slice number

Figure 6.11. Plotted output using GWBasic for assessing transfer of cold-injured L.
monocytogenes from an inoculated slicer blade (6 log CFU/blade) to turkey and
salami

 
 

 

4 — —o—Observed
0 3.5 ~ ------- Predicted
,3 3 d . .
g 2.5 j f, =7.0x10'4
3 1: f2 =0.08
a ' 1 R2=065
o 1 . .
-' 1
0.5 1
0 1"“? *‘r 1 k 1 ""‘TTT—l" ' "*‘r‘ __'"'—-l
0 5 10 15 20 25 30

Slice number

158

Figure 6.12. Plotted output using GWBasic for assessing transfer of L.
monocytogenes from an inoculated slicer blade (6 log CFU/blade; 6 h incubation,
22°C/78% RH) to turkey and salami

   

4 1 —o—Observed
a, 3.5 ‘1 ------- Predicted
:2 3 1‘
£ 2.5 ‘1 _3
a 2 1 f1 = 1.0X10
0 . _
g, 1.5 l f2-0.11
_l 1 7 R2=0.80

0.5 1
0 — , - ~ ~

5 1O 15 20 25 3O
Slice number

0

Figure 6.13. Plotted output using GWBasic for assessing transfer of L.
monocytogenes from an inoculated slicer blade (6 log CFU/blade; 24 h incubation,
22°C/78% RH) to turkey and salami

 

4 —+—Observed
a, 3.5 i ------- Predicted
=2 31
a 2.5 .
E 21 f,=2.7x10‘4
‘é, 1.? 5; f2 =0.13
4 f R2=0.2
0.5 1 8
0 - — ~ ~— .

10 15 20 25 3O
Slice number

O
01

159

6.5 DISCUSSION

Predictive modeling of microbial pathogens during food production and storage
has been approached using various mathematical models and methods, including
empirical modeling or mechanistic mathematical translation of various factors including
attachment properties and metabolic functions (Bemaerts et al., 2004). To date, Vorst
(2005b) has developed the only model to predict L. monocytogenes transfer during slicing
of RTE delicatessen meats on a commercial delicatessen slicer. The model predicts the
number of CFU transferred to any given slice, as well as the number CFU lost to the
environment through aerosols and bacterial death. Assumptions made in this model
were: a) the number of Listeria transferred (CF U) to any particular slice is a fraction “fl”
of the number of Listeria on the blade just before each slice, b) the number of Listeria
transferred to the surrounding areas during the slicing of each slice is a different fraction,
“f;” of the number of Listeria on the blade just before slicing, and c) “No,” the number of
Listeria cells on the blade that are available for transfer before any slicing begins is
known. The fractions “ 1” and “ 2” are expected to be constant because the degree of
adhesion between Listeria and the blade/meat surface stabilizes after the first slice.
Under the conditions tested by Vorst (2005b), this model had a correlation coefficient
varying from R2 = 0.40 when Slicing salami, to over 0.90 when slicing turkey or bologna

with a slicer blade inoculated at 8 log CFU/blade (Vorst, 2005b).

In this study, which tested the validity of the aforementioned model, a better
correlation coefficient was obtained for transfer to salami due to the larger number of
salami replicates resulting in an increased number of data points. Salami seems to

represent a different transfer scenario than turkey or bologna, due to being a high-fat,

160

low-moisture product. Lin et al. (2006) conducted a study in which the blade of a
commercial-scale meat Slicer used to slice roast turkey breast, salami and bologna was
inoculated to contain L. monocytogenes at levels of 1, 2, or 3 log CFU/blade (1 and 2 log
CFU/blade inoculum used with turkey only). In their study, more equipment samples
were positive for L. monocytogenes after slicing salami (8 samples) than turkey (3
samples) or bologna (1 sample), which supports a longer residence time for L.
monocytogenes on fat-coated slicers as suggested by Vorst et al. (2006; Lin et al., 2006).
Although fat caused Listeria to remain on the surface longer when slicing salami, the

model was accurate when more replicates were analyzed.

Furthermore, the model was fairly accurate in predicting transfer under all of the
tested conditions (0.77 S R2 S 0.94), despite the fact that the transfer data for all of the
modeled conditions were significantly different (Chapter 5). The only exception was for
the prediction of previously cold-injured cells (R2 = 0.65), in which the predicted values
showed a greater deviation from the observed values. In all scenarios modeled, the
transfer of L. monocytogenes to the surroundings (,3), was much greater than the amount
of L. monocytogenes transferred to the slices of meat ([1, Table 6.1). This indicates that
there is a risk of L. monocytogenes remaining in the delicatessen environment on surfaces

and equipment surrounding the slicer.

The model had previously been used to predict transfer of L. monocytogenes from
a slicer blade inoculated at levels of 3, 5 and 8 log CFU/blade (V orst, 2005b). However,
in this study, the slicer blade was inoculated at 6 log CFU/blade, and L. monocytogenes
was desiccated to the stainless steel surface for either 6 or 24 h prior to slicing turkey or

i salami. Despite these additional variables that greatly affected the numbers of L.

161

monocytogenes that remained viable and able to be transferred, the model was still

remarkably accurate.

Empirical (curve fitting) models predict populations based on previously obtained
experimental data, which offers more accurately predicted environmental populations.
However, many empirical models based on empirical data do not account for underlying
factors that influence the results and in some cases may be dependent on specific

environmental or laboratory conditions.

This work confirms the findings by Vorst et al. (2005) that the greatest number of
Listeria (> 90%) will be found in the first 15 slices of delicatessen meats after mechanical
or knife slicing. Despite the Vorst (2005b) model being an empirical model, it appears to
be accurate for certain underlying microbiological mechanisms that may affect survival
(cold-injury and desiccation over time) and may affect attachment and persistence on
surfaces (biofilm forming ability). However, in order to be truly applicable to real-
world situations, the model needs to be improved and refined to predict low-level Listeria
transfer, as would be expected with more realistic inoculum levels (< 3 log CFU/blade),
especially in a long-term continuous transfer scenario, such as one would expect when

Slicing salami.

162

CONCLUSIONS AND FUTURE RECOMMENDATIONS

Cross-contamination of RTE meats by Listeria monocytogenes poses a health risk
to the public, and a safety and financial concern to food processors and food retail
establishments. Results from this research demonstrate that L. monocytogenes can
survive under desiccated conditions on a delicatessen slicer or knives for up to 24 h and
contaminate product sliced during this time period. Prior injury of L. monocytogenes
due to exposure to refiigeration temperatures or chlorine does not significantly inhibit
this transfer, and in the case of exposure to cold, can actually enhance subsequent transfer
to delicatessen meats.

The first objective of this research, to assess the biofilm formation by L.
monocytogenes, demonstrated that considerable variation exists in the ability of different
L. monocytogenes isolates to form biofilms. Differences were also observed in the
biofilm forming ability of strains of different serotypes and isolates from different
sources. Food and environmental isolates were significantly better at forming biofilms
than clinical and veterinary isolates. Lineage I strains were significantly better at forming
biofilms than Lineage 111 strains. Significant differences were also observed based on
serotype, with serotype 1/2b forming significantly stronger biofilms than serotypes 1/2a
and 4b. Further research needs to be conducted as to the genetic traits of strong biofilm
forming strains of L. monocytogenes, to determine whether this is an evolutionary
advantage and how it may relate to virulence of strains.

The second objective of this research, to evaluate biofilm formation on

. polystyrene by selected strains of L. monocytogenes following cold injury, cold

163

starvation, acid injury and chlorine injury, demonstrated that certain types of injury (cold
injury and cold starvation) enhance biofilm formation as compared to uninjured cells.
However, oxidative injuries (acid injury and chlorine injury) inhibit subsequent biofilm
formation. Further research is required to better understand the mechanism of injury
induced by exposure to chlorine, and any subsequent effects it may have on cell
physiology. In addition, the nature of L. monocytogenes cell surface hydrophobicity and
the role that injury may play in altering this hydrophobicity needs to be determined along
with the potential of attachment and subsequent biofilm formation on surfaces. Current
methods yield widely variable and contradictory results with new methods for the
determination of cell surface characteristics needed if the role of attachment in biofilm
formation is ever to be truly understood.

The third and fourth objectives of this research were quite similar, in that they
both required the determination of sequential transfer of L. monocytogenes from stainless
steel knives or slicer blades to product sliced with contaminated blades. Overall, biofilm-
forming ability had a significant effect on L. monocytogenes transfer, with strong biofilm
forming strains transferring to products in higher numbers than weak biofilm formers.
Additionally, cold-inj ured cells of L. monocytogenes transferred in higher numbers to
delicatessen meats than uninjured or chlorine-injured strains. In both cases, enhanced
survival of strong biofilm formers and cold-injured listeriae were thought to explain the
higher levels of transfer observed. However, as in the second objective, cell surface
hydrophobicity and its effect on interactions with stainless steel may play a role in
enhancing or inhibiting bacterial transfer to products. Higher numbers of L.

monocytogenes transferred to delicatessen meats after 6 h of desiccation, however,

164

transfer still occurred after 24 h, thus raising concern that even under unfavorable
environmental conditions, L. monocytogenes can survive in environmental niches to later
contaminate products. Product composition had a significant effect on transfer, with
higher numbers of listeriae transferred to turkey than to salami. F at from the salami
seemed to retain L. monocytogenes to the slicer surface, and further research into the
protective effect of fat on localization and survival of listeriae on stainless steel surfaces
under desiccated conditions may provide insight into specific risks associated with cross-
contamination in the processing and handling of products with different fat and moisture
contents.

