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STUDY OF SPORICIDAL PROPERTIES OF CROSSLINKED
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DEEBIKA BALU

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STUDY OF SPORICIDAL PROPERTIES OF CROSSLINKED POLYELECTROLYTE
MU LTILAYERS

By

DEEBIKA BALU

A THESIS

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

MASTER OF SCIENCE
Clinical Laboratory Science

2010

ABSTRACT

STUDY OF SPORICIDAL PROPERTIES OF CROSSLINKED
POLYELECTROLYTE MULTILAYERS

By

DEEBIKA BALU

Polyelectrolyte multilayers (PEM) have become a highly studied class of materials due to
the range of their applicability in many areas of research, including biology, chemistry
and materials science. Recent advances in surface coatings have enabled modification of
PEM surfaces to provide desirable properties such as controlled release, super-
hydrophobicity, biocompatibility, antifouling and antibacterial properties. In the past
decade, antimicrobial PEM coatings have been investigated as a safer alternative to the
traditional disinfection methods that usually involve application of hazardous chemicals
onto the surface to be cleaned. These antimicrobial coatings could be applied to common
surfaces prone to colonization of bacteria (such as bench tops, faucet handles, etc) to

supplement routine sanitization protocols by providing sustained antimicrobial activity.

Vegetative bacteria (such as Escherichia coli) are more susceptible to antimicrobial
agents than bacterial species that form spores. Hence, the antimicrobial activity of PEM
coatings fabricated using Layer by Layer (LbL) technique were assayed using Bacillus
anthracis spores (Sterne strain). In this thesis, the sporicidal effect of various
polyelectrolyte multilayer coatings containing cross-linked polymers immersed in bleach

have been evaluated as potential augmentation to existing disinfection methods.

ACKNOWLEDGEMENTS

This research would not have been possible without the constant support and help I
received from many people over the past two years. Although I might not have
mentioned explicitly the names of all the people who have made me possible to come this

far, I do remember their contributions to my research and life.

First I would like to thank my research adviser Dr. Stephen Cendrowski, who gave me a
research project for me to work. His appreciation coupled with critical evaluation of my
work has enabled me to present my research project as a thesis. I would like to thank my
academic advisor and my committee member Dr. David Thorne for his time and help
which helped me to select the right courses that would be helpful for my future years as a
researcher. I would also like to thank him for his pertinent questions and useful feedback
regarding my thesis progress. I would like to my other committee member thank Dr.
Steven Kaganove for his critical evaluation and support. I would specifically like to thank

him for helping me to interpret complex FTIR spectra.

The unwavering support of my family and friends during these years at MSU enabled me
to survive through all these years of graduate school. I would like to thank my parents
and my husband Saravan for letting me study whatever and wherever I want. My husband
had a number of indirect contributions to my research life. I am thankful to him for the

technical, emotional and (logistics!) support he provided.

iii

I am grateful to Keri Nice and Elena Sias for their all their help in making my transition
into the new research work easier than what I had expected. I also would like to mention
the lovely times with Carmen Yu who made it less creepy to work late hours in Kedzie
Hall. I would also like to acknowledge a number of staff at BLD who made the two years
in the program fly by. Most of all I enjoyed taking the online courses in BLD department
from Dr. Gerlach and Dr. Thorne, which made me get a new perspective on molecular

pathology, immunology and cell biology.

I would like to thank the BLD department for accepting me into their program which
enabled me to take a number of biochemistry courses and complete my research project. I
would like to also thank Dr. Keith Lookingland, Dr. John Goudreau and Dr. Seong-Woon
Yu for their help and support in providing recommendation letters for my PhD programs.
I would like to thank them for an excellent research opportunity they had provided for
about a year on neurodegenerative disease mechanisms. It enabled my development as an
independent researcher. I would also like to thanks their lab members for their
encouragement and technical support for my research work. Last, I would like to thanks
the Office of Radiation, Chemical and Biological Safety (ORCBS) for providing me with
a graduate assistantship. I learnt a number of things that I used to neglect when I was a
researcher! My special thanks to my closer colleagues at the ORCBS: Elvet Potter,
Genevieve Cottrell and my supervisor Bob Ceru for providing an amiable work

environment and for understanding my graduate school work schedule.

iv

TABLE OF CONTENTS

 

 

 

 

 

 

LIST OF TABLES- - - ..... -- - - -_ - - - - - VII
LIST OF FIGURES - - -- ..... - - VIII
CHAPTER 1 INTRODUCTION - - -- - ........ - _- - - 1
1.1 HOSPITAL ACQUIRED INFECTIONS .................................................................. 1
1.2 ORGANIZATION OF THESIS ................................................................................ 3
CHAPTER 2 SPORES AS A HEALTH HAZARD - - -- ...... - 4
2.1 INTRODUCTION ..................................................................................................... 4
2.2 STRUCTURE OF A SPORE ..................................................................................... 4

2. 2.] Spore Core ......................................................................................................... 6
2.2.2 Inner Membrane ................................................................................................. 7
2.2.3 Cortex and germ cell wall .................................................................................. 8
2.2.4 Outer Membrane ................................................................................................ 9
2.2.5 Coat .................................................................................................................... 9

2. 2. 6 Exosporium ........................................................................................................ 9

2. 2. 7 Concluding remarks ......................................................................................... 10

2.3 SPORULATION AND GERMINATION .............................................................. 1 1
2.3.1 Sporulation ....................................................................................................... 11
2.3.2 The process of germination .............................................................................. 12

2.4 ANTIMICROBIAL AGENTS ................................................................................ 13
2.4.1 Introduction ...................................................................................................... 13
2.4.2 Definition of antimicrobial agents ................................................................... 13
2.4.3 Common antimicrobials and their mode of action .......................................... 15
2.4.4 Concluding remarks ......................................................................................... 19
CHAPTER 3 BIOFILMS AND ANTIMICROBIAL PEM(S)--- - _ 23
3.1 INTRODUCTION: ................................................................................................. 23
3.2 BACTERIAL BIOFILMS AND SPORES .............................................................. 23
3.3 ANTIMICROBIAL PEM(S) ................................................................................... 25
3.3.1 Adhesion-resistant PEMS ................................................................................. 25
3.3.2 Biocide leaching PEMS .................................................................................... 27
3.3.3 Contact killing PEMS ....................................................................................... 27

3.4 FABRICATION OF PEMS ..................................................................................... 29
3.4.] Properties of pH tunable PEMS ....................................................................... 30
3.4.2 Properties of N-halamies grafted onto PAMAM dendrimers .......................... 31
CHAPTER 4 EXPERIMENTAL METHODS AND RESULTS 35
4.1 INTRODUCTION ................................................................................................... 35
4.2 MATERIALS AND METHODS ............................................................................ 35
4.2.1 PEM construction materials ............................................................................ 35

4.2.2 Fabrication procedure ..................................................................................... 36

4.2.3 Testing the coatings for antimicrobial efficiency ............................................. 37
4.2.4 Calculating antimicrobial efi‘iciency ................................................................ 37
4.2.5 Fourier Transform Infrared (F TIR) spectroscopy ........................................... 38
4.3 RESULTS .................................................................................................................. 38

4. 3. 1 [PAA/PAH]5 bilayers with varying topmost layer that contacts the spores 38
4.3.2 Efl'ect of [FAA/PAH] and [ PSS/PAH] bilayers with PAA or PSS as topmost

 

 

 

 

layer and heat treatment ........................................................................................... 42
4.3.3 Effect of number of bilayers ............................................................................. 45
4.3.4 Coatings formulated with other common polyelectrolytes .............................. 48
4.3.5 F T IR spectra analysis ...................................................................................... 52
4.3.6 Shelf life issues ................................................................................................. 56
4.3.7 Conclusions ...................................................................................................... 58
4.3.8 Future work ...................................................................................................... 59
APPENDIX A - - 61
PROTOCOL FOR PREPARING BHI-AGAR PLATES .............................................. 61
APPENDIX B _ 62
PROTOCOL FOR SPORE INCUBATION IN PP PLATES AND FILTER
MEMBRANES ............................................................................................................. 62
APPENDIX C - - - _ _ - -- _ 64
TEST TO DETERMINE THE SPORICIDAL COMPONENT IN THE COATINGS .. 64
APPENDIX D -- 67
CALCULATIONS FOR % REDUCTION IN CFU VALUES ..................................... 67
BIBLIOGRAPHY ............................. - - 70

 

vi

LIST OF TABLES

Table 2.2 Sporistatic agents and their mechanism of action [42] ..................................... 20
Table 2.3 Sporicidal agents and their mechanism of action [42] ...................................... 21
Table 4.1 PEM Coatings tested with at least five [PAA/PAH] bilayers ........................... 39
Table 4.2 PEM Coatings with 2, 5 and 10 bilayers with PAA as topmost layer .............. 43
Table 4.3 PEM coatings with varying number of bilayers ............................................... 46

Table 4.4 PEM Coatings [PSS/PAH] bilayers and [PAA/PAH] with PSS as topmost layer
........................................................................................................................................... 49

Table 4.5 PEM coatings with variants containing having PEI, G4PAMAM and HBPU. 51

Table 4.6 Coatings that has shown evidence of antimicrobial activity compared to the
control coatings (without bleach) ...................................................................................... 59

Table C.1 PEM coatings tested to determine sporicidal agent .......................................... 65
Table D.1 Table of actual spore counts obtained during testing the coatings 3A-4B ....... 67

Table D2 Actual spore counts for coatings 3A-4B ........................................................... 68

vii

LIST OF FIGURES

IMAGES IN THIS THESIS ARE PRESENTED IN COLOR

Figure 2.1 Scanning Electron Microscopy (SEM) images of: A. Bacillus cereus; B.
Bacillus subtilis ; and C. Bacillus anthracis. The exosporium of Bacillus cereus is visible
as a loosely bound membrane on the spore surface. The endospores structure is not easily
visible in the SEM image. Bacillus anthracis endospores are seen under phase contrast
microscopy as lighter areas, because they are dehydrated and therefore more refractile.
Photo credit: Larry Stauffer, Oregon State Public Health Laboratory ................................ 4

Figure 2.2 Schematic diagram showing the internal structure of an endospore. The size of
the layers has not been drawn according to scale and the thickness of each layer varies
across species. ..................................................................................................................... 5

Figure 2.3 Electron Micrograph image of a bacterial endospore [14] ................................ 5

Figure 2.4 Life cycle of Bacillus subtilis. Note that this diagram depicts key stages in the
Sporulation process [17] .................................................................................................... 12

Figure 3.1 Scanning electron micrographs of biofilms formed on common surfaces. a)
Scanning electron micrograph of an infected catheter showing dense and complex biofilm
on the extra luminal surface [52]. b) Scanning electron micrograph of a Staphylococcus
biofilm on the inner surface of a needleless connector. Photo credits: Jance Carr, CDC,
Atlanta, GA (USA) [53] .................................................................................................... 25

Figure 3.2 Structures of common polyelectrolytes used in this study. a) PEI b) PAH c)
PSS (1) PAA [68] ............................................................................................................... 30

Figure 3.3 A schematic of layer by Layer deposition technique. The polyelectrolyte
multilayers are synthesized by a repeated charging of the surface with oppositely charged
ions until a desired number of layers is deposited. Each step results in a reversal of
surface charge allowing the next layer to be deposited [46] ............................................. 31

Figure 3.4 Structure of 5,5 dimethyl hydantoin and its halogenated derivative — 1,3
dichloro- 5,5, dimethyl hydantoin. .................................................................................... 33

Figure 3.5 Schematic representations of a a) dendrimer and b) a hyperbranched polymer
molecule. The core is represented by the grey molecule in the center with multiple
functional end groups. ....................................................................................................... 33

Figure 3.6 A diagram of PEM assembly as an antimicrobial surface. Polyelectrolyte

layers are coated onto the substrate. A final layer is applied made of a PAMAM
dendrimer with hydantoin appended to the end-group. .................................................... 34

viii

Figure 4.1 Spore counts in PEM coatings (Set 1, 2). Spore count data represent mean +/-
SD. of three independent experiments. ............................................................................ 40
Figure 4.2 Spore counts in PEM/G4-PAMAM coatings (Set 3, 4, 5). Spore count data
represent mean +/- SD. of three independent experiments. The plot also has the data
represented as set numbers (3, 4, 5) along with topmost layer of the PEM that contacts the
spores. The set of coatings with “B” in the coating ID contain alanine and inosine
(germinants). ..................................................................................................................... 41

Figure 4.3 Spore counts in PEM coatings without or with PAA as topmost surface that
contacts the spores. Spore count data represent mean +/- SD. of three independent
experiments. ...................................................................................................................... 44

