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CHARACTERIZATION OF TETRACYCLINE EFFLUX GENES
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ENVIRONMENTAL FACTORS CONTROLLING THEIR
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presented by

BRIAN MARK CAMPBELL

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CHARACTERIZATION OF TETRACYCLINE EFFLUX GENES IN SOIL
BACTERIA AND AN ANALYSIS OF ENVIRONMENTAL FACTORS
CONTROLLING THEIR EXPRESSION
By

Brian Mark Campbell

A THESIS

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

MASTER OF SCIENCE
Microbiology & Molecular Genetics

2008

ABSTRACT
CHARACTERIZATION OF TETRACYCLINE EFFLUX GENES IN SOIL
BACTERIA AND AN ANALYSIS OF ENVIRONMENTAL FACTORS
CONTROLLING THEIR EXPRESSION
By
Brian Mark Campbell
Un-altered tetracycline (TC) residues disseminate to soil environments through
the practice of applying manure to cropland as fertilizer from animals receiving sub-
therapeutic doses of tetracycline for growth promotion purposes. Such practice has
created concern that these residues are enhancing the development and transfer of TC
resistance in soil environments. However, multiple knowledge gaps exist that need to be
examined to address such concerns. First, few investigators have studied tetracycline
resistance in soils by molecular methods. Second, it is unknown if TC residues present in
the soil environment are bioavailable to microbial cells to exert a selective pressure.
Two separate sets of experiments were conducted to bridge these gaps. TC
resistance was examined in an agricultural soil, collected one week after manure
application by both culture dependent and independent methods. Novel findings include
the discovery of tet gene variants tet(30), and tet(31) in soil, and the first observations of
tet(A) in Microbacterium Spp., tet(Y) in Pseudomonas Spp., tet(C) and tet(3 l) in
Stenotrophomonas Spp., and tet(A) and tet(C) in Thermomonas spp.
In the second set of experiments bioavailability of cation chelated TC in solution

was examined with a tetracycline bioreporter containing a gfl) gene fused to a tetracycline

inducible promoter. Both TC-Mg2+ and TC-Ca2+ chelation significantly reduced TC

efflux gene expression whereas monovalent cations (N a+ and K+) had no effect.

This work is dedicated to my two grandfathers, George Campbell and Carl McLaughlin,

who instilled in me the value of knowledge and hard work.

iii

ACKNOWLEDGEMENTS

There are a number of people who deserve thanks for helping me throughout my
time as a graduate student. In no particular order I would like to thank the following
people affiliated with Michigan State University; Uri Levine, Zarraz Lee, Yujing Ding,
Erick Cardenas, J ie Deng, Woo Jun Sul, Yu Yang, Ryan Penton, Jeff Urquart, and Drs.
Jorge Rodriguez, Dion Antonopolous, Peter Bergholz, Stephan Gantner, Carlos
Rodriguez-Minguela, Hui Li, Brian Teppen, Robert Britton, Stephanie Eichorst, Deborah
Himes, Mary Beth Leigh, and Jennifer Auchtung. Without their expertise none of this
would have been possible. Special thanks go to Dr. Louis King, Dr. Jeff Landgraf, and
Shari Tjugum-Holland of the Research Technology Support Facility for their help with
various projects. My family, friends, and specifically Rachel Kanine are especially
deserving of thanks. Their continued support throughout my time as a graduate student
was essential. Credit for this work also goes to current and previous members of my
research guidance committee, Dr. Syed Hashsham, Dr. Jay Lennon, Dr. Thomas Schmidt,
and Dr. Vince Young. I am grateful to have been able to work with you. I would like to
thank the United States Department of Agriculture, and Michigan State University whose
financial support made this work possible.