The final objective of this research was to validate a model developed by Vorst et
al. (2005) to predict L. monocytogenes transfer during slicing of delicatessen meats. The
model performed well, with correlation coefficients ranging from 0.65 -- 0.94. However,
the model has only been tested and shown to perform accurately when used to predict the
transfer of unreasonably high inoculum levels (103 CF U/blade and greater). Research has
shown that contaminated delicatessen meats generally contain < 1 CFU/ g of L.
monocytogenes (Gombas et al., 2003). New methods must be developed to detect and
quantify low levels of L. monocytogenes in order to determine whether the models
developed can provide accurate predictions for transfer of L. monocytogenes under these
more realistic levels. Alternatively, it may be possible to compensate for the difficulty of
quantifying low levels of L. monocytogenes by pooling samples and replicating
experiments a numerous times to obtain enough data to generate reasonably accurate

transfer curves.

165

Overall, this research Shows that food processors and retailers need to be aware
that L. monocytogenes is able to survive unfavorable conditions and persist in the
environment. Biofilm formation can be used as a predictor for survival of L.
monocytogenes in the environment with weak biofilm-producing strains being most
vulnerable to environmental stress. Many processors are aware and concerned about
issues with biofilms, but this research shows that sites associated with biofilm
development are not the only sites that could be colonized by persistent strains of L.
monocytogenes—these strong biofilm forming strains can still survive longer than their
weak biofilm forming counterparts when present in niches that will not support biofilm
development. Furthermore, the practice of storing delicatessen slicers at refrigerator
temperatures overnight to prevent the development of unpleasant odors may be indicative
of inadequate sanitation of delicatessen slicers. The odor-causing product residues may
harbor L. monocytogenes, and the exposure to lower temperatures may enhance L.
monocytogenes survival and subsequent transfer to product. Equipment designed to
facilitate cleaning and sanitizing is vital in combating persistence of L. monocytogenes in
the environment, and adequate and frequent sanitation is the only certain means to reduce
L. monocytogenes post-processing contamination. Given the risk of L. monocytogenes
transfer to a variety of products from delicatessen slicers, it may be prudent for operators
of delicatessens to only use products obtained from facilities operating under UDSA L.
monocytogenes control strategies Alternative 1 and Alternative 2, since the measures in

both of these alternatives are more stringent than Alternative 3.

166

APPENDIX I

167

KNIFE TRANSFER DATA
Table Al.l. Listeria monocytogenes transfer from knife blades to turkey (6 log

CFU/blade)
biofilm forming CFU per time

slice ability slice (h) rep
1 strong 1 12424 6 1
2 strong 152396.16 6 1
3 strong 93089.28 6 1
4 strong 28072 6 1
5 strong 12506.56 6 1
6 strong 3946.16 6 1
7 strong 3898.2 6 1
8 strong 894 6 1
9 strong 1 1236.96 6 1
10 strong 5530.8 6 1
1 1 strong 281.32 6 1
12 strong 210.4 6 1
13 strong 174.8 6 1
14 strong 153.92 6 1
15 strong 103.8 6 1
16 strong 444.72 6 1
1 strong 116724 6 2
2 strong 9430 6 2
3 strong 74358 6 2
4 strong 8301 .48 6 2
5 strong 231 1 .96 6 2
6 strong 953.16 6 2
7 strong 1005.72 6 2
8 strong 162.4 6 2
9 strong 4188.8 6 2
10 strong 351.12 6 2
1 1 strong 19.96 6 2
12 strong 154.56 6 2
13 strong 813.6 6 2
14 strong 599.2 6 2
15 strong 5110.56 6 2
16 strong 451.36 6 2
1 strong 163254 6 3
strong 29760 6 3

3 strong 4985.76 6 3
4 strong 3528.32 6 3
5 strong 376 6 3
6 strong 367.2 6 3
7 strong 3154.56 6 3
8 strong 870.24 6 3
9 strong 0 6 3
10 strong 131.6 6 3
11 strong 1438.4 6 3
12 strong 39.12 6 3

168

Table A1.1. (Cont’d)

13
14

15
16
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
1
2
3
4
5
6
7
8
9
10

d—L—l A—L—b—l-A
N-‘OCDQNO’U’I-5CDN—KCDOIDODN

strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong

224.4
225.12
131.6
5953.76
35616
59462.4
1468.8
37712.64
13319.6
3337.32
3030.2
4919.2
424.2
154
46.8
50.16
153.6
54.64
23.24
36.4
16944
11340
1431.36
2592.8
1718.72
989.4
83.04
851.44
16.88
35.44
22.68
21.12
159.36
19.68

0

41.84
1407.44
1350.72
365.76
169.2

0

0

57.24

0

0

17.76
17.28
22

169

wwwwwwwwwwwwNNNNNNNNNNNNNNNN—‘Ad-fi-‘d—fi—‘A-‘A—h—fiA-i-lwwww

Table Al.l. (Cont’d)

13
14

strong
strong
strong
strong
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak

190.4
65.04
20.48
24.52
78820
6725.92
4380.8
1747.2
564.96
250.08
104
46.72

0

0

20.6

0

0

15.84
17.2
15.12
12620
2646.68
2125.92
623.2
313.04
376.2
145.6
88.16
148.68
70.8
40.8
20.4
74.4
154.56
17.44
14.96
145632
9525.6
2461.76
6369.56
2311.92
1362.4
3056.4
1531.6
1364.56
186.48
78.8
342.68

NNNN
mmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmbAAA

170

wwwwwwwwwO-JQQNNNNNNNNNNNNNNNN—‘A—Sé—‘A—SA—AA-fi—tA-‘i—fiwwww

Table Al.l. (Cont’d)

13
14

15
16
1
2
3
4
5
6
7
8
9
10

weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak

22.92
25.84
25.6
22.88
14800
2880
2427.8
3605.12
1444
1009.8
1167
1065
712.92
1320
369.6
223.2
13.48
98.84
214.4
35.52
224.64
1313.2
21.2
22.4
22.08

003030)

24

24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24

171

wwwwmwwwwwwwNNNNNNNNNNNNNNNNA-l—l—lA—l—téAAAA—A-l-A-fiwwww

 

Table Al.l. (Cont’d)

13
14
15
16

weak
weak
weak
weak

100.32
20.72
20.16

18.6

24
24
24
24

172

00000300

Table Al.2. Listeria monocytogenes transfer from knife blades to turkey (8 log
CF U/blade)

biofilm forming CFU per time
slice ability slice (h) rep
1 strong 22070160 6 1
2 strong 2560000 6 1
3 strong 6119008 6 1
4 strong 509081 .076 6 1
5 strong 12471296 6 1
6 strong 1 1 13763.2 6 1
7 strong 13416 6 1
8 strong 35046 6 1
9 strong 78336 6 1
10 strong 1943928 6 1
11 strong 27121.6 6 1
12 strong 161.2 6 1
13 strong 1 1 520 6 1
14 strong 1729.2 6 1
15 strong 3984.8 6 1
16 strong 7387.6 6 1
1 strong 21517200 6 2
2 strong 15916080 6 2
3 strong 594093312 6 2
4 strong 404088 6 2
5 strong 2074528 6 2
6 strong 644504 6 2
7 strong 3560112 6 2
8 strong 102810 6 2
9 strong 70676.8 6 2
10 strong 495232 6 2
11 strong 85248 6 2
12 strong 56358.4 6 2
13 strong 1482384 6 2
14 strong 4727.2 6 2
15 strong 4658.8 6 2
16 strong 2077216 6 2
1 strong 3973320 6 3
2 strong 5529600 6 3
3 strong 18201456 6 3
4 strong 1524936 6 3
5 strong 7484176 6 3
6 strong 2596800 6 3
7 strong 141198 6 3
8 strong 15690 6 3
9 strong 10416 6 3
10 strong 3338.8 6 3
11 strong 777.6 6 3
12 strong 182.4 6 3
13 strong 217.2 6 3
14 strong 432 6 3

173

 

Al.2.
15

16
1
2
3
4
5
6
7
8
9

10

4.3.3.3.: AAA—3.34.3 4.3.3.3.;
AQNAOCDCDN(DUI-bOON-‘OOIwadoomNGCfl$wNémeww

(Cont’d)
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong
strong

1468.8
1302
716352
20736
12672
22924
2270.4
194
1346.4
269.2
323.6
336
212.8
179.6
234
249.6
236.4
205.6
31524800
1059100
1674112
59889.2
1210824
8377.6
54810.4
1067.2
4826
560.8
2224.8
546.4
243.6
450.4
6260
7134
10438.884
160
176.8
440
202.8
204.4
191.6
189.2
177.2
164.8
245.6
176.8
181.2