Figure 4.4 Spore counts in PEM coatings with varying number of bilayers (2, 5, or 7
[PAA/PAH] bilayers. Spore count data represent mean +/- SD. of three independent
experiments. ...................................................................................................................... 45

Figure 4.5 Spore counts in PEM coatings with varying number of bilayer. Spore count
data represent mean +/- SD. of three independent experiments. ..................................... 47

Figure 4.6 A plot showing the effect of varying the bilayers on sporicidal activity.
[PAA/PAH] coating variants with varying number of bilayers were tested for sporicidal
activity. The difference in sporicidal activity between the sporicidal coatings and their
respective controls were plotted against the number of bilayers. ..................................... 48

Figure 4.7 Spore counts in PEM coatings. Spore count data represent mean +/- SD. of
three independent experiments. ........................................................................................ 50

Figure 4.8 Spore counts in PEM coatings with varying topmost layer. Spore count data
represent, mean +/- SD. of three independent experiments. These set of coatings have
four [PAA/PAH] bilayers and differ in the layer that contacts the spores ........................ 52

Figure 4.9 FTIR-ERS spectra of [PAH/PAA]5 multilayer films on gold (a) as prepared,
(b) after heating at 120 °C overnight, and (c) after heating at 180 °C overnight. ............ 53

Figure 4.10 FTIR-ERS spectra of a [PAH/PAA]5 multilayer film on gold (a) as-prepared,
(b) after heating at 120 °C overnight, and (c) after heating at 180 °C overnight. ............ 54

Figure 4.11 FTIR-ERS spectra of a [PAH/PAA]5 multilayer film on gold. (a) After
heating at 120 °C overnight, and (b) after treatment with NaOCl. ................................... 55

Figure 4.12 FTIR-ERS spectra expanded around the carbonyl region of a [PAH/PAA]5
multilayer film on gold. (a) after heating at 120 °C overnight, and (b) after treatment with
NaOCl. .............................................................................................................................. 56

Figure 4.13 PEM coatings tested for shelf-life issues. Sporicidal coatings with five
[PAA/PAH] bilayers having either PAA or G4PAMAM/PAA as the topmost layer were

ix

retested for sporicidal activity after 6 months from initial testing. Spore count data
represent mean +/- SD. of three independent experiments ............................................... 57

Figure C.1 Spore counts in PEM coatings ......................................................................... 65

Figure C.2 Spores sticking onto pipet tips could be visualized when stained with
methylene blue ................................................................................................................... 66

CHAPTER 1 INTRODUCTION

1.1 HOSPITAL ACQUIRED INFECTIONS

Hospital-acquired infections (HAIs), also called nosocomial infections, are infections
acquired by a patient in the hospital and are not related to the condition for which the
patient was originally admitted [1]. It is estimated that about 1.7 million HAIs occurred in
the United States in 2002, of which 78% were due to respiratory tract, urinary tract,
bloodstream, and surgical site infections [1]. Thus, HAIs have a significant impact on
both the patient health as well as the health care system. For the patient, HAI results in an
increased length of stay due to additional medical procedures. In severe cases, HAIs can
lead to septicemia or death [2]. For the health care system, such infections increase
treatment costs. HAIs increase the cost per patient by $1,909 to $38,656 and increase
length of stay in the intensive care unit by 4.3 to 15.6 days [3,4,5]. A significant
percentage of these infections can be attributed to bacterial colonization and biofilm
formation on critical items such as surgical instruments, cardiac catheters and urinary
catheters. In addition to these invasive instruments, environmental surfaces such as bed
pans, bed rails, sinks, patient furniture and floors can also be potential sources of

infection [6].

Biofilms that develop preferentially on surfaces of implants, bed rails and sinks result
from bacterial colonization of these inert surfaces, and they contain aggregates of
bacterial colonies within organic matter synthesized by these organisms to form a

complex multi-cellular structure [7]. Biofilms acts as a barrier to common disinfectants

such as chlorine and hydrogen peroxide by neutralizing these chemicals in the surface
layers, thereby limiting the diffusion of the disinfectant into the deeper layers [8]. Hence
one of the key strategies to prevent HAIs is to prevent biofilm formation. Disinfectants
such as hypochlorites and hydrogen peroxides are commonly used to prevent biofilm
formation on non-medical devices and are also the primary choice for post-contamination
applications in case of accidental exposure to biohazards. These traditional measures
require constant vigilance and training of personnel for routine disinfections and
emergencies. Also, these disinfectants are highly corrosive or toxic, which endangers the
health of the personnel involved in such procedures, as well as other individuals in the
proximity of the disinfected area at risk [9]. Thus applying the disinfectant in a form that
is safe to use for the personnel and the environment, as well as preventing biofilm
formation is clearly a better alternative to the traditional disinfection methods.
Furthermore, if the contact surface to which the bacteria adhere were self-disinfecting, it
could supplement routine sanitization protocols by providing sustained antimicrobial

activity.

Over the past decade, surfaces of multilayered polyelectrolyte films have been modified
to provide desirable properties such as controlled release, hydrophobicity,
biocompatibility [10], antifouling properties [11] and antibacterial properties. A readily-
available formulation of silver-zinc zeolite (AgION Technologies, Inc, Wakefield, MA)
has proven effective against a variety of pathogens in a variety of environments, and has
been incorporated in a number of materials for use in healthcare [12]. Most of these

antimicrobial products are produced by chemical surface modification techniques [13].

Layer-by-layer (LbL) assembled polyelectrolyte multilayers (PEM) have also been
shown to provide effective antibacterial properties. For example Chua et al. have
successfully produced an antibacterial coating on titanium alloy, used for orthopedic
implants [14]. Common materials such as stainless steel and glass have also been used as
substrates for a number of antimicrobial PEM. Thus, the LbL process has been used to
create PEMS ideally suited for controlling bacterial adhesion, and it is also amenable to

incorporation of a broad range of polymers, small biomolecules [15] and even cells [16].

The major goal of this research was to identify important characteristics that influence
sporicidal properties of PEM such as the time of contact, number of layers and
composition of the multilayers. Thermal cross-linking of PEMS, assembled using LbL
technique was investigated as an alternative to chemically modified surfaces. By thermal
cross-linking, the PEM coatings can remain intact at physiological pH ranges. The PEMS
were assessed for sporicidal activity using Bacillus anthracis Sterne 34F 2 spores, an

attenuated strain as a model organism.
1.2 ORGANIZATION OF THESIS

The first chapter provides a brief overview of pathogenesis, sporulation and germination
mechanisms of Bacillus anthracis. The second chapter details the basic structure of a
spore, requirements for spore formation, Sporulation and germination mechanisms. The
third chapter discusses the strategies for disinfection and also provides an overview of
PEM coatings. The fourth chapter discusses the results obtained during the study and

future directions for the research.

CHAPTER 2 SPORES AS A HEALTH HAZARD

2.1 INTRODUCTION

Bacterial spores (Fig 2.1) are highly specialized cell types designed for survival under
adverse environmental conditions. Among specialized differentiated cell types, such as
cysts and exospores, the endospores formed inside the bacteria are the most resistant [17].
Spores remain dormant and resist adverse conditions, such as starvation, high
temperatures, ionizing radiation, mechanical abrasion, chemical solvents, detergents,
hydrolytic enzymes, desiccation, pH extremes and antibiotics [17]. Their metabolic

dormancy combined with their resistance enables their survival for long periods of time.

 

a b c

Figure 2.1 Scanning Electron Microscopy (SEM) images of: a) Bacillus cereus; b)
Bacillus subtilis ; and c) Bacillus anthracis. The exosporium of Bacillus cereus is Visible
as a loosely bound membrane on the spore surface. The endospores structure is not easily
visible in the SEM image. Bacillus anthracis endospores are seen under phase contrast
microscopy as lighter areas, because they are dehydrated and therefore more refractile.
Photo credit: Larry Stauffer, Oregon State Public Health Laboratory.

2.2 STRUCTURE OF A SPORE

The resistance of a spore to extreme conditions is due to the presence of multiple layers.

The concentric layers starting from the core are the inner membrane, germ cell wall,

cortex, outer membrane, and the spore coat (Figure 2.2). An electron micrograph image

of bacterial endospores shows multiple layers of varying density (Figure 2.3).

Lytic enzymes

Cortex

Germination receptors
Inner membrane

Outer membrane

Spore coat

 

Figure 2.2 Schematic diagram showing the internal structure of an endospore. The size of

the layers has not been drawn according to scale and the thickness of each layer varies
across species.

   
 
  
     
     
  

  

  

    
      
  

   
   

     
 

       
       

       

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Figure 2.3 Electron Micrograph image of a bacterial endospore [14]

2. 2. 1 Spore Core

The core forms the innermost part of the spore similar to the cytoplasm of vegetative
cells containing essential proteins and nucleic acids. The most distinguishing
characteristic of the core is its very low water content, which is about 30-50%. In
comparison, the cytoplasmic water content of a normal vegetative cell is about 70-88%
[18]. The low water content in the core plays an important role in longevity and
dormancy of the spore [18]. A number of models have been proposed to discuss the state
of the cytoplasm. Cowen et al suggest a “glassy state” that promotes dormancy and
resistance [19]. Differential scanning calorimetry (DSC) and nuclear magnetic resonance
(N MR) studies on B. subtilis spores indicate that the dormancy is due to the low moisture
content and glassy state of the core [20]. The two peaks in the DSC thermograrns of
hydrated spores correspond to the activation stage at 60°C, and the killing stage at 120
°C. NMR spectroscopy studies on calcium dipicolinic acid (Ca DPA) in the core suggest
that the CaDPA is present in an amorphous solid-like environment [20]. However, others
have proposed the idea that core water freely crosses the membrane [21, 22]. Phosphorus

is present only in the core and change in molecular mobility of radio-labeled phosphorus

(31P) was observed when the spores were hydrated (or dehydrated), whereas no change
was observed in molecular mobility of dipicolinic acid (DPA) (another core-specific
molecule). Hence, these models propose the existence of DPA immobilized in a water-

insoluble network of macromolecules. Further research is needed to reveal the internal

structure of a spore and how the environment contributes to the structural stability.

Apart from low water content, the core lacks most of the high energy compounds such as

ATP and NADH that are usually present in the cytoplasm of a vegetative cell [l8]and is
characterized by low pH levels (6.3-6.5) [23]. Pyridine 2,6-dicarboxylic acid (commonly
called dipicolinic acid or DPA) has been shown to have an important role in spore
resistance, although the exact mechanism is not yet completely understood [24]. The
DPA present in the core chelates with divalent ions such as calcium, and this complex
accounts for about 15% of the dry weight of the spore. Calcium ions and DPA are
secreted during germination, and may play a role in subsequent germination steps [25].
The core also houses small acid soluble proteins (SASP), similar to histone proteins in
eukaryotic cells, that bind to the DNA [26], condensing DNA to protect it against

damages that may occur during harsh conditions [27].

2.2.2 Inner Membrane

The inner membrane of a spore, like the plasma membrane of a eukaryotic cell, is a
selectively permeable membrane surrounding the core. The inner membrane contains
germination receptor proteins, which play an important role during the first stages of
germination. They have been shown to play a role in signal transduction across the

membranes to other parts of the spore, upon activation of germination.

An intact inner membrane is essential for germination [28]. Upon germination under
favorable conditions, the inner membrane becomes the cytoplasmic membrane of the
vegetative cell. During germination the inner membrane swells to double its size without
membrane lipid synthesis. Hence, it has been proposed that the inner membrane of the

spore must be in a highly compressed state due to decreased lipid membrane mobility

[29]. Oxidizing agents such as chlorine dioxide, hydrogen peroxide, ozone, and sodium
hypochlorite may result in inner membrane damage that contributes to spore killing upon

germination.
2. 2.3 Cortex and germ cell wall

The cortex is a thick cell wall around the inner membrane and is mainly composed of
modified peptidoglycan (PG). It plays an important role in maintaining the core in a
highly dehydrated state [20]. The structure of the cortex is highly conserved among
species, and has been suggested to play an important role in heat resistance. There are
two major modifications to PG in the spore cortex that appear to be important in
germination [30]. First, only 3% of the muramic acid present in PG of the cortex exists in
the cross-linked state, in comparison to 40% in the vegetative cell wall [30]. Second,
most of the muramic acid is modified to a muramic-B-lactam structure [30]. Muramic-B-
lactam acts as a specific target for lytic enzymes that are activated during germination

and the lower cross-linking enables easier outgrowth.