Lastly, and most importantly, I would like to thanks Dr. James Tiedje. His
guidance and support as an advisor allowed me to grow as a student and a person and

made this work possible.

iv

TABLE OF CONTENTS

LIST OF TABLES ................................................................................. vii

LIST OF FIGURES .............................................................................. viii

CHAPTER I: TETRACYCLINES AND TETRACYCLINE RESISTANCE IN

THE ENVIRONMENT ............................................................................ 1
Discovery, Structure, and Mechanism of Action ................................................. 1
Applications of Tetracyclines ....................................................................... 1
Animal Husbandry ................................................................................. 2
Human Therapy .................................................................................... 3
Aquaculture .......................................................................................... 4
Plant Agriculture .................................................................................... 5
Development of Resistance to Tetracyclines ...................................................... 6
Mechanisms of Resistance ......................................................................... 6
Evolutionary Origins of Tetracycline Resistance Genes ...................................... 9
Horizontal Transfer ............................................................................... 21
Cross-resistance, Co-selection, and Fitness ...................................................... 23
Cross-resistance ................................................................................... 24
Co-selection ........................................................................................ 24
Fitness Effects ...................................................................................... 25
Dissemination of Resistance in the Environment ............................................... 26
Air Transport ....................................................................................... 27
Water Transport ................................................................................... 31
Aquaculture ...................................................................................... 31
Surface Water .................................................................................... 37
Ground Water .................................................................................... 41
Wastewater Treatment Plants ................................................................. 43
Terrestrial Transport ............................................................................... 45
Soil ................................................................................................ 45
Plant ............................................................................................... 49
Significance of Research ........................................................................... 51
Economic Impacts ................................................................................ 52
Knowledge Gaps .................................................................................. 53
Objectives ........................................................................................... 55
References ............................................................................................ 56

CHAPTER II: CHARACTERIZATION OF TETRACYCLINE EFFLUX PUMPS
IN A MANURED AGRICULTURAL SOIL BY BOTH CULTURE DEPENDENT

AND INDEPENDENT METHODS ............................................................ 78
Introduction ........................................................................................... 78
Materials and Methods .............................................................................. 81

Sample ............................................................................................... 81

Soil Microcosms .................................................................................. 81

DNA Extraction, Purification, Sizing, and Cloning .......................................... 82
Restriction Analysis and Library Storage ...................................................... 83
Construction of Control Strain EPI300 M-397 ................................................ 83
PCR Screen for Clone 397 ....................................................................... 84
Functional Screen of Soil F osmid Library and Reduction of Redundancy. . . . . . . . . . ..85
Sub-Cloning of Fosmids TCl, TC3, TC5, and TC6 .......................................... 88
Cultivation of Tetracycline Resistant Soil Isolates ........................................... 89
PCR of Tetracycline Resistance and 168 rRNA Genes ...................................... 90
Sequencing of Fosmid Ends and 16S rRNA Genes ........................................... 90
Results and Discussion ............................................................................. 91
F osmid Library Construction .................................................................... 91
Sequence and Function Based Screening for Cloned Tetracycline Resistance
Genes ................................................................................................ 91
Tetracycline Resistance Genes Present on F osmids .......................................... 92
Other Genes of Interest Isolated on F osmid Clones TCl — TC7 .......................... 101
Tetracycline Resistant Bacterial Isolates ..................................................... 102
Conclusions and Future Directions .............................................................. 110
References .......................................................................................... 1 16

CHAPTER III: AN ANALYSIS OF TETRACYCLINE — CATION CHELATION
AND ITS RELATION TO BIOAVAILABILITY AND TETRACYCLINE EFFLUX

GENE EXPRESSION ............................................................................ 121
Introduction ......................................................................................... 121
Materials and Methods ............................................................................ 128
Bacterial Strains and Culture Conditions ..................................................... 128
Promoter Activity Calculations ................................................................ 130
Analysis of Intracellular Tetracycline ......................................................... 130
Reagents and Chemicals ..................................................................... 130
Sample Preparation ........................................................................... 131
Solid Phase Extraction ........................................................................ 131
Liquid Chromatography-Mass Spectrometry .............................................. 132
Epifluorescent Microscopy ..................................................................... 132
Results and Discussion ............................................................................ 134
Verification of Functionality of Bioreporter .................................................. 134
Determining the Range of Linear Response in Promoter Activity at pH 5.0, 7.0 and
9.0 .................................................................................................. 134
Cation Chelation Effect on Promoter Activity ............................................... 136
Effect of Magnesium Chloride on Tetracycline Uptake .................................... 138
Modulations of Promoter Activity by Addition of Magnesium Chloride or EDTA
Mid-Culture ....................................................................................... 142
A Closer Comparison of the effect of Magnesium and Calcium at pH 7.0 .............. 148
Conclusions and Future Directions .............................................................. 154
References .......................................................................................... 162

vi

LIST OF TABLES

Table 1.1. Bacterial Isolates from Water Environments with Tetracycline
Resistance Genes .................................................................................... 10