0

24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24

174

wwwwwwwwaQQQWNNNNNNNNNNNNNNNN—t—fi-b—b—‘d—|—I~—B—l—l—8-B-b-l-AOJQ

Table Al.2. (Cont’d)

15

_s_s_n_s_s_s.s 4.3.5—3.54.; -l
N—imm-bWNAOCDmNQUI-hWN-lbmolbWN-‘OCDQNC’U’ItfiwN-ia)

strong
strong
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
lNeak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak

0

0
2138704488
21 5654.796
4716

1 00805.76
3612

82820
4552.4
18792
204.4

1 170

73008

203.6

1406972
4062024576
1016966016

152191.656
71870.4
74157.6
11037.6

167.2
4084.8
383.2
1422

770.4

2851.2

231 .2
4277456928
8180704
7533472
1 14646
97006
18422.8
32126.4
21708
7000
2129.6
63369.6
15023008
1033292
3420928

24

N
mmmmmmmm030305030)mammmmmmmmmmammmmmmmmmmmmmmmmmmmma

175

wwwwwwww0000030)wWNNNNNNNNNNNNNNNNé-fié-b-t—tA—t—lA—t-A-i-l-fi-fiww

Table A12

15

16
1
2
3
4
5
6
7
8
9

10

weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak
weak

. (Cont’d)

18701.6
27434.4
38169.792
14728.8
2102.4
1865.6

415.2
219.6

9652
305.6
277.2

8481.6

627.6

20908.8

12506.4
90032.544
13270.4

1332
2532
268.8
209.6
230.8
263.6
186.8
253.6
233.2
0
186.4
264.8
192.8
0

209347.776
91566.4

2178

17971.2
2766.4
2467.2

261.6
218.8
228.8
859.2
396.8
366.4
383.2

7488

24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24

176

wwwwwwwwwwwwwwNNNNNNNNNNNNNNNN—t-A-A-K44-84—84—34—3-‘4-80003

Table Al.2. (Cont’d)
15 weak 5588.8 24 3
16 weak 402.4 24 3

177

APPENDIX II

178

SLICER TRANSFER DATA

“10” CFU in “Rep” column indicates negative count, positive by enrichment

Table A2.]. Listeria monocytogenes transfer from slicer to turkey (strong biofilm
former/uninjured/6 h incubation)

Strong, 6 h

Turkey

Slice

Total CFU

transferred

A

_s
009N020!

11
12
13
14
15

16
1 7
18
1 9
20
21
22
23
24
25
26
27
28
29
30

Rep1
10

1 924

1044
234.3

65.88
67.68
62.4
16.8
48.48
41.2
32.8
17.04
10

10
25.2

2920.
92

10.2
10.32
10.36

10
10
10
10.52
10
10
10.36
20.64
10.64
21.52
10

6695.
32

Rep 3
29.4
7992.3

2200.0

1371.6
1232.8

395.2
459.2
1064
402.56
159.6
514.8
183.04
68.88
183.04
91.84

57.4
45.92
34.44
78.96
11.28
22.88
116.8

57.4
162.4
80.92
114.4
46.08
22.96
66.96

22.08
17289.
28

Rep 4
61.2
13299.
12
15755.

4734.5
1464.1

789.36
504
386.08
177.84
213.36
304.8
203.2
71.12
111.32
82.24

491.2
135.52
92.16
31.2
41.12
227.92
124.8
61.2
62.88
62.64
72.8
30.96
20.32
50.6
10.24
39673.

Average
3.35E+01

7.74E+03
6.33E+03
2.11E+03

9.21 E+02
4.17E+02
3.42E+02
4.89E+02
2.10E+02
1.38E+02
2.84E+02
1.34E+02
5.00E+01
1.01E+02
6.64E+01

1.16E+03
6.39E+01
4.56E+01
4.02E+01
2.08E+01
8.69E+01
8.39E+01
4.30E+01
7.84E+01
5.1ZE+01
6.59E+01
3.26E+01
1.80E+01
4.64E+01
1.41 E+01

Rep %
Injury

179

Rep 1
Healthy
13.28

1027
198.36
52.92

14.64
30.08
23.4
25.2
0
8.24
8.2

0

0

0
16.8

2814.88
0

0

31.08
10.6

0

10.48

4295.92
35.836972
69

Rep 3
Healthy

0
3166.6

1100.04

702

476.56
156
324.8
560
152.32
148.2
251.68
68.64
103.32
91.52
80.36

11.48
11.48
0
11.28
11.28
0
23.36
11.48
46.4
46.24
0
23.04
57.4
22.32
0

7657.8

55.707814

32

Rep 4
Healthy

40.8
10417.12
12946.56

3850.64

1195.2
526.24
403.2
243.84
168.48
111.76
274.32
152.4
60.96
141.68
20.56

417.52
135.52
102.4
72.8
41.12
145.04
156
40.8
62.88
41.76
62.4
51.6
50.8
20.24
0

31954.64
19.455756
25

 

Table A2.2. Listeria monocytogenes transfer fi'om slicer to turkey (weak biofilm
former/uninj med! 6 h incubation)

Weak, 6 h,
Turkey

Slice

0301 -h (A) N _s

AO‘OGV

AA

1 3
14
1 5
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
Total CFU
transferred

Rep
1

11.3
2
191.
34
48.6
4
20.6
4
20.7

_A
OOOOOOOOOOOOOOOO

355.

Rep 2 Rep 3
15.2 0
4447. 1304.
52 16
796.4 696.3
8 2
371.4 380.1
8 6
182.8
8 88
52.4 21.36
96.12 20.96
42.4 0
216.8 10.48
107.6 10.52
32.28 0
21 .44 0
53.6 10
21.36 10.8
10.56 0
31.8 10
10.52 10
31.32 0
10 0
10 0
10.48 0
10.4 0
51 10.84
10 0
10 0
10 10
10 0
10 10
10 0
10 0
6703. 2603.
64 6

Average
8.84E+00
1 .98E+03
5.14E+02
2.57E+02

9.72E+01
2.46E+01

4.26E+01
1.41E+01
7.945+01
3.94E+01
1.41E+01

1.09E+01
2.12E+01
1.41E+01
6.85E+00
1.39E+01
6.84E+00
1.04E+01
3.33E+00
3.33E+00
3.49E+00
3.47E+00
2.06E+01
3.33E+00
3.33E+00
6.67E+00
3.33E+00
6.67E+00
3.33E+00
3.33E-l-00

Rep %
Injury

180

Rep 1
Healthy

0
52.32

24.32

OOOOOOOOOOOOOOOOOOO 00000 OO O

76.64
78.467071
25

Rep 2
Healthy

0
2332.44
356.32
251

50.8
20.96

32.04

53
75.88
32.28
32.28

21.44

..h
.0
a:
coo

10.5

OOOOOOOAOONOOOOO

3330.64
50.31 5947
75

Rep 3
Healthy

0
364.8
152.32
52.8
8.8

S0
co
no

OOOOOOOOOOOOOOOOOOO DOC

Table A2.3. Listeria monocytogenes transfer from slicer to turkey (strong biofilm
former/uninjured/24 h incubation)

Strong, 24 h,
Turkey

Slice

#0) N 4

0'1

18
19
20

21
22
23
24
25
26
27
28
29
30

Total CFU
transferred

Rep 1
0
1 867.
92
639.8
4

272
55.44
73.8

16.8
16.72
8.12
16.24
61.04
10
8.8

10

0
17.36
16.96
10

10

0

0000000000

3121.
04

Rep
3
0
145.
04

70
10.6
31.4

4
10.4
8

_L
9
Oh

00000

0000000000 OOOOOOO

277.

E

Rep
4
22.8

286
107.
2

8.2

u-l .
OOOOOOOOOG

Average
7.60E+00

7.66E+02

2.72E+02
9.69E+01

2.90E+01
2.81 E+01

1.24E+01
5.57E+00
2.71 E+00
5.41 E+00
2.37E+01
6.67E+00
6.27E+00

6.77E+00
2.96E+00
9.12E+00
9.05E+00
3.33E+00
6.67E+00
3.33E+00

3.43E+00
3.33E+00
0.00E+00
0.00E+00
0.00E+00
0.00E+00
0.00E+00
0.00E+00
3.33E+00
0.00E+00

Rep %
Injury

181

Healthy

0

984.64

309.6
104

55.44
16.4

25.2
0
16.24
8.12
8.72
0

8.8

0
0

9°
a.
000

ooooo'ooomoo 000

1561.8
49.958988

03

Healthy

0
0

60
10.6

10.48

_s
O
A.

oooooooooo ooooooo oooooo'

91.48

67.088789

75

Rep 4
Healthy

0

549.12

96.48
24.6

0

0000000 0

10.3

00000000 05000000

..3
.0
o}:

691

Table A2.4. Listeria monocytogenes transfer from slicer to turkey (weak biofilm
forrner/uninjured/24 h incubation)

Weak, 24 h,
Turkey

Slice

N4

(DQNO) (”#00

11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30

Total CFU
transferred

Rep Rep

1
26.7
6

0

N

. A
GNOOOOOOOOOOOOOOOOOOOOO 0000 000

8}

2

Rep
3
23.0

11.76 4

91.56

34.44
21.36

263.