Early stages of spore germination involve changes in permeability of the membrane,
resulting in redistribution of ions and water in the spore. It has been proposed that the
ions and water may activate the lytic enzymes in the spore coat, resulting in degradation
of the cortex PG [30]. The inner part of the cortex called the germ cell wall, lacks
muramic acid lactam and muramic acid alanine in PG that are characteristic of the cortex.

The germ cell wall forms the primordial cell wall of the freshly germinated cell [31].

2. 2.4 Outer Membrane

The outer membrane separates the cortex from the spore coat and is an essential structure
for spore formation [32]. Recent work has suggested that the outer membrane acts as a
major permeability barrier in spores [33]. An intact outer membrane is required for

dormancy in B. megaterium spores [34].
2. 2.5 Coat

The inner layer of the coat harbors the lytic enzymes that degrade the cortex during
germination [17]. The coat has also been shown to be resistant to UV-radiation and to a
variety of chemicals (including oxidizing agents such as hypochlorite) [35]. Atomic
Force Microscopy studies of B. thuringiensis have revealed that the spore coat expands
and shrinks in response to changes in relative humidity [22]. Several coat proteins are
involved in germination by facilitating the passage of germinant molecules through the

coat [36].
2. 2. 6 Exosporium

The exosporium is a species-specific structure. For example, Bacillus cereus and Bacillus
anthracis possess an exosporium while Bacillus subtilis does not. The exosporium is
essential for maintaining the hydrophobicity of the spore [3 7]. Substrate and spore
hydrophobicity, and surface tension contribute to non-specific interactions which may
eventually result in bacterial adhesion to substrates [37]. Bacterial adhesion has been a
major concern for the medical, pharmaceutical and food industries. The ability of

Clostridium spores to adhere to surfaces and resist high temperatures compromises the

sterility of the processed food and results in food- borne illness. Also, bacterial transfer
fiom the environment to hospital surfaces such as catheters used in patients has been

associated with increase in hospital acquired infections.

The exosporium is visible as a loose membrane-like structure around the coat (Fig 1.1).
Spore resistance is further enhanced by the presence of a manganese oxidizing enzyme in
the exosporium that creates a “metal shield” over the internal layers [3 8]. Certain
enzymes in the exosporium have been shown to be involved in germination [39]. The
homolog of exosporium protein (Est) in Bacillus subtilis is required for proper
assembly of the coat [40], and it has been suggested that the exosporium could be
considered as a specialized coat layer. It should be noted that Est is required for

adherence of the exosporium to the coat.

2. 2. 7 Concluding remarks

Bacterial spore formation is the ultimate survival mechanism, and it has been studied for
more than a century. The resistance of bacterial spores is due to the presence of multiple
protective layers over a highly dehydrated core (Fig 2.2). Several spore-forming bacteria
such as Bacillus and Clostridium spp. have pathogenic properties that present a health
hazard. Dormant bacillus spores are extremely difficult to destroy due to their resistance
to heat, UV radiation, alkylating agents and oxidizing agents [18]. Given that spore
formation is essential for both survival and dispersal of the bacteria, spores clearly are a
major problem in settings that require sterility and hygiene especially in healthcare and

food industries. A brief discussion of Sporulation and germination processes in bacteria is

10

presented in the following sections.

2.3 SPORULATION AND GERMINATION

2. 3.1 Sporulation

Sporulation is defined as the process of spore formation, and hasbeen well documented
in B. subtilis. Like other bacillus species, the B. subtilis exists in two forms: a vegetative
cell and a dormant endospore (formed by Sporulation). In the presence of sufficient
nutrients and other favorable conditions (physiological temperature, pH), the vegetative
cell undergoes division. However, unfavorable conditions (such as high temperature, high

population density) trigger the bacterial cell to undergo spore formation.

An overview of the process is illustrated in Fig 2.4 [32]. Asymmetric cell division of the

bacterial cell (Stage II - Fig 2.4) is triggered by phosphorylation of transcription regulator
SpoOA along with CH — an RNA polymerase co-transcriptional factor. This produces two
distinct cells, the smaller pre-spore that ultimately develops into the spore, and the
mother cell that ultimately undergoes programmed lysis). Sigma factors in the mother
cell (OE) and the prespore (O'F) trigger expression of distinct genes involved in
Sporulation. This process is followed by engulfinent of the pre-spore by the mother cell
(Stage 111). After engulfinent, changes in gene expression and morphogenesis occur in the
pre-spore leading to formation of distinct cell membrane, cell wall, cortex and spore coat

(Stage IV-V). The lysis of the mother cell (Stage VI, VII) releases the mature spore into

the environment.

11

2.3.2 The process of germination

/ O<~\ Coat
Stage V

Growth 0 Spore Coat
/\ Stage VI, VII
Maturation, cell lysis
Cortex

SPORULATION Cell wall

Cell membrane

Medial Polar Divrsion ,
Division . G Prespore __ .\ Stage IV
l I Cortex
S
eptum L2
\d
Mother cell

  

Stage 11 Stage III
Asymmetric Engulfment
cell division

Figure 2.4 Life cycle of Bacillus subtilis. Note that this diagram depicts key stages in the
Sporulation process [17]

A gerrninant molecule binds to germinant receptors and this leads to rapid, irreversible
changes in the spore, ultimately resulting in a fully germinated bacterial cell. In natural
conditions, nutrients such as amino acids and sugars activate the GerA receptors in the
inner membrane [39]. Ca2+ and DPA are released from the core as it becomes partially

rehydrated [3 5]. Further rehydration is facilitated by the degradation of the cortex by lytic

enzymes. Rehydration restores the normal metabolic state of the bacterial cells via

restoration of enzymatic activity

2.4 ANTIMICROBIAL AGENTS

2.4.1 Introduction

As described in the previous sections, spore formation allows the bacteria to exist in a
protected state in adverse conditions. For example, Bacillus cereus food poisoning occurs
because heat-resistant endospores survive cooking or pasteurization. The spores
germinate when food is inadequately refiigerated and release toxins when the food is
ingested. However, food poisoning due to spores may be avoided by cooking and storing
food at right temperatures. Since hospital environments demand good hygiene,
disinfection of contaminated surfaces may be achieved by chemical methods that usually
require a combination of one or more chemicals that are bactericidal or sporicidal agents.
However, preventive decontamination of an entire building on a regular basis may not be
possible. Typical chemicals used in decontamination are based on bleach, chlorinated
solvents or corrosive chemicals. Most of the bactericidal agents (such as phenol, alcohol

and aldehydes) are not sporicidal even when applied to the surface for a long time [9].

2. 4.2 Definition of antimicrobial agents

Commonly used antimicrobial agents may be physical conditions, chemical agents, or
combinations of both. Physical antimicrobial agents include moist heat, dry heat,

ultraviolet and ionizing radiation, and high pressure to reduce microbial activity [41].

13

Depending on their intended use, chemical agents may be classified as antibiotic agents,
antiseptics, food preservatives, sanitizers, disinfectants or sterilizers (sporicides) [41].
Antibiotics such as penicillin are drugs taken orally or intravenously to treat diseases of
humans, animals and plants. Antiseptics are substances applied topically to living tissues
to prevent or arrest the growth or action of microorganisms. Food preservatives are
chemicals used in foods, alone or along with physical agents, to extend the shelf life of
food products. A sanitizer is commonly used to reduce, but not necessarily to eliminate,
the number of bacterial contaminants to levels that are considered safe by public health
regulatory agencies. Disinfectants are chemical or physical agents that inactivate disease
causing microorganisms, but not necessarily their spores. Sterilizing agents or sporicidal

agents eliminate all forms of microbial life, including bacterial spores [9].

Based on their specific activity against bacterial spores, rather than their intended area of
application, antibacterial agents may be broadly classified as bactericidal or Sporistatic
agents such as phenols, quaternary ammonium compounds, alcohols, organic acids and
esters; or sporicidal agents such as glutaraldehyde, formaldehyde, peroxygens, iodine
compounds, fumigating chemicals (e.g., ethylene oxide), chlorine releasing compounds
(e.g., hypochlorite, chlorine gas, halogens) [9]. Bactericidal agents are effective against
vegetative bacteria by inhibiting germination or outgrowth (Sporistatic) while sporicidal
agents are effective against spores that resist many harsh chemical and physical

conditions.

14

Biocides vary in their chemical structures and also in their mode of action. However,
their responses when lethal concentrations are used show many similarities. Biocides
must reach and interact with their microbial target site(s) to be effective. Although most
biocides are nonspecific, some antimicrobial agents interact with specific enzymes or
cellular components. Irrespective of the type of microbe, it is highly likely that there
exists a common sequence of events leading to reduction in microbial population. The
end effect mostly depends on the interaction of the disinfectant with the cell surface and
its penetration into the cell. After its penetration, the disinfectant may have a target site(s)
of action. For example, gluteraldehyde interacts with the hydrophobic spore coat
resulting in increased penetration into inner layers [42]. Thus, the outermost layers of
microbial cells can have a significant effect on their relative susceptibility to antiseptics

and disinfectants.
2.4.3 Common antimicrobials and their mode of action

In this section, the mechanisms of antimicrobial action of a range of chemical agents used
as antiseptics, disinfectants or both, are discussed. Finally, the mechanism of action of
common sporistatic and sporicidal compounds has been tabulated (Table 2.1 and Table

2.2).

2.4.3.1 Acids

Organic (benzoic, acetic and citric) and inorganic acids (sulfuric, hydrochloric) acids
have been used widely as preservatives and in cosmetics and food processing industries
and to a lesser extent as commercial disinfectants. For example, benzoic acid is usually

used in a salt form (sodium benzoate) in food products as a preservative and acetic acid is

15

commonly used as a preservative in the form of vinegar, which contains about 4% acetic
acid. In general, short chain acids (methyl and ethyl derivatives) are less effective than

long chained acids but have higher solubility in water.

The antimicrobial activity of acids and their derivatives depend on their solubility in
water or lipid, which in turn influences their end effect when applied as chemical
formulations [41]. These acids disrupt the structure of membranes and proteins thereby
interfering with the cellular uptake of substrate molecules in bacteria, eventually result in
killing by interfering with key metabolic processes [43]. Since they remain effective in a
wide range of pH (pH 4.0 to 8.0), they are attractive for use as preservatives. Most of
these acid derivates are non toxic and non corrosive in the concentrations in which they

are typically used [42].

2.4.3.2 Alcohols

Alcohol based disinfectants containing 60 — 90% ethyl alcohol or isopropyl alcohol are
commonly used for rapid broad spectrum activity against vegetative bacteria [42]. Their
use is restricted to hard surface disinfection and skin antiseptic since they lack sporicidal
activity [44]. They are usually applied in formulations containing other disinfectants
(e.g., chlorohexidine), which does not evaporate and remains on the skin to render anti-
microbial activity. It is hypothesized that alcohols cause denaturation of proteins,
consequently disrupting the membranes. This could result in disruption of metabolic

processes across the membranes, and eventually lysis of the cell.

16

2.4.3.3 Aldehydes

Broad spectrum antimicrobials such as gluteraldehyde and formaldehyde target proteins
and nucleic acids leading to inhibition of important metabolic processes. Although
formaldehyde is both sporicidal and bactericidal, it is not as effective as gluteraldehyde.
Both have been shown to be effective under alkaline conditions [42]. Formaldehyde
interacts with the amide and amino groups in proteins, disrupting the membrane and
penetrating into the layers. It is also considered mutagenic since it extensively interacts

with nucleic acids [42].

2.4.3.4 Quaternary Ammonium Compounds

The Quartemary Ammonium Compounds (QACS) are derived from exhaustive alkylation
of amines. The chain length of the alkyl groups influences the antimicrobial properties, as
well as solubility of the compound. Alkyl groups containing 11 to 17 carbons have been
shown to have the highest antimicrobial activity [44]. Cationic surfactants such as QACS
are the disinfectants of choice for disinfection of non critical surfaces and for
preoperative disinfection of intact skin. Upon adsorption and penetration into the cell
wall, the long alkyl chains of the QACs react with phospholipids and proteins in the
cytoplasmic membrane [42]. This degrades the membranes and eventually results in cell
wall lysis due to lytic enzymes. In this process, there is loss of cellular components as
well as cell wall degradation. QACS being sporistatic, inhibit the outgrowth of vegetative

cell from germinated spore.

2.4.3.5 Chlorine Compounds.

Chlorine and iodine compounds are the most commonly used chemicals in clinical

17

settings as disinfectants and as antiseptics. Chlorine compounds are broadly classified as
chlorine gas, hypochlorites (e.g., sodium hypochlorite) and chlorine releasing agents
(e.g., dichlorodimethyl hydantoin and chloramines) [9]. Their activity is dependent on

their solubility, available chlorine and the pH of the environment.