Table 1.2. Bacterial Isolates from Soil Environments with Tetracycline Resistance

Genes ................................................................................................. 1 2
Table 1.3. Tetracycline Resistant Bacteria Isolated from Concentrated Animal

Feeding Operation (CAFO) Environments ................................................... 14
Table 1.4. Tetracycline Resistant Bacteria Isolated from Food .......................... 15
Table 1.5. Tetracycline Resistant Bacteria Isolated from Animal Sources ............ 16

Table 1.6. Tetracycline Antibiotic Concentrations found in Soils Under Field

Conditions ........................................................................................... 48
Table 2.1. Site # 21 Soil (Michigan Farm) ................................................... 79
Table 2.2. Primers Used .......................................................................... 86
Table 2.3. Fosmid Sequences with Homology to Genes Listed ......................... 100

Table 2.4. Tetracycline Resistant Colonies Cultured. C% is the confidence for genus
assignment with a 95% confidence threshold set for RDP’S seqmatch tool. Best type
match are the closest type culture relatives. S. S. is the similarity score. It corresponds to
the number of unique oligomers shared between the query and a given RDP sequence
divided by the lowest number of oligos in either of the two sequences. L is the length of
the partial 168 sequence ........................................................................... 112

Table 3.1. Liquid Chromatography Separation Gradient for Analysis of
Intracellular Tetracycline ...................................................................... 133

vii

LIST OF FIGURES

Figure 1.1. Pathways of tetracycline residues and resistant microbes to and between
environmental reservoirs. TCs indicates direct human input of tetracyclines into
primary reservoirs. Gray shading represents primary reservoirs whereas no shading
indicates secondary reservoirs which receive tetracyclines and resistant bacteria from
primary reservoirs ................................................................................... 28

Figure 1.2. Publications that Identified Tetracycline Resistance Genes in Bacterial
Isolates from Environmental, Food, and Animal Origin .................................. 50

Figure 2.1. Test of PCR Screening Sensitivity for Clone 397. EPI300TM host cells
containing the pCClFOSTM vector with a fragment of clone 397 were diluted with 99
randomly picked soil fosmid library clones to give positive to background ratios as

described. (a) 1 positive cell to 100 total cells. (b) 1/10th dilution of (a). (c) 1 positive to
1000 total cells. (d) 1/10th dilution of (c). (e) 1 positive to 10,000 total cells. (1) 1/10th

dilution of (e). (g) negative control including the 99 cells used as background. (h) 1/10th
dilution of (g). + cells = EPI3OOTM-397 cell lysate. + pCCFOSlTM-397 = vector DNA.
+ pCR4®-397 = original source of clone 397. — = negative PCR control. The ladder is
Invitrogen’s 100 bp ladder .......................................................................... 89

Figure 2.2. PCR Verification of Presence of Clones 397 and 492 in Soil DNA. 3%
Metaphor Agarose gel in 1X TAE stained with SYBR Safe. Ladder is Invitrogen’s 100
bp ladder. 1, 2, 3, & 4 refer to 4 separate soil DNA samples, + and — are positive and
negative controls respectively ..................................................................... 93

Figure 2.3. Verification of Presence of Clones 397 and 492 in Purified Soil DNA to
be Cloned. 3% Metaphor Agarose gel in 1X TAE stained with SYBR Safe. Ladder is
Invitrogen’s 100 bp ladder. 397 and 492 refer to sample DNA. + and — are positive and
negative controls respectively ..................................................................... 93