10.4

10.8
10.96
10.88

9.16
18.3

0

_s
O

0
0
10
10

..s
C

0
0
10
0
0

0

_L—l
0.;

—§

m—xooooooooo

\l
O
QCDOOOOOOOOOOOOOOOOOOOOONOOOQLJOON

Average

2.05E+01
3.36E+01

2.43E+01
7.12E+00
0.00E+00

3.39E+00
0.00E+00
3.33E+00
3.33E+00

3.43E+00
0.00E+00
3.33E+00
0.00E+00
0.00E+00
3.47E+00
0.00E+00
6.93E+00
3.65E+00
3.63E+00
3.67E+00
3.33E+00
0.00E+00
0.00E+00
0.00E+00
3.33E+00
0.00E+00
0.00E+00
0.00E+00
0.00E+00
0.00E+00

Rep %
Injury

182

Rep 1 Rep 2
Healthy Healthy

0 0
0 39.24
10 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
10 39.24
82.381959 85.088919
13 29

Rep 3
Healthy

15.36
9.16

18.32

OOOOOOOOOOOOOOOOOOOOO 0000 00

42.84
39.627959
41

Table A2.5. Listeria monocytogenes transfer from slicer to turkey (strong biofilm
former/cold-injured/6 h incubation)

Strong, 6h, Cold,

Turkey

Slice

20
21
22
23
24
25
26
27
28
29

30
Total
CF U
transferre
d

Rep1
0

16450.56
1 1992.16
3483.36
691 .44
699.48
480
133.28
130.56
249.24
787.64
563.04
484.8
444.4
88.88
408

784

424
678.72
1042.76
650.72
335.4
595.84
572.32
3056
4968.72
4032
5008
281 1.84
2516.52

64563.68

Rep 2
38

32284.8
27953.28
18422.4
13432.32
7808.24
2466.2
2600.64
3015.36
572.88
630.48
361.2
216
77.76
537.6
101.76
221.52
140.8
86

42.8
78.12
60.48
68.16
25.32
77.04
17.04
10
17.04
10

10

1113832
4

Rep 3
0

11652.48
7666.56
1581.84

520.56
375.96
245
226.32
180.72
80
202.4
200
246
205.6
204.8
295.8
247.68
237.36
41.6
137.28
53.2
231.44
246.72
41.6
20.72
30.96
51.6
10.16
51.6
31.2

25317.16

Average
1 2.66667

20129.28
15870.67
7829.2
4881 .44
2961 .227
1063.733
986.7467
1 108.88
300.7067
540.1733
374.7467
315.6
242.5867
277.0933
268.52
417.7333
267.3867
268.7733
407.6133
260.68
209.1067
303.5733
213.08
1051 .253
1672.24
1364.533
1678.4
957.8133
852.5733

Rep %
Injury

183

Rep1
Healthy
0
1 1880.9
6

9717.12
2180.64
627.12
546.72
344
148.96
145.92
241.2
373.52
408
323.2
218.16
113.12
336

560

360
597.92
1416.88
564.48
288.6
486.08
431.2
1360
5145.6
1680
3072
2763.36
2138.64

48469.4
24.9277
6124

Rep 2
Healthy

26.6

26309.28
25767.36
15061.12
13780.8
6888.64
2758.8
2151.36
2047.68
616.28
562.32
395.6
190.08
86.4
385.28
144.16
178.92
132
103.2
34.24
43.4
43.2
25.56
16.88
77.04
34.08
25.56
8.52

8.68
16.96

97920
12.08731
224

Rep 3
Healthy

0

10357.76
4820.64
1469.52

616.96
424.16
254.8
147.6
110.44
90
101.2
180
108.24
133.64
122.88
183.6
144.48
123.84
62.4
73.92
21.28
178.84
205.6
10.4

0

0
41.28
0

0

10.4

19993.88
21.02637
105

 

Table A2.6. Listeria monocytogenes transfer from slicer to turkey (weak biofilm
former/cold-injured/6 h incubation)

Weak, 6h, Cold, Turkey

Slice

Total CFU
transferred

.J

N

001-5 00

10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30

Rep1
3488
1046
64
3948.
16
1086.
8
200
257
2108
4
1566
1357
2

10
954
4272
4304
318
10
8224
2136
2128
10

0
1056
424
4272
2128
10
1056
10
104
10
2064
1705
28

8.2
10
8.28
16.64
8.44
8.36
10

0

10

10
8.04
8.24
10

10

10

10
6950.
52

Rep 3
5.6

8923.2
8256.4
8
8951.0
4
2412
2214
1630.7
2

519.68

762.72
325.44
526.64
866.4
726.4
479.12
229
334.08
338.92
82.44
83.16
157.08
503.8
162.72
36
53.28
8.96
10
26.52
54.48
10
44.8
38734.
68

Average
16.01333

821 5.92
4357.653

3377.627
913.3333
850.6

638.0933
244.3733

315.72
122.8533
223.6667
308.5067

283.68

173

82.4
142.1067
122.8533
40.12
33.86667
55.14667
174.7867
68.37333
29.57333
28.18667
9

9.6
15.50667
24.96

10
25.14667

Rep %
ImUW'

184

Rep1
Healthy
17.44

2724.4
783.68

79.04
20
61.68

20.08
20.88

41.76
10.56
10.6
32.04
10.76
0
10.8
10.28
10.68
21.28

0000000000

10.28
10.32

3906.56
77.091386
75

Rep 2
Healthy
0

2042.16
393.96

47.52
40
40.4

48.48
48.72

24.36
16.56
0
16.4
40.8
0

0
8.24

00000000000000

2767.6
60.181396
5

Rep 3
Healthy
5.6

6138.08
6386.08

3594.24
1602
1 323

716.8
224

390.44
207.92
463.08
665.76
553.88
171.76
164.88
287.68
109.92
137.4
83.16
101.64
311.44
117.52
18
44.4
26.88
0

0
18.16
9
53.76

23926.48
38.229824
02

 

Table A2.7. Listeria monocytogenes transfer from slicer to turkey (strong biofilm
former/cold-injured/24 h incubation)

Strong, 24h. Cold, Turkey

Slice

10
11
12
13

14
15
16
17

18
19

20
21
22
23
24
25
26
27
28
29

30
Total CFU
transferred

Rep1
10

12932.
24

4351 .2
2377.4
4

157.76
419.4
158.44

75.2
56.64
9.52
76.48
28.56
9.6

9.48
10

10
19.12

10
10

10

10
9.76
19.68
9.76
9.8
184.68
286.8
35.68
9.48

10
21326.
72

Rep 2

19972.
10016.
6420.4
1970.3

2152.2
1185.9

497.04
371.28
142.08
268.8
133.8
151.64

63
45.4
172.52
63

10
36.8

36.48
119.08
9.2
45.4
9.04
10

10
9.08

18.08
10
43967.
16

Average
9.346667

1 1558.4
5475.853
3340
855.72
894.16
514.7067

241.9733
164.0133

128.2
146.8267
104.2533
85.34667

138.88
143.7867
98.6
81.09333

41.57333
78.26667

54.21333
52.50667
12.69333

47.08
16.26667

26.12
74.85333
124.5467
27.85333
34.89333
16.46667

Rep %
Injury

185

Rep1
Heanhy

0
9642.64
2817.92
1067.04

139.2
382.12
83.88

9.4

0
9.52
19.12
0

0

9.48

000

00

9.64
0
0
0

0

9.8
97.2
105.16
35.68
9.48
19.04

14466.32

32.168097

11

Rep 2
Healthy

0
16293.6
8560.32
4798.64
2308.88
1679.56

854.56

427.28
176.8
177.6

116.48

107.04

107.04

54
45.4
208.84
18

46.6
27.6

64.12
36.8
18.16

18.16
9.08
18

0

0

36172.56

17.728231

71

Rep 3
Healthy

0
985.36

1166.88

848.16

268.8
120.12
118.04

90.4
54.96
93.2
28.56
103.4
37.92

133.84
144.6
66.08
37.92

95.2
56.4

29.04
18.96
38.24
9.52
0

0
9.96
29.16
0
57.84
0

4642.56
45.198545

73

 

Table A2.8. Listeria monocytogenes transfer from slicer to turkey (weak biofilm

former/cold-injured/24 h incubation)

Weak, 24h, Cold, Turkey

Slice

.5

_s
000V00’l

11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29

30
Total CF U
transferred

Rep1
10
425.0
4
228.4
8
178.5
6

18.4
27.24
18.56

9.44

10
10
9.4
10
9.52
9.52
10

0
9.84
10

0
9.84
10
10
10
10

0

10
10
10
19.6

0
1103.
44

Rep
2

0
59.0
4
60.4
8

9.32

10
9.32
10
10
9.52
10
10
10
10
10

0

0

10

0

10

0

0

10

0

0
0
0
10
10
0

0
277.
68

Rep 3
18.16
6316.