As a strong oxidizer, chlorine gas reacts with any organic matter present, which can
decrease its efficacy as a antimicrobial. For example, chlorine gas oxidizes organic and
phenolic compounds in the environment before it interacts with microbial surfaces [45].
Since it is toxic and corrosive it has limited applications as commercial disinfectant [41].
On the other hand, sodium hypochlorite is relatively safer to handle than gaseous
chlorine, and hence is widely used in clinical settings. Sodium hypochlorite (commonly
known as bleach) is widely used for cleaning inert surfaces. The active agent in sodium
hypochlorite is hypochlorous acid, which is formed by hydrolysis of hypochlorite ion.
Hypochlorous acid is a weak acid, but a powerful oxidant, hence making the
hypochlorites bactericidal and sporicidal [41]. Due to its low molecular weight and
neutral charge, hypochlorous acid readily interacts with the cell wall and penetrates
bacterial cells. The activity of the hypochlorous acid is a function of pH and temperature

[41].

Chlorine-releasing compounds such as N-halamines are organic or inorganic compounds
in which oxidative halogens are attached to nitrogen. In aqueous solutions, chloramines

undergo hydrolysis in varying degrees to form hypochlorites [41]. Monomeric n-

18

halamines such as 1,3 dichloro- 5,5, dimethyl hydantoin have been widely used as water

soluble alternatives to chlorine based disinfectants [41].

2.4.3.6 Iodine Compounds.

Iodine solutions are widely used as antiseptics because they are effective against bacteria,
virus, spores and fungi [9]. However, their efficacy is limited by the fact that they are
irritants and are highly unstable in aqueous solutions. They cannot be used to disinfect
hard surfaces and skin since they cause excessive irritation and staining. Such limitations
were overcome by the development of iodophors or iodine releasing compounds similar
to chlorine-releasing agents such as chloramines [42]. They act as reservoirs of active
iodine and are used as antiseptics and disinfectants. Even at low concentrations, iodine
has rapid antimicrobial activity. It is hypothesized that iodine penetrates the cell wall and
binds to sulfur containing amino acids, nucleotides and fatty acids, resulting in metabolic

inhibition and eventually cell death [42].
2. 4.4 Concluding remarks

Most of the sporicidal agents are sporistatic at lower concentrations. Non-porous surfaces
in hospitals such as floors, faucets and bed rails can be decontaminated using liquid
antimicrobial products that may be poured or sprayed onto the area. One of the most
common chemicals used for this purposes is 10% household bleach solution. The type of
surface and the extent of contamination determine the minimum time the surface needs to
be exposed to the antimicrobial agent. Tables 2.1 and 2.2 describe the mechanism of

action for common antimicrobial agents used for disinfection.

l9

Table 2.1 Sporistatic agents and their mechanism of action [42]

 

Sporistatic agents

Mechanism of action

 

Organic acids such as

benzoic acid, citric acid

Aliphatic alcohols such as

ethanol

Chlorhexidine salts (CHX),

(QACS)

Phenol and Cresol

Interfere with the cellular uptake of substrate molecules
by disruption of tertiary and quaternary protein

structures and membranes.

Disrupt membrane structure and permeability; inhibit
protein synthesis and enzymes involved in glycolysis

and phospholipid synthesis

Ionic interaction with phospholipids in the membrane

result in membrane disruption

Penetrate the phospholipid membrane and disrupt

membrane integrity

 

20

Table 2.2 Sporicidal agents and their mechanism of action [42]

 

Sporicidal agents Mechanism of action

 

Targets DNA and proteins that have amino, amide and
Gluteraldehyde thiol groups; cross link amino groups resulting in

metabolic inhibition and cell wall damage

React with amino and thiol groups in amino acids
Iodine compounds disrupting protein synthesis. Also target carbon bonds in

fatty acids resulting in membrane immobilization

Target on the cell wall and amino acids in proteins,
Chlorine releasing agents

oxidize thiol groups in proteins and/ or chlorinate
(CRA) and hypochlorites

nucleotides in vegetative bacterial DNA

Peroxygens such as Target thiol groups in proteins and oxidize them resulting

hydrogen peroxide in metabolic inhibition of enzymes

 

Although a wide variety of sporicidal agents is available, most of the chemicals have the
disadvantage of being corrosive or toxic at low concentrations. For example,
gluteraldehyde is highly effective as a sporicidal agent. However, it is highly toxic even
at concentrations as low as 2% which is normally used for disinfection [42]. Hydrogen
peroxide is highly unstable and decomposes readily, while hypochlorite solution is

inactivated by organic matter [9]. Ethylene oxide is hazardous since it penetrates organic

21

porous materials, which limit its use as a disinfectant in hospital environments [9].
Despite the disadvantage of being hazardous, these sporicidal agents are the primary
choice for bacterial or spore disinfection, because they can be bactericidal or sporistatic at

low concentrations [9].

22

CHAPTER 3 BIOFILMS AND ANTIMICROBIAL PEM(S)

3.1 INTRODUCTION:

The interaction between bacteria and surfaces has recently evolved as an area of
extensive research potential. Using a variety of surface systems that include surface
grafted molecules and nanoparticles, researchers have determined surface parameters that
resist bacterial attachment, maximize antimicrobial efficiency and prevent biofilm
formation. Polyelectrolyte multilayers (PEMS) provide a simple and versatile platform for
exploring such surface interactions. Due to the wide range of materials that can be
incorporated into them ranging from small biomolecules such as DNA and proteins to
cells, PEMs also make excellent candidates for biomaterials [46]. In addition, precise
control can be exercised over their material properties such as thickness of the layers,
surface roughness, and charge. The high stability of the multilayers under physiological
conditions offers an opportunity for exploring many aspects of bacterial cell control.
Major concepts about antimicrobial PEM coatings are introduced in this chapter before
delving into the applications explored in this thesis. The chapter also emphasizes the

performance of PEM surfaces containing biocides as antimicrobial coatings.

3.2 BACTERIAL BIOFILMS AND SPORES

Bacterial biofilms are complex multicellular structures consisting of one or more
bacterial species or fungal species in a polysaccharide matrix. These biofilms act as
important reservoirs for pathogens, and biofilm growth may provide microbes with

survival advantages in adverse environments. The structure of a biofilm depends on

23

various parameters such as nutrient availability and surface properties. In anaerobic flow
chemical reactors, microbes form granular sludges that settle to the bottom since there are
no walls to adhere [47]. Hence, surfaces as well as nutrient availability limit the
formation of biofilms. In bacteria such as Vibrio cholerae, a nutrient-rich environment
has been shown to promote biofilm formation [48]. Most biofilms have a heterogeneous
structure consisting of cellular aggregates interspersed throughout a matrix that varies in
density. This structure creates channels for water to flow in and out of the matrix. Particle
tracking techniques and in situ measurements of metabolic products such as oxygen, have
been utilized to demonstrate the presence of interstitial voids in the matrix that provide
means for circulating as well as exchanging metabolic products with the environment

[49].

In addition to biofilms, bacterial spores (discussed in Chapter 2) can resist adverse
environmental conditions. The combination of these two highly resistant structures is
hypothesized to contribute to an ultimate survival mechanism employed by certain
opportunistic bacterial species such as Pseudomonas aeroginosa and Streptococcus
epidermidis [50]. Furthermore, biofilm formation on surfaces is an important

consideration in healthcare and food processing industries [51].

Biofilms not only protect the bacterial species residing in them, but also provide a
protective niche for the bacteria that resist antimicrobial agents. The first is the physical

fi'amework of the biofilm that resists antimicrobial agents. The thick exopolysaccharide

24

presents an impenetrable barrier that prevents diffusion of chemical disinfectants. The
biological interaction between various bacterial colonies in a biofilm enhances their
survival since these biofilms are typically composed of many species of bacteria that
possess a range of antimicrobial susceptibilities. The persistence of hospital—acquired
infections in even developed countries indicates a lack of efficient disinfection strategies

and the need for improved antimicrobial technologies.

 

a) b)

Figure 3.1 Scanning electron micrographs of biofilms formed on common surfaces. a)
Scanning electron micrograph of an infected catheter showing dense and complex biofilm
on the extra luminal surface [52]. b) Scanning electron micrograph of a Staphylococcus
biofilm on the inner surface of a needleless connector. Photo credits: Jance Carr, CDC,
Atlanta, GA (USA) [53]

3.3 ANTIMICROBIAL PEM(S)

3.3. 1 Adhesion-resistant PEMS

Most of the current disinfection strategies in healthcare and food processing industries

consist of physical and chemical disinfectants. To address problems such as biofilm

25

formation, which increases microbial load, and to avoid applying high concentrations of
chemical disinfectants to surfaces that come in contact with human skin, alternative
disinfection strategies have been proposed. Initial attachment of bacteria to surfaces
involves formation of weak and reversible van der Walls, electrostatic and hydrophobic
interactions with the surface. At this point mere rinsing of the surface can remove
bacteria. The second step in bacterial attachment involves specific interactions of the
bacteria with the surface using pili or fimbriae that enables them to be in very close

proximity.

One of the earlier approaches was to limit colonization of bacteria by using conjugated
polypeptides to resist bacterial adhesion since biofilm formation depends on stable
attachment of bacteria to surfaces [54]. Poly(ethylene glycol) (PEG) grafted onto
surfaces directly or as one of the multilayers along with poly (L-glutamic acid) (PGA)
has been shown to reduce attachment of E. Coli even in the presence of nutrients [55].
Poly(methylmethacrylate) (PMMA) coated intraocular lenses, modified by linking of
hyaluronan chemical group to the surface has been shown to effectively reduce surface
adhesion of Staphylococcus epidermidis. The mechanism behind these adhesion resistant
PMMAs has been attributed to hydrophilic properties of the hyaluronan layer [56].
Biodegradable properties have been utilized to produce adhesion-resistant PEMS
containing antibiotics that prevent attachment due to top-down erosion of the multilayers

[57].

26

3. 3.2 Biocide leaching PEMS

PEMS can also be designed as coatings that leach antimicrobial compounds. In one of the
studies by Feng et a1, silver nanoparticles were embedded onto stainless steel surface
containing multilayers of poly (acrylic acid) (PAA)/ quaternized polyethylenimine (PEI)
[58]. The biocidal activity of silver ions is due to their binding to thiol groups present in
cell membranes, thereby disrupting permeability. Thus the silver particles bind to DNA
resulting in metabolic inhibition [58]. Hammond et al. have worked on PEM assemblies
that control the release of Gentamicin (an antibiotic) [57]. Various parameters such as

the number of bilayers and their composition influence antimicrobial activity.

3. 3.3 Contact killing PEMS

Some variants of PEMS have been engineered to kill bacteria when they come in contact
with the surface. When immobilized onto surfaces, chitosan (a cationic antimicrobial
polysaccharide) has been shown to bind to negatively charged bacterial cell membranes
resulting in leakage of ion channels [59].Long hydrophobic polycations have been grafted
onto various substrates to kill pathogenic bacteria on contact [59]. These polycations
(e.g., PEI) reduce the microbial population by damaging the bacterial cell membranes
[59, 60]. Various studies have been performed to optimize parameters such as contact
time, chain length and concentration of the polycations [59, 60, 61]. Chemical
disinfectants such as QACS have been widely used to create non-leaching biocidal
surface coatings. The antimicrobial mechanism of QACS was discussed in detail in
section 2.4.3.4. PEM assemblies containing charged polymers with grafted QACs have

been shown to be effective against Gram positive bacteria [62]. However, such coatings

27

have their own disadvantages. These coatings can lose their efficacy when the microbial
load increases due to constant use. Increasing the concentrations of hazardous chemicals
such as QACS to achieve better antimicrobial activity is not recommended. Hence,
covalently cross-linking polymers that makes the polycations to retain their antimicrobial
properties in a permanent and non-leachable fashion when placed in an environment (for
example bodily fluids, high moisture area, sterile contact surfaces) has been investigated.
F urtherrnore, n-halamine siloxane coatings has been recently investigated as excellent
antimicrobial coatings on textiles and as polymeric surface coatings that can be
replenished when the surface is sprayed with commercially available household bleach

solution [63,64].

Antimicrobial PEM surface coatings that reinforce surface sterility between disinfection
cycles have been developed [64]. Apart from the wide range of materials that can be
incorporated, [46] these PEMS are also biocompatible and environment friendly.
Biological materials such as proteins [65], DNA [15], polysaccharides [66] and enzymes
[67] have been incorporated into multilayers for drug delivery applications. Unlike self
assembled monolayers and Langmuir-Blodgett films, PEMS manufactured with the
Layer-by—Layer (LbL) deposition technique have clear advantages such as the wide
variety of substrates that allow the growth of a polymer multilayer and control over the
multilayer properties. Glass, polymers and metals are being increasingly used as

substrates.