Figure 2.4. Average Insert Size of Soil Fosmid Library. Gel showing the EcoRI
restriction patterns of fourteen randomly chosen fosmid clones. The outermost ladder is
Invitrogen’s 7. phage digest ladder, the middle ladder is NEB’s Low Range PFG ladder
and the innermost ladder is Invitrogen’s 1 Kb DNA ladder. Average insert size equals
29.94 Kb. Library size was between 27,000 and 30,000 clones .............................. 94

Figure 2.5. Size and Tetracycline Minimum Inhibitory Concentration of Isolated
Fosmid Clones. The EcoRI restriction patterns, average insert sizes, and relative
minimum inhibitory concentrations for 7 tetracycline resistant fosmid clones isolated by a
functional screen. Clone T7 is also highly resistant to kanamycin ........................... 95

Figure 2.6. PCR Verification of the Presence of tet Genes on Fosmids. Each gene
amplified and the proper amplicon size is listed on each gel corresponding to various

viii

fosmids. The well to the right of each positive amplicon is a negative control (except for
tet(3l) which is to the left). The ladder in each gel is Invitrogen’s 1 Kb DNA ladder....98

Figure 2.7. Accumulation of Tetracycline Resistant Colonies on Various Media
Types ................................................................................................ 106

Figure 2.8. Accumulation of Colonies on LB Plates with 5 ug/mL Tetracycline.
Symbols represent different soil microcosm conditions. n/a refers to no tetracycline
added ................................................................................................ 107

Figure 2.9. Accumulation of Colonies on 1/10th R2A Plates with 5 pg/mL
Tetracycline. Symbols represent different soil microcosm conditions. n/a refers to no
tetracycline added ................................................................................. 108

Figure 2.10. Accumulation of Colonies on Soil Media Plates with 5 ug/mL
Tetracycline. Symbols represent different soil microcosm conditions. n/a refers to no
tetracycline added ................................................................................. 109

Figure 3.1. Epifluorescent Microscopy Images of the Tetracycline Bioreporter. a)
uninduced b) induced with 0.1 ug/mL TC and c) induced with 1.0 ug/mL TC ........... 126

Figure 3.2. Schematic of Bioreporter Cells. Tetracycline enters the cell, de-represses
the TetR repressor protein from the Ptet(A) promoter, and activates gypmut3 transcription.
When the GFP protein is translated and matures the cell gives off a fluorescent signal
proportional to the amount of tetracycline that entered the cell. ............................ 127

Figure 3.3. Fluorescent Images of pTGM Bioreporter in the Presence of Increasing
Magnesium Chloride Concentrations ........................................................ 135

Figure 3.4. Range of Linearity of Promoter Activity at pH 5.0. Triplicate samples,
error bars = 1 standard deviation ................................................................ 139

Figure 3.5. Range of Linearity of Promoter Activity at pH 7.0. Triplicate samples,
error bars = 1 standard deviation ................................................................ 140

Figure 3.6. Range of Linearity of Promoter Activity at pH 9.0. Triplicate samples,
error bars = 1 standard deviation ................................................................ 141

Figure 3.7. Promoter Activity Data for Magnesium at pH 5, 7, and 9. % promoter
activity on the Y axis, mM concentration of corresponding cation on the X axis.
Performed in triplicate, error bars represent 1 standard deviation ........................... 143

Figure 3.8. Promoter Activity Data for Calcium at pH 5, 7, and 9. % promoter

activity on the Y axis, mM concentration of corresponding cation on the X axis.
Performed in triplicate, error bars represent 1 standard deviation ........................... 144

ix

Figure 3.9. Promoter Activity Data for Sodium at pH 5, 7, and 9. % promoter
activity on the Y axis, mM concentration of corresponding cation on the X axis.
Performed in triplicate, error bars represent 1 standard deviation ........................... 145

Figure 3.10. Promoter Activity Data for Potassium at pH 5, 7, and 9. % promoter
activity on the Y axis, mM concentration of corresponding cation on the X axis.
Performed in triplicate, error bars represent 1 standard deviation ........................... 146

Figure 3.11. Promoter Activity of Fully Induced pTGM (0.1 ug/mL TC) versus
pH .................................................................................................... 147