8
2157.
84
533.3
2
138.2
4

61.44
15.36
38.4
23.28
8.04
10

10

10

10
16.08
10

10

10

10

0

10

10
8.16
8.2
10

10
8.12
10

10

0
9491.
44

Average

9.386667
2266.96
815.6
240.4

55.54667
32.66667
14.64
19.28
14.26667
9.346667
9.8

10

9.84

9.84
8.693333
3.333333
9.946667
6.666667
6.666667
3.28
6.666667
10
6.053333
6.066667
3.333333
6.666667
9.373333
10
9.866667
0

Rep %
Injury

186

Rep1
Healthy

0
151.8
76.16
69.44

0
9.08
9.28

00000000000000000000000

315.76
71.384035

38

Rep 2
Healthy

0
19.68

00000000000000000000000000 0 0

19.68

92.912705

27

Rep 3
Healthy

9.08

3033.6
1010.88

238.8

38.4
23.04
15.36
30.72

7.76

8.04

N.“
820000

on
oiooooooooo

16.32
8.12
0

0

0

4472.32
52.880490

21

Table A2.9. Listeria monocytogenes transfer from slicer to turkey (strong biofilm

former/chlorine-injured/6 h incubation)

Strong, 6h, Chlorine,

Turkey

Slice

Total CFU

transferred

0301500194

00 0N

12
13
14
15

16
17
18
19
20
21
22
23
24
25
26
27
28
29
30

Rep1
1058
12636.
56
13672.
96

10480
12378.
56
5901.3
6
5635.2
8
6215.6
5030.2
4
4664
3247.8
4
3358.0
8
2654.2
4
1721.4
4
1305.9
2
1104.2
4
291.04
346.04
224.64
144.84
25.32
73.8
101.76
42.4
50.16
50.4
42
25.2
33.28
16.64
91579.
64

Rep 2
8.36
5019.6
8
3017.1
2
1738.2
4

264
450.84

253.12
165.6

18.64
55.92

83.16
46.6
27.96
9.48
47.6

47.2
9.44
38.24
28.32
18.96
10
47.6
58.08
38.08
19.12
9.6
19.36
19.6
10

10
11589.
92

Average
43.84

9483.547
8036.88
7246.507
5028.853
2480.92

2132.64
2234.76

1745.68
1596.947

1 151.293

1 176.92
933.2667
598.0933
475.2933

413.44
108.2
133.4

107.96
62.56

30.25333
59.41333
72.32
43.06667
44.74667
215.64
135.8133
42

19.92
16.92

Rep %
Injury

187

Rep1
Heaflhy

0

382
580. 16
264
428.24
96.48

194.88
131.2

135.04
101.76

84.8
59.36
59.36

8.48

2559.12
97.205579

76

Rep 2
Healthy

0
586.24
340.08
170.24

35.2
26.52

36.16
27.6

0
18.64

9.24
0
0
0
9.52

0000000001000000

1268.76
89.052901

14

Rep 3
Healthy

2.48

3798.64
1281.84
1176.24

142.88
53.76

15.44
0

15.68
0

15.36
15.76

.‘1
<9

G
.3
00001000000 0 0 0

112.56
49.44
0

0

0

6696.16
80.559126

6

Table A2.10. Listeria monocytogenes transfer from slicer to turkey (weak biofilm

former/chlorine-injured/6 h incubation)

Weak, 6h, Chlorine,

Turkey

Slice

Total CFU

transferred

01wa

00%0

10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30

Rep1
7.24
10677.
12

3044.8
616.2
290.88

152
130.56
65.28
40.8
40.8
16.16
16.16
49.92
24.84
10

42
25.32
10
33.92
8.4

0
8.44

_L
0000000

10
15330.
84

Rep 2
0
759.9
2
170.5
6

61.2
13.92

6.92
0
14.08
0

cox:
N .‘1 .‘l' .‘1 .
bOOOO-hOOOOtObOOONOO

\l
#0

1108.
44

Rep 3
6.48
5751 .

1 524.
24
605.4

167.0
113.2

84
49

28.96
42.24
27.84
35.2
7.28
7.12

7.16
14.32
0
7.32
0

0

0
14.48
14.88
14.8
0

0
14.48

7.28
8628.
52

Average
4.573333

5729.573
1579.867
427.6133

157.28

90.73333
71 .52
42.78667
41.6
23.25333
19.46667
17.06667
28.37333
10.70667
5.706667
16.46667
10.82667
10.58667
23.84
5.24

0
2.813333
5.8
4.826667
4.96
4.933333
0
2.466667
4.826667
8.226667

Rep %
Injury

188

Rep1
Heanhy
0
402

8.8

0

0000000000000000000000000 0

49
99.680382
81

Rep 2
Healthy

0
392.84
52.48
0

0

6.92

o)
8

466.4
57.922846
52

Rep 3
Healthy

0
89.44
6.96

0

00000000000000000000000V0 0

103.4
98.801648
49

Table A2.11. Listeria monocytogenes transfer from slicer to turkey (strong biofilm
former/chlorine-injured/24 h incubation)

Strong, 24h, Chlorine,

Turkey

Slice

A

00

A

003N001

10
1 1
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
Total CFU
transferred

Rep1
848
1902.
8
1033.
68
3354
6
1084
6
5536
0
1384
0
696

00000000000VV000§000

3485.
28

Rep 2 Rep 3
10 10
979.0 2400.
4 24
350.8 686.7
8 2
250.3

118.8 2
67.2 65.16
57.68 7.4
8.04 7.48
8.2 22.56
8.44 22.56
10 10

10 7.52

10 10

10 7.56
8.6 10
10 15.28

10 10

10 10
8.64 10
10 10

10 10

10 10

10 10

10 15.68

10 7.84

10 10

10 10

10 10

10 10

10 10

10 10
1815. 3686.
52 32

Average
9.493333

1760.693
690.4267
234.76

80.17333
40.14667
5.173333
14.86667
10.33333
8.986667
5.84
6.666667
5.853333
8.48
8.426667
6.666667
6.666667
8.546667
9
6.666667
6.666667
6.666667
8.56
5.946667
6.666667
6.666667
6.666667
6.666667
6.666667
6.666667

Rep "/0
Injury

189

Rep 1
Healthy

F”
3000

00000000000000000000000000

6.84

Rep 2
Healthy

0

33.76

00000000000000000000000000 0 0

33.76

99.803746 98.140477

04

66

Rep 3
Healthy

0

759.2

153.92

65.56

14.48
7.4
14.96
7.52
0

0

.‘l
01
N

0000000000000000000

1030.56
72.043664

14

 

Table A2.12. Listeria monocytogenes transfer from slicer to turkey (weak biofilm
former/chlorine-injured/24 h incubation)

Weak, 24h, Chlorine,

Turkey

Slice

Total CF U

transferred

N

_L
ocoooxtoacnhw

11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30

Rep1
0

227.92

52.16
81.64
6.48
6.52
46.48
26.88
26.88
244.8
132.24
48.44
13.92
7

10

10

21

10

_s
0

00000000000

982.36

Rep
2
0
26.0
8

6.92
10
10
10
10
10

0
10
10

0

0
10
10

0
10

0

0

0

0
10

0
10
10

0

0

0

0
10

173

Rep 3 Average

0
1659.
68
353.2
8

95.68
59.2
37.4

15.12
15.2
7.72
7.68
7.68

0
637.8933

137.4533
62.44
25.22667
17.97333
23.86667
17.36

1 1.53333
87.49333
49.97333
16.14667
4.64
5.666667
6.666667
3.333333
10.33333
3.333333
3.333333
0

0
3.333333
0
3.333333
3.333333
0

0

0
2.706667
3.333333

Rep %
Injury

190

Rep 1
Healthy
0
0
0
0
0
0
0
0
0
0
0
6.92
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
6.92
99.295573

92

Rep 2
Healthy

0 0000000000000000000000000000 0 0

100

Rep 3
Healthy

0000000000000000000000000000 0 0

7.48
99.670013
59

 

Table A2.13. Listeria monocytogenes transfer from slicer to salami (strong biofilm
former/uninj ured/6 h incubation)