28

3.4 FABRICATION OF PEMS

Polyelectrolyte multilayers are typically assembled using a LbL process [46] (Fig 2.2)
involving iterative adsorption and rinsing steps. The substrate is dipped into the first
polyelectrolyte solution for a sufficient period of time to allow the polyelectrolyte to
adsorb to the surface. This is followed by a rinsing step that removes excess unbound
polymer. The substrate is then dipped into a polyelectrolyte solution of opposite charge,
which adsorbs to the surface due to electrostatic attraction and neutralizes the existing
surface charge, resulting in a reversal of the previous surface charge. The process is
repeated until the desired number of layers is deposited. The chemical structures of some

common polyectrolytes is shown in Fig 3.2.

The most common polycations used for the preparations of these multilayers includes
polyethyleneimine (PEI), poly allylarnine hydrochloride (PAH) and polydiallyl
dimethylammonium chloride (PDDA). Commonly used polyanions include poly sodium
4-styrenesulfonate (PSS), poly ethenesulfonic acid (PBS) and poly acrylic acid (PAA)

[68].

29

Ch“
H
\+N H
or H n
so; ml+
a) b)

c) (1)

Figure 3.2 Structures of common polyelectrolytes used in this study. a) PSS b) PEI c)
PAH d) PAA [68]

3. 4.1 Properties of pH tunable PEMS

A particularly versatile approach to PEM fabrication involves use of weak
polyelectrolytes, such as PAH and PAA. Varying the pH of the processing solution
changes the deposited thickness, morphology and swelling characteristics of the PEM
film [69]. However, one disadvantage of their pH dependence is that exposure to acidic
conditions breaks the electrostatic interactions between cationic and anionic layers
resulting in water soluble film [69]. A particularly useful method for stabilizing PAA and

PAH is by amide formation between the amine group of PAH and carboxylic group of

30

PAA multilayers is thermal [70] or chemical methods [71], resulting in cross-linked

films. After cross-linking, the films exhibit enhanced stability towards changes in pH.

Polyanion
wash

——>

 

Figure 3.3 A schematic of Layer-by-Layer deposition. The polyelectrolyte multilayers are
synthesized by a repeated charging of the surface with oppositely charged ions until a
desired number of layers is deposited. Each step results in a reversal of surface charge
allowing the next layer to be deposited [46].

3. 4.2 Properties of N-halamies grafted onto PAIIIAM dendrimers

In addition to PAA/PAH bilayered coatings with sporicidal agents, N-halamine
functional groups have been incorporated into polyamidoamine (PAMAM) dendrimers
that provide additional advantages over the conventional PEM. The n-halamines are

capable of regeneration by simply exposing the molecules to free chlorine, resulting in a

31

broad spectrum antimicrobial. Furthermore, the stability of N-Cl bonds on chlorinated
halamines contributes to the durability and stability of the antimicrobial properties of the
polymer [72]. N-halamines are inorganic and organic compounds in which oxidative
halogens are attached to nitrogen. In aqueous solution, chloramines and bromarnines
undergo hydrolysis in varying degrees to form hypochlorites and hypobromites, which
are sporicidal agents [72]. Monomeric N-halamines such as 1,3-dichloro-5,5-dimethyl
hydantoin and 3-bromo-l-chloro-5,5-dimethylhydantoin have been widely used as

disinfectants [72].

PAMAM dendrimers, unlike classic polymers, are known for their high degree of
molecular uniformity, specific size and shape characteristics, and a high density of
reactive surface groups. The number of chemical bonds through which the core can be
connected to the external parts of the molecule is defined as functionality of the core.
Through the bonds of the core, the layers of branching units (monomers) are attached to
the core. Analogous to preparing a PEM, the synthesis of a PAMAM dendrimer consists
of a series of repetitive steps starting with a central initiator core. If all of these branching
units are attached to the molecule perfectly, a dendrimer is formed. A large number of
“imperfections” results in a hyperbranched polymer structure (Figure 3.5). Each series of
steps produces a new "generation" with a larger molecular diameter twice the number of
reactive surface sites, and approximately twice the molecular weight of the preceding
generation [73,74]. In our project, the end group of a G4-PAMAM dendrimer
(Generation 4 PAMAM) was modified to possess a 5, S-dimethyl hydantoin. The G4

PAMAM- hydantoin could also be grafted on a multilayer of PAH/ PAA or PAH/PSS.

32

H o H O

\ /
N / N
03K 0/
i

H H

Figure 3.4 Structure of 5,5 dimethyl hydantoin and its halogenated derivative — 1,3
dichloro- 5,5, dimethyl hydantoin [72].

   

b)

Figure 3.5 Schematic representations of a a) dendrimer and b) a hyperbranched polymer
molecule. The core is represented by the grey molecule in the center with multiple
functional end groups [73].

A typical assembly of the polyelectrolyte coatings constructed for our experiments is
shown in Fig 3.6. After fabrication of the multilayers, the coatings were thermally cross
linked, and finally immersed in bleach solution for activation of the N-halamine

functional groups (if it was to incorporated into the PEMS). Unoccupied charged sites

33

within the interior voids of the PEM assembly are used to accommodate small molecules
such as CaDPA, L-alanine and L-inosine that can act as gerrninant molecules. The
coatings are expected encapsulate and deliver small germinant molecules to the surface of
entrapped spores. The amount of germinants that can be encapsulated in the coatings can
be controlled based on thickness of the PEM and the size of the dendritic layers. The
PEM are preferred over the dendrimer layers since PEMS are easy and inexpensive

compared to the dendrimer layers.
G4-PAMAM
hydantoin
’o .' 0 J—e—p PEM multilayers

Substrate

 

Figure 3.6 A diagram of PEM assembly as an antimicrobial surface [Personal
communication ~ Dr. Steven Kaganove]. Polyelectrolyte layers are coated onto the
substrate. A final layer is applied made of a PAMAM dendrimer with hydantoin
appended to the end-group.

34

CHAPTER 4 EXPERIMENTAL METHODS AND RESULTS

4.1 INTRODUCTION

LbL technique was used to synthesize a number of multilayer coatings where different
combinations of cationic and anionic polymers, number of bilayers and the presence or
absence of germinants and sporicidal agents were systematically varied. Most of the
coatings house germinants and sporicidal components in the “cargo spaces” of dendritic
polymers (G4PAMAM) or within multiple layers of polyelectrolytes. In few of these
coatings, L-Alanine/inesine is used as a germinant and N-halamines attached to end
group of PAMA dendrimer are used as sporicidal component. 96-well polypropylene
plates were used as substrates for antimicrobial surfaces. Each coating variant was tested

for antimicrobial efficiency and

4.2 MATERIALS AND METHODS

4. 2. 1 PEM construction materials

Polyacrylic acid (PAA) (MW = 240,000) was obtained from Acres Organic (Morris
Plains, NJ) as a 25% aqueous solution. Pelyallylarnine hydrochloride (PAH) (MW =
70,000) and Pelyethyleneimine (PEI) (MW = 25,000) were obtained from Sigma Aldrich
(St. Louis, MO). Generation 4 PAMAM (G4PAMAM) dendrimer was obtained from
Dendritech Inc. (Midland, MI) as a dilute methanol solution. All polyectrolytes with the
exception of G4PAMAM were used without further purification. G4 PAMAM used as
the starting material for the synthesis of hydantoin-PAMAM, was subsequently purified

by ultrafiltratien. Commercially available bleach was purchased in department stores. All

35

polyelectrolyte coating solutions were prepared as 10‘2 M solutions (based on the repeat
unit molecular weight) in Milli-Q water, and pH was adjusted to neutral pH with either
hydrochloric acid or sodium hydroxide. The PEM substrates used were black
polypropylene (PP) plates (96 wells, 360uL volume “round-bottom” plates), purchased

from Fisher Scientific (Pittsburgh, PA).
4. 2.2 Fabrication procedure

F or fabricating a PEM substrate, each PP plate was evenly cut into three equal pieces
with 32 wells in each piece. This was done to ensure immersion in approximately 175 mL
of coating solution. Polyelectrolyte multilayer (PEM) coatings were deposited on
polypropylene plates by alternating sequential immersion in 0.01M aqueous solutions of
cationic and anionic polyelectrolytes. The pH values of 0.01 M PAA and 0.01 M PAH
solutions were adjusted to 3.5 and 8.5 respectively. 10% household bleach solution (pH 7
or specified) was prepared fresh for coatings. The concentration of G4PAMAM
derivatives was 1mg/mL. Depending on the type of PEM composition, 500 mL of coating
solutions were prepared. The PEM films were deposited as thin films ever the PP surface
by manual dipping. Clean PP plates were immersed in a polyelectrolyte solution for 20
min, followed by several rinses in Milli-Q water. The substrates were then immersed into
the oppositely charged polyelectrolyte solution for 20 min and subjected to the same
rinsing procedure. After the desired number of layers was assembled, the substrates were
dried in air, and subsequently heated to 120 °C overnight for thermal cross-linking. The
crosslinking temperature was necessarily limited to 120 °C for the polypropylene

micreplates since they would melt at higher temperatures. Antimicrobial coatings were

36

fabricated by bleach immersion. Bleach immersed PEMS were obtained when the cross-
linked PEMS were immersed in 10% household bleach solution for 20 min followed by

the same rinsing procedure after 24 hours.

4. 2.3 Testing the coatings for antimicrobial efficiency.

The protocol used for testing the efficacy of these coating is described in Appendix B of
the thesis. An outline of the process is summarized in this section. Coatings containing
biocides or germinants will be at the optimum concentration when little to no water is
present. Thus, an aqueous suspension of the Bacillus anthracis spores (with pre-
determined CFU/mL) was applied onto the coated wells, and the suspension was dried
under vacuum. The plate was sealed for 24 hours. After the incubation time, the spores
were recovered using 200uL of 0.04%BSA/ 0.03% Thiosulfate solution. The 0.04% BSA
has been shown to aid in enhanced recovery of the spores from the coated wells
(Appendix C). To prevent transfer of residual bleach on the coatings to the BHI agar
plates, thiosulfate solution that neutralizes the residual chlorine was used. The diluted
suspensions of collected spores were plated onto BHI-agar plates (Appendix A) by
diluting the 200uL working stock 10,000 fold. All dilutions were plated to obtain

accurate spore counts.

4. 2.4 Calculating antimicrobial efliciency

The PEM coatings containing polymer layer (or G4-PAMAM) immersed in bleach were
tested for their efficacy in reducing the number of Bacillus anthracis spores (streme

strain) from a pre-determined initial concentration (measured in CFU/mL) after an

37

incubation period of 24 hours. The sporicidal coatings were tested for their perferrnances
by calculating the difference in logarithmic CFU/mL values between the sporicidal
coatings and the respective controls. Each sporicidal coating was compared to its
respective control for all the experiments. The sporicidal coatings differ fi'em their
control coatings only in the presence or absence of a sporicidal agent. A log difference of
at least 2 was expected for the sporicidal coatings, which indicates that the coatings
reduced the spore counts by 99% (See Appendix D for calculations). For statistical
analysis, an experiment is defined when the spores were incubated for 24 hours in a

single well of a 96-well polypropylene plate.

4.2.5 Fourier Transform Infrared (F T IR) spectroscopy

Five bilayers [PAA/PAH] films were built onto geld substrates for FTIR analysis. The
effect of additional PAA layer (as the topmost layer that would contact the spores) and
cross-linking induced by heat treatment of the PEMS at two temperatures (120°C and
180°C) on the absorption spectra of the PEM layers was studied. All IR spectra were
recorded using an ATI Mattson Research Series 1 spectrophotometer with a liquid

nitrogen-cooled MCT detector. Typically spectra were acquired using an average of 32

-1 ,
scans at a 4cm resolutron.

4.3 Results

4. 3. 1 [PAA/PAH]5 bilayers with varying topmost layer that contacm the spores

Coating variants that have at least five bilayers of [PAA/PAH] were fabricated (Table

4.1) and tested for sporicidal activity. Within this group, were five sets of coatings; each

38

with different variants as the topmost layers. Each of the multilayers had both control and
sporicidal variants. Each set also had two controls differing in the presence or absence of
germinants (L-alanine and inosine). The sporicidal coatings had either sporicidal agents
alone or a combination of germinants and sporicidal agent. It has been reported that
vegetative bacteria (germinated spores) are more susceptible to antimicrobial chemicals
compared to spores. Thus we tested if the presence of chemical germinants along with
sporicidal agents has any significant increase in sporicidal effect. The log (CFU/mL) for

each coating was plotted to estimate the perferrnance of the coatings.