Figure 3.12. Promoter Activity of Cells Grown in 0.1 ug/mL TC (White Bar) and
0.1 ug/mL TC + 10 mM Magnesium Chloride (Black Bar). Error bars represent one
standard deviation .................................................................................. 149

Figure 3.13. Intracellular TC Concentration (Measured by LC-MS/MS) of Cells
Grown in the Presence of 0.1 ug/mL TC (White Bar) or 0.1 pg/mL TC + 10 mM
Magnesium Chloride (Black Bar). Error bars represent one standard deviation ....... 150

Figure 3.14. Addition of 10 mM Magnesium Chloride and 10 mM EDTA Mid-
Growth. Experiment done in triplicate, error bars represent one standard deviation. . . 152

Figure 3.15. Comparison of Effects of Calcium and Magnesium on Promoter
Activity. The black square and triangle represent the theoretical cation concentration to
achieve a 70% reduction in promoter activity based on equation 1 (Calcium) and 2
(Magnesium). Error bars represent one standard deviation .................................. 155

Images in this thesis are presented in color.

CHAPTER I

TETRACYCLIN ES AND TETRACYCLINE RESISTANCE IN THE
ENVIRONMENT

DISCOVERY, STRUCTURE, AND MECHANISM OF ACTION

In 1948 the first tetracycline compound, aureomycin, was isolated from a soil-
dwelling Actinomycete, Streptomyces aureofaciens (Duggar, 1948). Soon after in 1950
Finlay and coworkers isolated terramycin, the first tetracycline to have its structure
solved, from S. rimosus (Durckheimer, 1975). The tetracyclines are four-ring molecules
with partial conjugation and a carboxyamide functional group (Sarmah, Meyer, & Boxall,
2006). Tetracycline has three distinct functional groups: tricarbonyl methane (pKa 3.3);
dimethyl ammonium cation (pKa 9.6); and phenolic diketone (pKa 7.7) (Sarmah et al.,
2006). There are a number of structural features important for antibacterial activity.
These include the (a) stereochemical configurations at the 4a, 12a (A-B ring junction),
and 4 (dimethylamino group) positions, and conservation of the ketO-enol system (11, 12,
and 12a) in proximity to the phenolic D ring (Chopra & Roberts, 2001). For a review of
tetracycline structures see (Oka, Ito, & Matsumoto, 2000). This compound and other
derivatives inhibit bacterial translation by binding to the 308 ribosomal sub-unit. More
Specifically, tetracyclines exert their bacteriostatic effect by binding and inhibiting
accommodation of amino-acyl tRNA into the A Site (Mitscher, 1978). The tetracyclines
have a broad antibacterial spectrum, broader than any other known antibiotic at their time
of discovery, and as such they were quickly applied for a variety of purposes.

APPLICATIONS OF TETRACYCLINES
It has been estimated that total antibiotic consumption worldwide lies between

100,000 — 200,000 tons (Kummerer, 2003). Worldwide tetracycline (TC) usage

encompasses a wide variety of purposes including human therapy, aquaculture, animal
husbandry, and plant agriculture. Accurate estimations of antibiotic usage are
notoriously difficult to obtain, however it is known that agricultural purposes represent a
large percentage of usage. For example, it has been estimated that 70% of the 16 million
kg of antibiotics used in the United States (U .S.) is for non-therapeutic purposes (Sarmah
etaL,2006)

Especially concerning is the fact that developing countries, which account for
25% of world meat production, have poor or absent policies regarding antibiotic use in
agriculture (Witte, 1998). In African countries such as Tanzania and Uganda veterinary
antimicrobials are easily accessible and under low levels of control from government
authorities (Sarmah et al., 2006). Worldwide usage of tetracyclines has created an
environment which is potentially enriching tetracycline resistant (TcR) bacteria. The
foundation for understanding this problem begins with an understanding of the ways in
which tetracyclines are commonly used.