Strong,6h
Salami
Slice Rep1 Rep2 Rep3 Average Rep1 Rep2 Rep 3
Healthy Healthy Healthy
1 44.28 167.28 78 9.65E+01 11.48 118.08 54.6
2 1555.2119064119232 1.31E+03 1728 1064.8 665.28
3 510.72 716.8 240 4.89E+02 468.16 918.4 201.6
4 220.92 252.56 115.2 1.96E+02 136.76 298.48 38.4
5193568190696 132.72 1.33E+03 231.44 1877.92 94.8
6 257.28 364.8 142.8 2.55E+02 160.8 288 95.2
7 266 190.4 218.04 2.25E+02 42.56 180.88 180.12
8 313.2 197.4 379.2 2.97E+02 108 75.2 208.56
9 97.92 199.92 171.36 1.56E+02 54.4 85.68 95.2
10 419.64 157.08 247.52 2.75E+02 86.08 64.68 285.6
11 21.36 120.12 66.08 6.92E+01 42.72 73.92 28.32
12 10.76 36.96 186.4 7.80E+01 10.76 46.2 111.84
13 84.48 62.44 149.76 9.89E+01 63.36 53.52 84.24
14 73.36 45.6 18.96 4.60E-I-01 52.4 45.6 18.96
15 116.6 64.12 19.12 6.66E+01 95.4 36.64 28.68
16 302.4 44.4 84.96 1.44E+02 356.4 35.52 66.08
17 53.8 45.2 106.04 6.83E+01 21.52 18.08 28.92
18 84.8 17.76 9.56 3.74E+01 53 44.4 0
19 30.96 61.32 9.52 3.39E+01 41.28 61.32 28.56
20 10.6 17.68 37.76 2.20E+01 21.2 0 0
21 51.6 16.96 18.24 2.8QE+01 30.96 8.48 9.12
22 10.48 26.52 45.2 2.74E+01 10.48 17.68 36.16
23 40.16 48 9.56 3.26E+01 20.08 38.4 28.68
24 30.48 28.32 10 2.29E+01 40.64 18.88 0
25 20.48 9.24 9.52 1.31E+01 51.2 9.24 19.04
26 41.6 17.76 77.12 4.55E+01 31.2 8.88 57.84
27 63.12 9.28 66.08 4.62E+01 0 9.28 18.88
28 20.8 10 27.72 1.95E+01 20.8 9.52 0
29 10 18.96 10 1.30E+01 31.44 28.44 18.56
30 51 66.64 38.4 520E+01 0 47.6 38.4
Total CFU 6749.68 6111.12 3917.16 4022.52 5583.72 2541.64
transferred
Rep% 4040428583 8.630169265 3511523655
Injury

191

 

Table A2.14. Listeria monocytogenes transfer from slicer to salami (weak biofilm
former/uninj ured/6 h incubation)

Weak, 6h Salami

Slice
1

001.50 M

0

10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29

30
Total CFU
transferred

Rep 1 Rep 2 Rep 3 Average

18.08 33 36.6
192.5

52.8 116.28 6
201.7

10.88 255.36 6
0 163.68 123.6
9.32 154.4 123
36.8 117 123
141.4

0 125.44 4
158.0

54.48 175.12 8
1874.5 198.7
6 358.2 2
1474.7 217.3
6 485.56 6

10 8 10

9 0 10
8.44 8.08 10
10 8.12 10
4.56 10 10
10 16.16 10

0 10 8.48

10 10 16.8
10 16.4 8.44
8.72 10 10
10 16.4 8.4
10 8.08 10

0 8.04 10

10 8.12 10
10 40.6 10
10 10 8.16

0 16.08 0

0 0 0

10 8.12 24.84
10 16.08 16.56
2212.3 1717.
3682.4 2 8

2.92E+01
1.21 E+02

1.56E+02
9.58E+01
9.56E+01
9.23E+01

8.90E+01
1.29E+02
8.10E+02

7.26E+02
9.33E+00
6.33E+00
8.84E+00
9.37E+00
8.19E+00
1.21E+01
6.16E+00
1.23E+01
1.16E+01
9.57E+00
1.16E+01
9.36E+00
6.01 E+00
9.37E+00
2.02E+01
9.39E+00
5.36E+00
0.00E+00
1.43E+01
1.42E+01

Rep %
Injury

192

0

21.12

10.88
27.72

27.6

9.08

18.56

9.24

000

4.56

00000000000000

8.48

137.24

0

24.48

114.24

148.8
38.6
15.6

39.2

63.68

2000000000000

9°

3

14.64

99.6

124.16

65.92
41
49.2

24.96
49.92

57.96

0:
9°
0:
N

9°
ooootoooooooo

N

8.3

000000

602.64

96.27308277 69.8054531 64.91791827

Table A2.15. Listeria monocytogenes transfer from slicer to salami (strong biofilm
former/uninj med/24 h incubation)

1.85E+02
7.92E+02
2.07E+02
1.58E+02
1.25E+02
2.87E+02
7.86E+01
5.83E+01
5.52E+01
1.63E+02
9.35E+00
9.57E+00
1.19E+01
1.30E+01
1.94E+01
6.67E+00
9.88E+00
1.26E+01
8.97E+00
6.67E+00
1.00E+01
6.67E+00
6.67E+00
1.88E+01
9.68E+00
1.00E+01
9.73E+00
6.48E+00
6.67E+00
6.67E+00

Strong, 24h
Salami
Slice Rep1 Rep 2 Rep 3 Average

1 460 34.76 60.48
2 1326179676 445.76
3 118.8 430.08 73.08
4 75.6 313.72 84.8
5 71.96 199.2 105.16
6 119.52 58.08 684.44
7 206.64 19.04 10
8 88.56 66.64 19.6
9 98.4 67.2 0
10 354.24 124.28 10
11 9.68 8.36 10
12 10 8.72 10
13 19.44 6.16 10
14 10 10 19.12
15 10 10 38.24
16 0 10 10
17 10 9.64 10
18 28.8 0 9.04
19 18.4 0 8.52
20 10 0 10
21 10 10 10
22 10 0 10
23 10 0 10
24 10 36.48 10
25 0 19.04 10
26 10 10 10
27 0 10 19.2
28 9.44 0 10
29 10 10 0
30 10 0 10

Total CFU 1932.08 3268.16 1727.44

transferred

Rep %
Injury

193

Healthy

441.6
70.2
129.6
54
71.96
49.8
78.72
59.04
68.88

354.24

0

0
38.88
9.72
0
19.44

0

ooomoooooooboo

@
.3

1463.44

18.96

1689.12
174.08
445.28
139.44

19.36
19.04
19.04
57.6
38.24
0
8.72
3.08
0

0

P
A
on

18.2

000000-50000000

2659.68

20.16

382.08

52.2
0
9.56
38.56
0

9.8

0

0

0

9.6

0

0
28.68
18.64

45.2
8.52

ooobooobooo

642.2

24.2557244 18.61842749 62.82360024

Table A2.16. Listeria monocytogenes transfer from slicer to salami (weak biofilm
former/uninjured/24 h incubation)

Weak, 24h

Salami

Slice

(”Nam-500194

NNNNNNNNNAAAAAAAAAA
mflmm#wN-¥OCOGJ\IC)UI#OONJOCD

29
30

Total CFU
transferred 16864.16

Rep 1

2653.28
4132.8
1397.28
1944.8
1261.44
1611.84
1060.8
901.68
792
1088.24

00000000000000000000

Rep 2

48.6
74.8
49.2
120.32
84
73.2
118.4
127.84
127.16
218.08
0

0

0

10

7.4
10

1130.28

Rep 3

167.16
156.56
148.32
247.2
158.84
106.6
254.2
549.4
369.84
254.72
10
8.16

I
A A
0

000000000001000000

A

2459.12

Average

9.56E+02
1 .45E+03
5.32E+02
7.71 E+02
5.01 E+02
5.97E+02
4.78E+02
5.26E+02
4.30E+02
5.20E+02
3.33E+00
2.72E+00
0.00E+00
3.33E+00
0.00E+00
5.83E+00
0.00E+00
0.00E+00
2.71 E+00
0.00E+00
2.43E+00
2.43E+00
2.43E+00
2.44E+00
6.67E+00
3.33E+00
4.88E+00
0.00E+00
2.47E+00
6.67E+00

Average °/0

Rep1
Heaflhy

1880.48
2394.4
1175.76
1343.68
1103.76
1296.48
884
601.12
739.2
892

00000000000000000000

12310.88

84

Rep 2
Healthy

64.8
13.6
29.52
112.8
14
43.92
74
52.64
67.32
255.68

00000000000000000000

728.28

32

Rep 3
Healthy

31.84
16.48

8.24
32.96
16.72

0'!

919°

Nah
0000000000000000000010 A00

211

26.999743 35.566408 91.419694

85

Table A2.17. Listeria monocytogenes transfer from slicer to salami (strong biofilm

former/cold-inj ured/6 h incubation)

Strong, 6h, Cold,

Salami

Total CFU
transferred

1 8.96

2 23.4

3 72

4 28.64

5 300.12

6 44.16

7 66.24

8 36

9 21.72
10 194.4
11 36.8
12 10
13 42.96
14 50.68
15 709.52
16 14.72
17 7.32
18 313.04
19 65.88
20 80.52
21 596.16
22 294.4
23 10
24 7.36
25 50.96
26 58.88
27 37
28 10
29 7.36
30 296

60.2
310.08
198.88

200
31.52
162.12
131.92
185.28
133.96
163.8
118.2
176
198
118.8
63.04
78.8
93.6
31.36
62.72
70.92
39.4
47.76
10
15.76
23.76
23.52
23.88
23.76
23.76
23.88

10
463.68
468.48

395.2
142.12
324.24
399.36
183.36

220.4
148.96
338.84
138.24
185.28
165.48

85.8
124.8
192
160.44

61.76

30.88
216.16
112.56
234.32
176.88

93.6
193
144.72
79.6
77.6
39

3495.2 2844.68 5606.76

Slice Rep1 Rep2 Rep3 Average

26.38667
265.72
246.4533
207.9467
157.92
176.84
199.1733
134.88
125.36
169.0533
164.6133
108.08
142.08
111.6533
286.12
72.77333
97.64
168.28
63.45333
60.77333
283.9067
151.5733
84.77333
66.66667
56.10667
91.8
68.53333
37.78667
36.24
119.6267