Table 4.1 PEM Coatings tested with at least five [PAA/PAH] bilayers

 

Coating
ID

1A [PAA/PAH]5/ l 20°C
13 [PAA/PAH]5/120°C/ Ala+Ino
2A [PAA/PAH]5/120°C/NaOCI
2B [PAA/PAH]5/120°C/NaOCl/ Ala+Ino
3A [PAA/PAH]5/PAA/120°C
3B [PAA/PAH]5/PAA/120°C/ Ala+In0
4A [PAA/PAH]5/PAA/120°C/NaOCl
4B [PAA/PAH]5/PAA/ 1 20°C/NaOCl/ Ala+In0
5A [PAA/PAH]5/PAA/hydantoin PAMAM/120°C
SB [PAA/PAH]5/PAA/hydantoin PAMAM/120°C/ Ala+In0
6A [PAA/PAH]5/PAA/hydantoin PAMAM/120°C/NaOCl
6B [PAA/PAH]5/PAA/hydantein PAMAM/120°C/NaOCl/ Ala+Ino
7A [PAA/PAH]5/[PAA/hydantoin PAMAM]/PAA/ 120°C
73 [PAA/PAH]5/[PAA/hydantoin PAMAM]/PAA/120°C/ Ala+In0
8A [PAA/PAH] 5/[PAA/hydant0in PAMAM]/PAA/1 20°C/NaOCI
8B [PAA/PAH]5/[PAA/hydantoinPAMAM]/PAA/ l 20°C/NaOCl/ Ala-I-Ino
9A [PAA/PAH] 5/[PAA/hydantoin PAMAM]2/ 120°C
93 [PAA/PAH]5/[PAA/hydant0in PAMAM]2/1200C/ Ala+lno
10A [PAA/PAH]5/[PAA/hydantoin PAMAM]2/1200C/NaOCI
10B [PAA/PAH]5/[PAA/hydantoin PAMAM]2/120°C/NaOCl/ Ala+Ine

Coating

 

 

 

 

 

 

 

 

 

39

The coatings variants were tested to see a) if the presence of additional layers ever the
five bilayers of PAA/PAH and b) the presence of germinants contribute to significant
sporicidal activity (Figure 4.1 and 4.2). In all the figures representing spore count data,
the numbers above the sporicidal coating bars indicate the difference in log (CFU/mL)
from their corresponding control coatings. For example the number above 4A (2.64) is
the difference in log CFU/mL values between the control 3A (=6.46) and sporicidal

coating 4A (=3 .92).

D Control Coatings w/e germinants u Sporicidal Coatings w/o germinants
Control Coatings with germinants Sporicidal Coatings with germinants

 

-o.12 0&7»

 

 

log (CFU/mL)

 

 

 

 

 

 

 

 

Coating ID

Figure 4.1 Spore counts in PEM coatings containing atleast 5 bilayers of [PAA/PAH]
after 24 hours of exposure. Additional layers built over the five bilayers are represented
as X where X = PAA (in coatings 3A, 38, 4A, and 4B). After the layers were built both
the control and the sporicidal coatings were heat treated to introduce amide bonds
between PAA and PAH. Sporicidal coatings were immersed in 10% bleach selutien.The
control and sporicidal coatings containing germinants are patterned as indicated above
the plot. The set of coatings with “B” in the coating ID contain alanine and inosine
(germinants). Spore count data represent mean +/- SD. of three independent experiments.

40

a Control Coatings w/o germinants u Sporicidal Coatings w/o germinants
Control Coatings with germinants Sporicidal Coatings with germinants

 

X = PAA/PAMAM X=PAA/PAMAM/PAA X=[PAA/PAMAM]2

 

0.03 -0.33

 

 

log (CFU/mL)

 

   

\‘
t
s
s
s
s
s
at
s

 

 

 

 

 

 

5A 5B 6A 6B 7A 7B 8A 8B 9A 9B 10A 10B
CoatingID

 

Figure 4.2 Spore counts in PEM coatings containing atleast 5 bilayers of [PAA/PAH]
after 24 hours of exposure. Additional layers built over the five bilayers are represented
as X where X = PAA/PAMAM (in coatings 5A, 5B, 6A, and 6B), X =
PAA/PAMAM/PAA (in coatings 7A, 7B, 8A, and 8B) and X = [PAA/PAMAM]2 (in
coatings 9A, 9B, 10A, and 10B)b. After the layers were built both the control and the
sporicidal coatings were heat treated to introduce amide bonds between PAA and PAH.
Sporicidal coatings were immersed in 10% bleach solution to provide antimicrobial
properties. The control coatings are represented in blue color and the sporicidal coatings
are represented in pink color. The control and sporicidal coatings containing germinants
are patterned as indicated above the plot. The set of coatings with “B” in the coating ID
contain alanine and inosine (germinants). Spore count data represent mean +/- SD. of
three independent experiments.

PEM coating variants had atleast five bilayers of PAA/PAH and differed in the
composition of the top most layer (the layer that is in direct contact with the incubated
spores). In all these coatings tested, there is no significant difference between CFU values
for coatings with sporicidal entities in the presence or absence of germinant. Hence,
coatings variants for further experiments did not have any germinants. The sporicidal

coatings henceforth were immersed in 10% bleach solution. A two leg difference of

41

CFU/mL corresponds to 99% reduction in the number of spores (See Appendix D). Beth
coatings 4A and 48 had more than 2.5 log decrease in their CFU values, which

corresponds to more than 99% killing.

It was also observed that the sporicidal coatings that had PAA as the topmost layer
performed better than the sporicidal coatings that did not have PAA as the topmost layer.
Coating 4A and 4B (fiver bilayered PEM with PAA as topmost layer) showed
significantly higher reduction in log CFU values than coating 2A and ZB. Even in coating
variants containing PAMAM, the coating variant that had PAA as the topmost layer
perferrned better than the other coatings consistently, which lead to further experiments
testing whether the presence of PAA as the topmost layer affects the efficacy of the
coatings. Reduction in spore counts from some of the PEM coatings containing G4
PAMAM and treated with bleach was also observed. Also when comparing the bilayered
PAMAM (coating ID: 10A, 10B) coating with the monolayer ones (coating ID: 6A, 6B),
there is no significant difference between the reduction in log CFU values. This may be
because the bilayer may sequester some bleach in them which may contribute to their

lower killing potential.

4. 3.2 Effect of [PAA/PAH] and [ PSS/PAH] bilayers with PAA or PSS as topmost layer

and heat treatment

A subsequent batch of coatings (Table 4.2) compared three different parameters: effect of
PAA as the topmost layer, effect of the number of PAA/PAH bilayers and effect of heat

treating the coatings at 120°C. Most coating variants were heat treated to introduce an

42

amide cross-linking between the PAA and PAH layers, thereby providing additional
stability to the multilayers. In this set of coatings tested, even numbered experiments are
treated with bleach and the odd number experiments are the corresponding controls

without bleach.

Table 4.2 PEM Coatings with 2, 5 and 10 bilayers with PAA as topmost layer

 

 

 

 

 

 

 

 

 

 

 

 

Ceplgrn g Coating
1 [PAA/PAH]2/120°C
2 [PAA/PAH]2/120°C/NaOCl
3 [PAA/PAH]2/PAA/120°C
4 [PAA/PAH]2/PAA/120°C/NaOCl
5 [PAA/PAH]5/ 120°C
6 [PAA/PAH]5/120°C/NaOCl
7 [PAA/PAH]5/PAA/120°C
8 [PAA/PAH]5/PAA/120°C/NaOCl
9 [PAA/PAH]5
10 [PAA/PAH]5/NaOCl
1 1 [PAA/PAH]5/PAA
l2 [PAA/PAH]5/PAA/NaOCl
13 [PAA/PAH]10/120°C
14 [PAA/PAH]10/120°C/NaOCl
15 [PAA/PAH]10/PAA/120°C
l6 [PAA/PAH]10/PAA/120°C/NaOCl

 

These variant sets (1-4, 5-8, and 13-16) differ in the number of bilayers. With each subset
the coatings on the right (3, 4; 7,8; 15,16) have PAA as the topmost layer while the other
variants in the same subgroup do not have PAA as topmost layer. Increasing the number
of bilayers in the coatings that have PAA as the topmost layer had a significant increase
in the difference between the log (CFU/mL) values of the individual bleach treated and
control coatings. However, the difference was the highest in 5 bilayer coatings. Further

experiments were performed to analyze the effect of bilayers on sporicidal activity.

43

El Control Coatings fl Sporicidal coatings
E Control coatings + PAA E Sporicidal coatings + PAA
Y = number of PAA/PAH bilayers

 

 

log (CFU/mL)

 

 

 

 

 

 

 

 

 

 

Figure 4.3 Spore counts in PEM coatings without or with PAA as topmost surface that
contacts the spores with varying number of PAA/PAH bilayers. Spore count data
represent mean +/- SD. of three independent experiments. Spore counts in PEM coatings
with [PAA/PAH]y/X after 24 hours of exposure. Additional layers built over the five
bilayers are represented as X where X = PAA (for coatings 3,4,7,8,15,16). The patterned
bars represent the leg CFU values of coating variants that contain an additional PAA
layer apart from the [PAA/PAH]Y bilayers.

As shown in Figure 4.3, except for coating variants for which Y = 2, the sporicidal
coatings for Y = 5, 10 with an additional PAA layer perferrn better than the
corresponding sporicidal coatings without PAA. Thus the presence of PAA as the

topmost layer that contacts the spores has a significant effect on sporicidal activity.

In the following set of coatings tested (Figure 4.4), even numbered experiments are
treated with bleach and the odd number experiments are the corresponding controls

without bleach. All the coating variants have five bilayers of [PAA/PAH] with set #2 and

#4 have PAA as the topmost layer; while set #1 and #3 does not have PAA as the topmost

layer. Sets 1 and 2 were heat treated while sets 3 and 4 were not heat treated.

D Control Coatings (heat treated) n Sporicidal Coatings (heat treated)
iiiiiii Control Coatings (no heat) WSporicidal Coatings (no heat)

#3 #4

 

 

 

_0:037 . -70.71

 

 

 

 

 

 

 

 

 

 

 

S 6 7 8 9 l 0 1 1 1 2
Coating #

 

Figure 4.4 Spore counts in PEM coatings with 5 or 10 [PAA/PAH] bilayers differing in
heat treatment. Spore count data represent mean +/- SD. of three independent
experiments.

The above results suggest that the synergistic effect of the PAA and the heat treatment
provides the highest reduction in log CFU values as noticed in the coating 7,8 (Figure
4.4). Hence heat treated and bleach treated coatings act as a better antimicrobial surface

than the respective controls.

4.3.3 Effect of number of bilayers

In all coatings tested these with PAA as the topmost layer performed better than coatings

with different topmost layers. [PAA/PAH] coatings with five bilayers have the maximum

45

reduction in spore counts compared with two or ten bilayers. To more thoroughly probe
the optimum number of bilayers for maximum sporicidal effect on B. anthracis spores,
the following coatings were be fabricated and tested. The following set of coatings differ
in the bilayer composition (Figure 4.5). The coatings 1-6 have 3, 4, 6, 7, 8 and 9 bilayers
of [PAA/PAH] with PAA topmost layer and heat treated. The relationship between
sporicidal activity and thickness was evaluated, where two through ten PAH/PAA

bilayers were thermally crosslinked, chlorinated, and exposed to spores.

Table 4.3 PEM coatings with varying number of bilayers

 

 

 

 

 

 

 

 

 

Coating ID Coating
Set 1A [PAA/PAH]3/PAA/120°C
Set 113 [PAA/PAH]3/PAA//120°C/NaOC1
Set 2A [PAA/PAH]4/PAA/120°C
Set 213 [PAA/PAH]4/PAA//120°C/NaOCl
Set 3A [PAA/PAH]6/PAA/120°C
Set 313 [PAA/PAH]6/PAA//IZO°C/NaOCl
Set 4A [PAA/PAH]7/PAA/120°C
Set 48 [PAA/PAH]7/PAA//120°C/NaOCl
Set 5A [PAA/PAH18/PAA/120‘E
Set 5B [PAA/PAH]8/PAA//120°C/NaOCl
Set 6A [PAA/PAH]9/PAA/120°C
Set 6B [PAA/PAH]9/PAA//120°C/NaOCl

 

46

 

I Control I Sporicidal

770.702 I 0.53 0.78 0.67 0.42 0.50
3 4 6 7 8 9

Number of PAA/PAH bilayers

 

e >1
O O

3"
0

log (CFU/mL)
g» 4:.
o o

.N
o

1'"
O

 

 

.9
o

Figure 4.5 Spore counts in PEM coatings with varying number of bilayer. Spore count
data represent mean +/- SD. of three independent experiments.

The data for the sporicidal activity of 2, 5 and 10 bilayers of [PAA/PAH] with PAA as
the topmost layer that contact the spores were taken from Section 4.3.2. As shown in
Figure 4.6, the results Show an increase in activity from three to five bilayers, followed
by a sharp drop off from six to ten bilayers. Thus, in all the coating variants tested the
five bilayered [PAA/PAH] with PAA as the topmost layer, heat treated overnight at a

temperature of 120°C has the maximum sporicidal activity.

47

 

t—Ih—I—d
v—NAG

A log (CFU/mL)
bx be

COO

9.
NA

 

 

3 4 5 6 7 8 9 10
Number of bilayers

Figure 4.6 A plot showing the effect of varying the bilayers on sporicidal activity.
[PAA/PAH] coating variants with varying number of bilayers were tested for sporicidal
activity. The difference in sporicidal activity between the sporicidal coatings and their
respective controls were plotted against the number of bilayers.

4. 3.4 Coatings formulated with other common polyelectrolytes

Several coating variants with other polyelectrolytes such as PEI and HBPU
(hyperbranched polyurethane) were assessed for sporicidal activity against B. anthracis
spores. They are polymers with densely branched structure and a large number of end
groups. Unlike dendritic polymers which have completely branched star-like topologies,
the hyperbranched polymers have imperfectly branched structures (Figure 3.5). To verify
that the bilayers containing [PAA/PAH] with PAA as the topmost layer indeed perform

better, coating variants that PSS as topmost layer were fabricated and tested (Table 4.4).

48

Table 4.4 PEM Coatings [PSS/PAH] bilayers and [PAA/PAH] with PSS as topmost layer

 

 

 

 

 

Coating ID Coating
l [PSS/PAH]5/PSS
2 [PSS/PAH]5/PSS/NaOCl
3 [PAA/PAH]5/PSS/ 120°C
4 [PAA/PAH]5/PSS/120°C/NaOCl
5 [PAA/PAH]5/120°C/PSS
6 [PAA/PAH] 5/ 120°C/PSS/NaOCl

 

The set of coatings fabricated above (Figure 4.7) differ in the bilayer composition. The
coatings 1 and 2 have [PSS/PAH] as bilayers. The coatings 3-6 have [PAA/PAH] as the
bilayers but differ application off an additional PSS layer before or after heat treatment at
120°C. PEMS made of strong polyelectrolytes such as PSS, remain charged over the
entire range of pH unlike weak polyelectrolytes such as PAA. The degree of ionization of
weak polyelectrolytes depends on the pH of the solution which in turn influences the
thickness of the film [75]. For example Shirateri et al. fabricated PAA/PAH films of
varying, thickness by assembling them in different solution pH. The solution pKa is
defined as the pH at which half of the polyelectrolyte’s units are charged. The solution
pKa of PAA and PAH is 6.5 and 8.8 respectively [76]. The 6.5/6.5 PAH/PAA form thin
layers Since both the polyelectrolytes are fully charged and hence are tightly bonded to
each other with very few free acid or amine groups. In contrast, the 3.5/3.5 PAH/PAA
films are very thick due to formation of loops and tails. This is because at a pH of 3.5
PAA is not fully charged and hence does form a flat surface ever the underlying PAH

layer.

49

D Control Coatings E Sporicidal Coatings

 

 

 

 

 

4 _J_ [ms/3.3.1115 i E [PAA/PAH]5 :

62 I -042 55 [PSS/120C] [120C/PSS] :
' E E; —0.07 :

6 : :l ' ’ 1
€5.84 I if :
55.6 I ll :
in I I: I
95-44 I i: .
35.2 E ii :
4.8a : i: E

I || l

4.64: a dark I

: 1 2 E; 3 4 5 6 l

I _______________ :CoatmgID

Figure 4.7 Spore counts in PEM coatings. Spore count data represent mean +/- SD. of
three independent experiments.

Assaying for sporicidal activity in coating variants listed in Table 4.4 suggests that
substitution of PAA (a weak polyelectrolyte) with PSS (a strong polyelectrolyte) as the
top layer resulted in films with little or no activity against spores after chlorination. This
was regardless of whether crosslinking was induced after (Coating ID: 3,4) or before

(Coating ID: 5,6) PSS deposition.

A few other coating variants with four bilayers of [PAA/PAH] that differ in layers
applied above them (Table 4.5) were fabricated to assay for sporicidal activity against

Bacillus anthracis spores. All the coatings were heat treated at 120°C that cresslink the

50

polyelectrolytes. The coatings are divided into three sets that vary in layers assembled
above the four bilayers of [PAA/PAH]. Within each set, one half of coatings differ form

the other in the presence of PAA as the topmost layer that contacts the spores.

Table 4.5 PEM coatings with variants containing having PEI, G4PAMAM and HBPU

 

 

 

 

 

 

 

Coating ID C::tt1:g Coating

773-56-7 [PAA/PAH]4/PAA/PEI/120°C

773-56-8 1 [PAA/PAH]4/PAA/PEI/120°C/NaOCl

773-56-9 [PAA/PAH]4/PAA/PEI/PAA/120°C
773-56-10 [PAA/PAH]4/PAA/PEI/PAA/120°C/NaOCl
773-56-1 1 [PAA/PAH]4/PAA/G4PAMAM/120°C
773-56-12 2 [PAA/PAH]4/PAA/G4PAMAM/120°C/NaOC1
773-56-13 [PAA/PAH]4/PAA/G4PAMAM/PAA/120°C
773-56-14 [PAA/PAH]4/PAA/G4PAMAM/PAA/120°C/NaOCl
773-56-15 [PAA/PAH]4/PAA/HBPU/120°C
773-56-16 3 [PAA/PAH]4lPAA/HBPU/120°CfNaOC1
773-56-17 [PAA/PAH]4/PAA/HBPU/PAA/120°C
773-56-18 [PAA/PAH]4/PAA/HBPU/PAA/120°C/NaOCl

 

51

a Control Coatings n Sporicidal Coatings

PAA/PEI PAA/ PAA/ PAA/ PAA PAA/HBPU
PEI/PAA G4 G4/PAA /HBPU /PAA

0.49 0.25 .43 0.36 0.32 0.05

7.0

 

    

6.0

5.0

4.0

log (CFU/mL)

3.0

2.0

 

 

7, 8 9,10 11 12, 13,14 15,16 17,18
Coatrng ID

Figure 4.8 Spore counts in PEM coatings with varying topmost layer. Spore count data
represent mean +/- SD. of three independent experiments. These set of coatings have
four [PAA/PAH] bilayers and differ in the layer that contacts the spores.

The coating variants tested for sporicidal activity (Figure 4.8) does not seem to show
Significant Sporicidal activity. Even though certain coatings (coating ID 10, 14 and 18)
have PAA as the topmost layer they did not perform better than their comparative

sporicidal coatings do not have PAA as the layer contacting the spores.

4. 3.5 F T IR spectra analysis

FTIR spectra were obtained fiom PEM films deposited on gold substrates under identical
conditions. Films on coated substrates were cross-linked by heating overnight at either
120 °C or 180 °C. After thermal cross-linking, the coated substrates were chlorinated by

immersion in dilute bleach adjusted to pH 7 for 20 minutes. FTIR spectra of the

52

multilayer films on gold indicates that only a small degree of crosslinking takes place at
120 °C since there is little difference in the spectra before and after heating (Figure 4.9).
Significant changes are evident after heating at 180 °C. However, the coatings
crosslinked at 120°C also Showed significant Sporicidal activity (Table 4.5). Figure 4.10

Shows the carbonyl region between 1800 and 1300 cm".

 

 

0.08 ‘
l
0.04 ‘
a
g 0.00 <
=
a
a b
8
.r: -0.04 .
<
C
-0.08 .
4000 3000 2000 1000

Wavenumbers (cm")

Figure 4.9 FTIR-ERS spectra of [PAH/PAA]5 multilayer films on gold (a) as prepared,
(b) after heating at 120 °C overnight, and (c) after heating at 180 °C overnight.

: <——————-—- ccxy
012 ;

  
   

COOH
Q 008 l
O
5 a
.n
L:
8 0.04
a
rt b
AmideI
0.00 -

4— Amide II

 

 

1800 1600 1400
Wavenumbers (cm'l)

Figure 4.10 FTIR-ERS spectra of a [PAH/PAA]5 multilayer film on gold (a) aS-prepared,
(b) after heating at 120 °C evemight, and (c) after heating at 180 °C overnight.

Peaks due to the —COOH carbonyl and carboxylate, asymmetric and symmetric stretches
(1701, 1572 and 1402 cm") are visible in both Figure 4.10a and b, and there is little
apparent difference between them. The COOH peak at 1701 cm'1 is smaller than the
carboxylate peaks because the films were washed with pH 6-7 water after each
polyelectrolyte deposition step. This results in an equilibrium shift in unreacted primary
amine groups present in the films towards carboxylate which may even obscure the
amide peaks. The extremely broad peak in Figure 4.10a and 4.10b between 3550 and
2530 cm'1 iS characteristic of —NH3+ stretching in ammonium carboxylates. After the gold

substrate is heated to 180 °C the carboxylate peak at 1572 cm'1 and the ~NH3Jr stretching

54

peak diminishes, and amide peaks at 1552 and 1660 cm‘1 predominate.

Figure 4.11 Shows Spectra of five bilayer PAH/PAA films heated to 120 °C on gold
before (a) and after (b) immersion in NaOCl. An expansion of the carbonyl region

between 1800 and 1300 cm“ is shown in Figure 4.12.

 

 

0.08 2
q, .
U
=
N
.C
L
8 0.00-
I: .
< b
-0.04 -
4000 3000 2000 1000

Wavenumbers (cm-l)

Figure 4.11 FTIR-ERS spectra of a [PAH/PAA]5 multilayer film on gold. (a) After
heating at 120 °C evemight, and (b) after treatment with NaOCl.

After chlorination the carboxylate peaks become greatly diminished in size and shift to
1565 and 1409 cm", while the pretenated acid peak shifts to 1709 cm". The -NH3+
stretching peak between 3550 and 2530 cm'1 is also no longer present. These Shifts can be
explained by conversion of primary amine —NH functional groups to —NCl. The absence

of basic primary amines Shifts the pH balance of the films, and the majority of

55

carboxylate groups are new pretenated. Hydrogen bonding between -NH3+ and carbonyl
groups is also eliminated. The Spectra in Figures 4.11 and 4.12 do not indicate the
presence of acid chloride functional groups, which would be expected to have carbonyl
stretch in the 1790-1850 cm'1 region. In addition to this, a new small peak at 809 cm'1
appears in spectrum in Figure 4.11, which is the region where N-Cl stretching has been

shown to occur in some known N-Chloramide [76].

 

 

008 ' COO
COOH
o 004- l
U
: .
N
.C
$-
3 a
.C
< one
.b
604,
1800 1600 1400

Wavenumbers (cm-l)
Figure 4.12 FTIR-ERS Spectra expanded around the carbonyl region of a [PAH/PAA]5
multilayer film on gold. (a) after heating at 120 °C evemight, and (b) after treatment with
NaOCl.

4. 3. 6 Shelf life issues

The Sporicidal activity of “promising” coatings was monitored over a course of 6 months
to determine if there are any shelf-life issues. The Significantly higher biocidal activity of

[PAH/PAA]5/PAA against spores compared to other PEM coatings was demonstrated on

56

different batches of similar coatings ever a course of 6 months. Two Specific coating
variants tested on 23 February, 2009 (Coating ID 4A and 8A: Table 4.1) were retested

sporicidal activity on 23 November, 2009 (Figure 4.13).

D Control Coatings tested in Feb 2009 u Sporicidal Coatings tested in Feb 2009
[E Control Coatings tested in Nov 20mg} Sporicidal Coatings tested in Nov 2009

 

7 T 71‘ hag!

    

99.99.000.990:

log(CFU/mL)

3

99.9990990990099900999.9
0.9009900909900090...

0.9990099900090000...