Average %
Injury

195

Rep1

Healthy

0

23.4
64.8
35.8
29.28
0
22.08
14.4
14.48
50.4
29.44
7.32
28.64
50.68
101.36
14.72
7.32
101.92
87.84
65.88
272.32
176.64
14.8

0

0
29.44
29.6
7.36

0
199.8

1479.72

Rep 2

Healthy

1

8.6
218.88
90.4
128
39.4
108.08
77.6
30.88
39.4
62.4
70.92
96
55.44
134.64
70.92
23.64
15.6

0
23.52
23.64
15.76
23.88
16

0

7.92

0

7.96
23.76
15.84
0
429.08

Rep 3

Healthy

0
463.68
387.96

608
224.4
270.2

384

168.08
205.2
188.16
236.4
115.2
200.72
189.12
124.8
156
107.52
61.12
30.88
77.2
123.52
136.68
145.44
176.88
78
162.12
136.68
111.44
69.84
39

5378.24

57.66422522 49.7630665 4.07579422

Table A2.18. Listeria monocytogenes transfer from slicer to salami (weak biofilm
former/cold-injured/6 h incubation)

Weak, 6h, Cold,
Salami
Slice Rep 1
1 10
2 1965.4
3 589
4 443.52
5 892.32
6 416.16
7 682
8 371.2
9 297.04
10 363.56
1 1 378.2
12 407.04
13 21 1.2
14 119.32
15 243.2
16 109.48
17 195.92
18 129.36
19 190.96
20 107.44
21 189.6
22 82.68
23 56.52
24 171.36
25 62.4
26 171.36
27 54.72
28 25. 12
29 49.28
30 24.64
Total CFU
transferred 9010

Rep 2

10
630.08

478.72

307.44
180
163.68
87.36
163.76
93.08
115.2

287.04
364
360.64
118.4
220.8
529.92
360.64
321.64
142.88
164.56
120.32
148

75.2
57.6
82.72
37.6
59.52
37.8
145.92
69.12

5933.64

Rep 3 Average

15.84
163.2
141.3
6
101.9
2
30.24
37.8
45.12
150.4
14.96

22.08
115.8
4

14.88
83.16
14.88
21.84
7.36
14.56
29.76
14.88
36.4
14.8
29.44
139.0
8

7.4
7.36
15.28
67.32
14.72
10

10
1391.
88

11.94667
919.56

403.0267

284.2933
367.52
205.88

271 .4933

228.4533

135.0267

166.9467

260.36
261.9733
218.3333

84.2
161.9467
215.5867
190.3733
160.2533

1 16.24

102.8

108.24

86.70667

90.26667
78.78667
50.82667
74.74667
60.52
25.88
68.4
34.58667

Rep % Inj

196

Rep1
Healthy
0
1 128.4

279

246.4
511.68
220.32

384.4

145
158
154.96

148.8
184.44
57.6
25.12
108.8
45.08
44.24
49.28
49.28
56.88
25.28
25.44

56.52
42.84
6.24
116.28
12.16
37.68
30.8
18.48

4369.4
51 .5049
9445

Rep 2
Healthy

0
372.32

359.04

226.92
216
59.52
36.4
156.64
28.64
57.6

191.36
182
228.16
88.8
125.12
272.32
147.2
209.44
112.8
74.8
120.32
74

67.68
50.4
37.6

30.08

29.76

60.48

30.72

53.76

3699.88
37.645694

72

Rep 3
Healthy

2.64
95.2

156.24

87.36
30.24
15.12
0
105.28
7.48
44.16

28.96
14.88
60.48
22.32
14.56
7.36
0
14.88
14.88
14.56
7.4
7.36

65.88
7.4
14.72
0
7.48
0

0

0

846.84

39. 1 58548

15

Table A2.19. Listeria monocytogenes transfer from slicer to salami (strong biofilm

former/cold—injured/24 h incubation)

Strong, 24h, Cold,

Salami

Slice

22
23
24
25
26
27
28
29
30
Total CFU
transferred

Rep
1

32.76
169.3
6
0
10
7.2
28.8
50.68
36.8
29.12
7.4
74.4
177.6
2977.
04
89.28
10
7.48
10
338.4
82.72

110.4
141.3
6
6118.
64

7.4
7.44
10
20.28
6.24
327.6
51.24
86.4
1102
6.04

Rep 2
203.36

14
35.6
7.12
6.88

49.28
13.76

10

10
7.24

21.48

10

13.76
34.8
6.88

42.48

28.16
7.16

21.48

20.52

14

7.12
20.76
54.72

35.4
27.84
53.76
26.72
21.24

10

835.52

Rep
3
372

32.6
39.36
20.88
25.76

13.6

10

34.2
46.76
20.64

10
28.16

48.44
61.2
27.52
10
20.76
13.68
10
27.84

20.64

20.76
10
6.8
10

0

6.8
6.84
10

10

975.2
4

Average

202.7067

71 .98667
24.98667
12.66667
13.28
30.56
24.81333
27
28.62667
1 1 .76
35.29333
71 .92

1013.08
61.76
14.8
19.98667
19.64
119.7467
38.06667
52.92

58.66667

2048.84
12.72
22.98667
18.46667
16.04
22.26667
120.3867
27.49333
35.46667

Rep % Inj

197

Rep1

Healthy

214.2

40.88
0

7.2
7.2

0
7.24
0
29.12
0
37.2
118.4

635.8
89.28
0
14.96
15.04
112.8
0
7.36

66.96

1540.88

0

0
14.72
27.04
0
226.8
7.32
21.6

3242

70.596877

94

Rep 2

Healthy

0

7
21.36
14.24
13.76
21.12
13.76

0

14.4

7.0

00500000

154.8

81.472615

86

Rep 3

Healthy

570.4

32.6
19.68
13.92
25.76

0
13.52
20.52

33.4

6.88

7.08
21 .12

55.36
81.6
6.88

27.84

13.84

0
0
0

13.76
6.92

00§00000

977.92

Table A2.20. Listeria monocytogenes transfer from slicer to salami (weak biofilm

former/cold-injured/24 h incubation)

Weak, 24h, Cold,

Salami

Slice

COGNCDCfi-b-OJN-A

10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30

Total CFU
transferred

Rep1

0

10
113.92
83.52
30.08
106.4
15.2
15.28
44.4
28
14.8
29.6
14.88
7.36
10
7.52
80.96
7.2
22.32
7.24
14.08
39.36
41.76
10
22.08
44.4
21.84
7.28
7.4
5.76

862.64

Rep 2

0
14.4
52.64
14.96
7.6
15.52
10
15.2
10
7.64
15.68
15.68
108.08
75.6
30.24
14.64
38.2
37.6
15.2
10

10
15.36
10

10

10

10

10

10
7.88
10

612.12

Rep 3

0

8.52
8.76

10

0

10

0

10

AA

.3
0000000000000000000000

87.28

Average

0
10.97333
58.44
36.16
12.56
43.97333
8.4
13.49333
18.13333
11.88
10.16
18.42667
40.98667
27.65333
13.41333
10.72
43.05333
14.93333
15.84
5.746667
8.026667
18.24
17.25333
6.666667
10.69333
18.13333
10.61333
5.76
5.093333
5.253333

Rep % Inj

198

Rep 1 Rep 2
Healthy Healthy
0 0
0 0
64.08 22.56
13.92 7.48
22.56 0
83.6 7.76
22.8 0
15.28 7.6
7.4 7.6
14 0
7.4 0
7.4 0
14.88 46.32
22.08 45.36
7.32 7.56
15.04 0
14.72 0
14.4 7.52
0 0
0 0
7.04 0
19.68 0
20.88 0
14.24 0
14.72 15.52
0 0
0 0
14.56 0
7.4 7.88
0 0
445.4 183.16
48.3678
0117 3

Rep 3
Healthy

8.5

0000000000000000000000000000N0

8.52

70.0777625 90.2383134

7

Table A2.21. Listeria monocytogenes transfer from slicer to salami (strong biofilm

former/chlorine-inj ured/6 h incubation)
Strong, 6h, Chlorine,

Salami

Slice

Total CFU

transferred

(a) N—h

(D \IOJU'IA

11
12
13

14
15
16
17
18
19
20

21
22
23
24
25
26
27

28

29
30

Rep 1

0
316.8
216.3

2
124.3

2

8
15.84
24.12

48.24

40.2
8
0
7.96
0

16

0

10

0

0
8.08
8

24

16
8.08
64.64
32.16
8.04
7.96

7.88

0

0
1020.
64

Rep
Rep 2 3

0 9.76
405.6 19.2

248.6
4 7.36
45.08 0
102.4 0
32.6 0
78.24 7.4
22.3
66.4 2
14.6
46.2 4
6.6 0
39.6 0
73.04 7.36
33.2 7.24
14.5
80.16 6
45.36 0
39.84 10
19.92 2.64
26.4 5.8
71.72 7.48
26.08 0
22.3
47.04 2
60.84 7.44
54.4 0
13.52 0
54.4 73.6
6.88 0
60.84 10
36.2
33.8 4
19.6
53.12 8
13.92 10
1885. 315.
84 04