0
9
9
9
9
0
9
0
0
9
9
9
9
0
9
0
9
0
0
9

0
9
0
9
9
9
9
0 .
9 0
9 .
. 0
9 9
0 0
0 9
9 9
0 9
0 9
9 9
0 0
9 9
9 9
9

0

9

0

9

0

9,

99

 

:2

9
09
099099.0990.990.909.90099909

 

:::.099990.909090009999009090

 

.9.9099.99009099909900999.99

 

A099009099990000999009

 

9

9

9

0

0

9

9 9
9 0
9 9
9 .
9 0
9 .
9 9
9 9
9 9
. 9
9 0
9 0
9 9
99 99
99 .0
09 9.
90

9.90.90.
0..
090

9

*0

 

 

 

 

Coating ID

Figure 4.13 PEM coatings tested for Shelf-life issues. Sporicidal coatings with five
[PAA/PAH] bilayers having either PAA or G4PAMAM/PAA as the topmost layer were
retested for Sporicidal activity after 6 months from initial testing. Spore count data
represent mean +/- SD. of three independent experiments. The color-filled and patterned
bars of same color for each coating variant represent the same coating variant tested in
February 2009 and November 2009 respectively.

As shown in Figure 4.13, both the coatings 4A and 8A showed significant sporicidal
activity (about 1.5 log reduction in CFU values). Coatings 3A and 7A are the
corresponding control coatings for the coatings 4A and 8A respectively. F and N refers

on the bars refers to February and November 2009 (the months the coatings were tested).

57

The coating variant that had G4PAMAM had higher sporicidal activity compared to the
variant with PAA as the topmost layer. This may be due to the sequestration of bleach
molecules by hydantoin molecules in the PEM, which results in better sporicidal activity

over a period of 6 months.
4. 3. 7 Conclusions

A number of PEM coatings were tested for sporicidal activity against Bacillus anthracis
steme spores. Coatings that reduced the spore counts by about roughly two legs
(equivalent to about 99% killing of the Spores) have been listed below in Table 4.5. The
difference in log (CFU/mL) values has been calculated, a difference of 1.5 or more is
taken as a measure of good sporicidal activity. The listed antimicrobial coatings could be
applied to “high infection rate areas” which are more prone to infection inside hospitals
(e. g. faucets, bed rails, elevator buttons, and electrical switches) as a preventive
decontarninant. These coatings are desirable because their reactive functionality can be

regenerated upon treatment with bleach.

58

Table 4.6 Coatings that has shown evidence of antimicrobial activity compared to the
control coatings (without bleach)

 

 

 

 

 

 

. Difference in log
CefaItDmg Coating CFU
4A [PAA/PAH]5/PAA/120°C/NaOCl (Figure 4.1) 2.64
[PAA/PAH]5/PAA/120°C/NaOCl/alanine&inosine
4B . 2.664
(Frgure 4.1)
[PAA/ PAH] 5/PAA/G4PAMAM/l 20°C/N aOCl
6A . 1.419
(F rgure 4.2)
[PAA/PAH]5/PAA/120°C/NaOCl (Figure 4.4) 1.529
8
[PAA/PAH]10/PAA/120°C/NaOCl (Figure 4.4) 1.134
16

 

 

 

 

 

The coatings could also be used in air filtration systems in research facilities and in
hospitals to prevent the spread of microorganisms. They are intended for use as a
preventive decontaminant as opposed to currently used pest decontamination agents.
Although the N-halamines that are used in these coatings may degrade ever time, they

can be regenerated by treatment with bleach.

4. 3.8 Future work

The thesis work has identified several promising coating variants that may be used in
“high infection rate areas”. Further work could better examine the sporicidal mechanism

of the germinated spores and whether the Sporicidal PEMS prevent biofilm formation.

59

This work was a pilot study in which the PEMS were fabricated on 96 well polyprelylene
plates using LbL method. To prove the versatility and efficiency of the LbL method,
sporicidal coatings may be fabricated on other materials such as glass and stainless steel
and assayed for sporicidal activity and biefilm growth. Substrates such as stainless steel
can heated to 180°C to introduce crosslinking in PEM bilayers and tested if there is any
significant improvement in reducing bacterial population compared to the substrates heat

treated at lower temperatures.

The protocol used in this thesis to test the coating variants for sporicidal activity tested
for sporicidal activity after 24 hours Since Significant sporicidal effect against Bacillus
anthracis Spores was observed only after 16 hours. Gerrninated spores have been shown
to be more susceptible to Sporicidal agents. Thus the activity of the sporicidal PEM
coatings could be tested with germinated spores. Furthermore, the sporicidal activity of
the PEMS could also be tested on common bacteria found in the hospital environment

such as E. Coli, S. epidermidis and S. aureus.

60

APPENDIX A

PROTOCOL FOR PREPARING BHI-AGAR PLATES

This protocol is used to prepare 500mL of the BHI agar solution, which Should always be

prepared in a 1L bottle, to prevent the solution from boiling off.

1. Add 18.5g Brain Heart Infusion (BHI) powder [Cat # 760 031, Biowerld] to 500mL of

sterile nanopure water.
2. Continuously heat and stir the solution until the solution starts to boil.

3. Add 7.5g agar of Bactoagar [Cat# 214010, BD] to the hot solution and stir

continuously until the agar is completely dissolved.
4. Sterilize the solution by auteclaving for 20 minutes.

5. Allow the solution to cool to room temperature, and pour it onto the desired culture

plates and cover the plates.

6. Transfer the plates to a biosafety hood and open the lids to allow solidification and

drying of the agar. Allow the plates to dry for no more than 10 minutes.

7. To store the plates for future use, cover the plates and keep refrigerated at 4°C.

61

APPENDIX B

PROTOCOL FOR SPORE INCUBATION IN PP PLATES AND FILTER
MEMBRANES

This protocol summarizes the steps to dry a layer of aqueous spore solution on a surface
coating, for determining the sporicidal qualities of the coatings being tested, which may

contain germinant or biocidal agents.

1. An aqueous solution of Spores containing 106 viable Spores is applied to a coated well

or 13 mm filter membrane. The total volume applied to a well in a 96 well plate is 50uL.
Immediately after application, the applied volume is re-pipetted to ensure that the well

bottom is covered.

2. Plate wells containing the 50uL spore solution are placed into a vacuum apparatus that
allows complete drying within 50 minutes (drying rate is about luL per minute). The
filter membranes are dried covered inside a Petri dish placed within a biosafety hood,

which takes about an hour to dry completely.

3. The optimum time required for the biocidal action is determined by setting up enough
wells or filter membranes to test a range of exposure times ( 2, 4, 6, 12, 18 and 24 hours).
Twenty four hours incubation time (~99% killing) was found to be optimum based on

initial experiments. Three replicates were performed per coating formulation.

4. Dried spores are recovered from the coated surfaces by application of
0.04%BSA/0.03% Sodium Thiosulfate solution. A well received 200uL and this voltune

is used to wash by pipetting repeatedly for 30 seconds. The BSA-Thiosulfate solution

62

forms bubbles, so the tip is irmnersed inside the well during the wash. Dried filter
membranes are placed in 1.5mL snap tubes and vortexed in 200uL BSA/Thiesulfate

solution for 30 seconds.

5. The washes are collected and 100uL is used to perform four lO-feld dilutions going
down to 10-3 dilution. BSA-thiosulfate solution is used to prepare the series dilution and

remaining volume from the wash is reserved.

6. 100uL of the dilution series and the remaining wash volumes are plated on pre dried
BHI-agar plates. The plates were spread with the spore solution using L-spreaders or loop

inoculation technique.

7. Incubate plates at 37°C, and enumerate colonies after 16-18 hours growth.

63

APPENDIX C

TEST TO DETERMINE THE SPORICIDAL COMPONENT IN THE
COATINGS

The coatings that we are currently testing are immersed in bleach to render the surface
sporicidal. We enumerate the Spores by plating them out on BHI-agar plate; hence the
spores may have residual chlorine that might prevent their normal growth even after the
Spores are washed with water. Hence we use BSA-Thiosulfate solution as the diluent
since the thiosulfate neutralizes the free chlorine ions and BSA washes out any spores
that get adhered to the surface of the coatings. This is essential to accurate determine if

the coatings are performing as expected.

An experiment was performed with selected coatings to determine the effect of BSA/
thiosulfate on spore recovery from coated wells and to determine optimum incubation
time to achieve about 98-99% antimicrobial activity. The spores were incubated in the
coated wells are diluted using sterile water for all experiments unless mentioned
otherwise. A batch of Spores was diluted using BSA/thiosulfate to test the effect of bleach

in the coatings. The coatings tested are listed below in Table Cl.

64

Table C.1 PEM coatings tested to determine sporicidal agent

 

 

Coating ID Coating
e [PAA/PAH]5/PAA/120°C
f [PAA/PAH]5/PAA/120°C/alanine&inesine
g [PAA/PAH]5/PAA/120°C/NaOC1
h [PAA/PAH]5/PAA/120°C/NaOC1/alanine&inosine

 

 

 

 

I 1 hour I 24 hour El 24 hour applied in BSA/Thio

LAJ>MO\\]OO

log(CFU/mL)

 

 

 

Coating ID g
Figure C.1 Spore counts in PEM coatings. Both the control and sporicidal coatings were
assayed for antimicrobial activity for 1 and 24 hours. When spores suspended in water
was used to determine sporicidal activity in coating g and h, the spore counts were
reduced by about 2 legs. However when the spores suspended in BSA/ thiosulfate

solution was used, the thiosulfate neutralizes the bleach in the PEM coatings and hence
no significant reduction in CFU values was observed.

There is no significant difference between the sporicidal coatings (g,h) and the control
(6,0 in 1 hour. However, 24 hour incubation produces about 2 log reduction in spore

counts in the sporicidal coatings. This effect is not observed when spores diluted in

65

BSA/thiosulfate solution were incubated in the test coatings for 24 hours. This is because
the thiosulfate neutralizes the chlorine ions released by the test coatings. BSA was used
Since spores sticking onto pipet tips can cause considerable errors while enumerating the

spores before and after applying them onto the wells of polypropylene plates (Figure A1).

 

Figure C.2 Spores sticking onto pipet tips could be visualized when stained with
methylene blue. a) pipet tip containing spores solution in water. The spores could easily
be seen sticking onto the inner surface of the pipet tip b) pipet tip was rinsed with water a
number of times to ensure that no spores were sticking onto the sides. However, some
spores could be seen sticking onto the surface 0) pipet tip was rinsed with BSA/
thiosulfate solution. The number of spores sticking onto the inner surface is considerably
less compared to the pipet tip rinsed with water.

66

APPENDIX D

CALCULATIONS FOR % REDUCTION IN CFU VALUES

All the results for bacterial spore counts in this thesis are presented as a log (CFU/mL).
An example has been shown below that summarizes calculations for determining the

efficacy of the coatings in reducing the number of viable spores.

Table D1 Table of actual spore counts obtained dming testing the coatings 3A-4B (Table

4.1)

 

 

 

 

 

Coating Duration (hr) # Spores CF U/mL log(CFU)

[D

358.5 3.59E+06 6.554

3A 24 455 4.55E+06 6.658

284 2.84E+06 6.453

270.5 2.71E+06 6.432

38 24 149.5 1.50E+06 6.174

390 3.90E+06 6.591

90.5 9.05E+03 3.956

4A 24 82 8.20E+03 3.913

75 7.50E+03 3.875

70 7.00E+03 3.845

48 24 80 8.00E+03 3.903

84 8.40E+03 3.924

 

 

 

 

 

 

 

Each coating was tested in triplicates. The number of spores obtained by counting the
colonies on BHI plate is the average of at least two different dilutions (See Apendix B for
complete procedure). For example the first CFU/mL value 358.5 is an average of 377

(Spore count from second 10-fold dilution) and 340 (Spore count fi'om third 10-fold

67

dilution). Each of these spore counts are converted to determine the individual CFU/mL.

The difference in log of the CFU/mL between the control and sporicidal coatings were to
determine the efficacy of the sporicidal coatings. For example, values obtained from
subtracting the leg (CFU/mL) values for Coating 4A from 3A; 4B from 38 were used to

determine if the sporicidal coatings are perferrning as expected.

Table D2 Actual spore counts for coatings 3A-4B

 

 

 

 

 

Coating ID Average Difference in log
log(CFU/mL) Calculations CFU
3A 6.555
3B 6.399
4A 3.915 =6.555-3.915 2.64
43 3.891 =6.399-3.891 2.508

 

 

 

 

 

 

The values 2.64 and 2.508 correspond to the decrease in log CFU values for the coatings
4A and 4B as shown in Fig 3.5. To convert the difference in log CFU/mL to percentage

reduction in number of viable spores, consider the following example:

. Let the initial CFU/mL be 1000 and the final CFU/mL be 10. This means that the

68

difference in the log (CFU/mL) values is log(1000) —- log (10) = 3-1 = 2.

Thus the percentage killing is = (initial — final) CFU/mL x 100 = 990*100/1000 = 99

 

initial

69

10.

11.

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