Average

4.88
212.4

157.44

56.46667

36.8
16.14667
36.58667

45.65333

33.68
4.866667
13.2
29.45333
13.48

36.90667
15.12
19.94667
7.52
10.73333
29.09333
11.36

31 .12
28.09333
20.82667
26.05333
53.38667
4.973333
26.26667

25.97333

24.26667
7.973333

Rep % Inj

199

Rep1
Healthy
0
40.32

13.52

8.88
0
7.92
16.08

0

00 0 0000000 0000000 00000

86.72
91 .5033
7043

Rep 2

Healthy

0 00 0 0000000 0000000 00000 0 0000 0 00

100

Rep 3

Healthy

0000000 0000000 00000 0 0000 0 00

0000

100

Table A2.22. Listeria monocytogenes transfer from slicer to salami (weak biofilm

former/chlorine-injured/6 h incubation)

Weak, 6h, Chlorine,
Salami

Slice

#wN

—L
000N001

11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27

28

29

30
Total CFU
transferred

Rep Rep
1 Rep2 3
0 896 0
21.1
2 10 171.6
21.6 54 17.28
10 5.12 5.8
14.0
8 0 10
7.32 0 10
10 11.52 5.68
10 0 10
10 10 0
10 10 10
7.6 5.68 10
10 0 10
10 0 5.72
10 0 0
10 0 10
10 0 0
0 284 10
10 0 10
10 28.6 10
10 0 10
10 10 0
0 0 10
10 10 0
10 10 0
10 0 0
10 10 0
10 0 10
14.4
8 0 0
10 0 0
0 0 0
286. 1354. 336.0
2 92 8

Average

298.6667

67.57333
30.96
6.973333

8.026667
5.773333
9.066667
6.666667
6.666667
10

7.76
6.666667
5.24
3.333333
6.666667
3.333333
98
6.666667
16.2
6.666667
6.666667
3.333333
6.666667
6.666667
3.333333
6.666667
6.666667

4.826667
3.333333
0

Rep °/0 Inj

200

Rep1
Healthy

0 000 00000000000000000000000 000 0

100

Rep 2
Healthy
0

5.72

000 00000000000000000000000 00

5.72
99.577834
85

Rep 3
Healthy

N
9°
mo

00000000000000000000000 00

000

28.6
91.490121
4

Table A2.23. Listeria monocytogenes transfer from slicer to salami (strong biofilm

former/chlorine-injured/24 h incubation)

Strong, 24 h,

Chlorine, Salami

Slice

(OQNOUIbQN—l

10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29

30
Total CFU
transferred

Rep 1

0

20.88

0

0

6.92

0

0

9’
co
N0

9’
aco

O)
(D
00000-50000000010000000

6.8

65.36

Rep
Rep 2 3

0 0
662.72 0
1286.56 0
0 0
0 0
598.4 7.44
136.04 22.44
0 21.96
7.08 7.4
0 0
7.16 0
0 10
7.2 0
7.24 0
0 0
0 0
0 0
7.2 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
7.16 10
0 0
7.04 0
7.04 0
0 0

2740.84 79.24

Average

0
227.8667
428.8533

0
2.306667
201.9467
52.82667

7.32
7.133333

0
2.386667
3.333333

2.4
2.413333

0
2.306667
3.333333

2.4

0
2.293333

0

0

0

0

2.28
5.72

0
2.346667
2.346667

0

% Injury
Rep

201

Rep 1
Healthy

0 000000000000000000000000000000

100

Rep 2
Healthy
0
82.84
1 72

000000000000000000000000000

254.84
90.702120
52

Rep 3
Healthy

0 000000000000000000000000000000

100

Table A2.24. Listeria monocytogenes transfer from slicer to salami (weak biofilm
former/chlorine-injured/24 h incubation)

Weak, 24h, Chlorine,

Salami
Rep Rep Rep 1 Rep 2 Rep 3
Slice Rep 1 2 3 Average Healthy Healthy Healthy
1 10 0 0 3.333333 0 0 0
2 0 5.52 0 1.84 0 0 0
3 10 5.76 0 5.253333 0 5.76 0
12.5
4 0 6 0 4.186667 0 0 0
5 6.92 6.36 0 4.426667 0 0 0
1045.
6 76 0 0 348.5867 220.16 0 0
7 0 0 0 0 0 0 0
8 0 6.4 0 2.133333 0 0 0
9 0 0 0 0 0 0 0
10 0 0 42 14 0 0 0
11 0 6.4 6.16 4.186667 0 0 0
12 0 0 6.04 2.013333 0 0 0
13 0 0 10 3.333333 0 0 0
14 0 0 0 0 0 0 0
15 0 0 6.28 2.093333 0 0 0
16 6.84 0 10 5.613333 0 0 0
17 6.88 0 0 2.293333 0 0 0
18 0 0 0 0 0 0 0
19 0 0 10 3.333333 0 0 0
20 0 0 10 3.333333 0 0 0
21 0 0 10 3.333333 0 0 0
22 6.88 6.48 6.16 6.506667 0 0 0
23 0 0 0 0 0 0 0
24 0 0 0 0 0 0 0
25 6.88 0 10 5.626667 0 0 0
26 0 0 10 3.333333 0 0 0
27 0 0 0 0 0 0 0
28 0 6.48 0 2.16 0 0 0
29 10 10 0 6.666667 0 6.4 0
30 0 0 0 0 0 0 0
Total CFU 1110. 65.9 136.
transferred 16 6 64 220.16 12.16 0
% Injury 80.168624 81.564584
Rep 34 6 100

202

APPENDIX III

203

SAMPLE MICROGRAPHS

Sample micrographs from viability staining using CSLM. Images in this
dissertation are presented in color.

Figure A3.1. Live/Dead micrograph of Listeria monocytogenes (strong biofilm
formers, cold-injured) afier 6 h of incubation on dry stainless steel

 

Figure A3.2. Live/Dead micrograph of L. monocytogenes (strong biofilm formers,
chlorine-injured) after I h of incubation on dry stainless steel

 

204

Figure A3.3. Live/Dead micrograph of L. monocytogenes (strong biofilm formers,
cold—injured) after 6 h of incubation on dry stainless steel

 

Figure A3.4. Live/Dead micrograph of L. monocytogenes (weak biofilm formers,
cold-injured) after 24 h of incubation on dry stainless steel

 

205

APPENDIX IV

206

Figure A4.1. An example of the GWBasic modeling program

‘8 ‘(SXIS? N*‘HXY)/(SX 2— N88 XX) 1 H: (S? H4$X)/N ’n 10 ope 8 b: intercept
R SQR((SX!SY- N-S KY)“2/(SX“2 N‘SXX)/(SY 2- Ni3 YY)) '1:0rr1:lation coefficient
' K: EXP(B) 1 R: EXPCH) ’fit parameters for cfu): ‘k* n
PRINT ”fraction left on blade duiing each 31 . :"10
F [NT cft transferred to 1‘t slice~”;K‘fl
" 1t: are inde endent of initial cfu‘u un blade”
initial cfu on hlnde'”;NB;“ then
~F1 f

’fractions t1an:;r1ed 1:0 neat an1 surroundings
”fraction transferred to neat during e11.1h slic EL” ,Fl
"fraction trans ferred to surrounding: during euith slice
NT ”fitted equatinn' all equiuult.nl) are:
" 1) 1n cfu(s )= '1H“*f * B

( ;H s'
“' :R;”:lu (":H/LOG(IB);”I:)"
”correlation coefficient for fit is ;R
"hit ENTER t:o see actual 1;: pr1:dicted dal a (IR at a tine)"
1 L=1118*(Q-1)I

11 van cfu predicted cfu”
FOR I—L TO L 9 F l)N TllLN STOP

PRINT 8(1) CFU(I) EXP(H*S(I)*B) 1 NEXT I
' 328

 

 

Figure A4. 2. GWBasic modeling program output when used to model transfer of
Listeria monocytogenes (108 CFU/blade initial inoculum level) to delicatessen meat

3 gluon cfu predicted cfu”
F I>N THEN STOP
S(l).CFU(I),EXP(H*S(l)+B) : NEXT 1
GOTO 328

traction left on blade during edLh slice: .8' 1947
cfu’ ‘ transferred to let slice: 1289712
above results are independent of initial cfu's on blade

if initial cfu' S on blade: 1E+98 then...
fraction transferred to neat during each slice: 1.289712E ”2
fraction transferred to surroundings during each slice = .152"082

fitted equations (31.1 equiualent) are:

1) ln cf11(s): .1888984 *3 +14.18S98

2) cfu(s)— 1448419 * .835194 7 “s

3) cfu(s)= 1448419 *e‘(-.18889@4 *3)

4) cfu(s)= 1448419 *IU‘(-7.821226E~82 *3)

correlation coefficient for fit is R: .9411926

hit ENTER to see actual us predicted data (18 at a tine)

 

 

207

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