Animal Husbandry. Tetracyclines are commonly used in animal husbandry for
purposes of prophylaxis, chemotherapy, and growth promotion. The bulk of tetracycline
usage in animal husbandry is for growth promotion, hence the focus of this discussion.
Growth promotion refers to the practice of mixing antimicrobials, such as tetracyclines,
into animal feed in order to rear a larger, healthier animal. The growth promoting
benefits of tetracyclines were first discovered in 1949 when chickens fed by-products of
tetracycline fermentation were found to grow faster than those not fed the antibiotic
(Phillips et al., 2004). After recognition of the beneficial effects, their application to

other animals quickly ensued. Today the greatest usage of tetracyclines is likely in swine

husbandry. In the United States there are 60 to 92 million swine and 40% are reported to
be fed chlortetracycline (CTC) for ~ 2 months during the production cycle (Sarmah et al.,
2006). The benefits of supplementing antibiotics into feed during swine production occur
throughout the entire growth stage of the pig, however, the greatest benefit is in young
pigs with an average growth rate improvement of 16.4% and an average increase in feed
utilization efficiency of 6.9% (Cromwell, 2002). Overall, animals with antibiotics in
their feed gain 4-5% more weight than those without (Witte, 1998).

Surprisingly the basis for why tetracyclines and other antibiotics achieve these
increases is still not well understood. Four mechanisms have been proposed for how
antibiotics improve animal growth: 1) inhibition of sub-clinical infections 2) reduction of
microbial metabolites that reduce animal growth 3) reduced utilization of nutrients by
microbes instead of the host animal and 4) nutrient uptake enhancement through thinner
intestinal walls associated with antibiotic fed animals (Gaskins, Collier, & Anderson,
2002)

Human Therapy. The most obvious use of the tetracyclines, in the eyes of the
public, is use in hmnans to treat infectious diseases. In the United States five
tetracyclines are used for human therapeutic purposes — demeclocycline (DMC),
doxycycline (DXC), minocycline (MCLN), oxytetracycline (OTC), and TC (Smilack,
1999). In 1997 wholesale cost of all TC prescriptions filled in US. retail pharmacies
totaled $400 million (Smilack, 1999).

Use in human therapy has generally declined because of increasing pathogen
resistance (Chopra & Roberts, 2001), however, there are still situations in which the

tetracyclines are useful. Tetracyclines are still the drug of choice for infections with

Chlamydia trachomatis (nongonococcal urethritis, pelvic inflammatory disease, and
lymphogranuloma venereum), Rickettsiae spp. (rocky mountain spotted fever, and
endemic and epidemic typhus Q fever), Borrelia recurrentis (Brucellosis, Lyme
borreliosis, Ehlrichosis, and Relapsing fever), Vibrio spp. (cholera), Helicobacter pylori
(gastritis and peptic ulcer disease), and Plasmodium falciparum (malaria) (Chopra &
Roberts, 2001; Smilack, 1999). Tetracycline is also still used for treatment of acne (Ross,
Eady, Cove, & Cunliffe, 1998).

There has also been development in new applications of tetracyclines. The
discovery the tetracycline can inhibit prion infectivity, infections associated with
Alzheimer’s disease and bovine Spongiform encephalopathy (mad cow disease), has
sparked interest in nontraditional uses (Tagliavini et al., 2000). The most recent
development in tetracycline therapy is the approval of tigecycline, a third generation
tetracycline derivative (more specifically a glycylcycline), for human therapy (Shlaes,
2006)

Aquaculture. In a span of 15 years, annual aquaculture production increased
from 16.8 million tons in 1990 to 52.9 million tons in 2005, and is expected to reach 172
million tons by 2015 (A. Sapkota et al., 2008). OTC is commonly used to treat a wide
range of bacterial infections in aquaculture. It is actually the most commonly used
antibiotic in aquaculture (A. Sapkota et al., 2008), with 92% of the top 13 aquaculture
producing countries employing its use. In the US, FDA approved uses for OTC include
treatment of disease in Pacific salmon (250 mg/kg*day for 4 days), salmonids (2.5-3.75
g/1001b*day for 10 days), catfish (same as salmonids), and lobster (1 g/lb medicated feed

for 5 days) (Shao, 2001). In countries other than the US. commercial Shrimp feeds are

commonly enriched with OTC (Graslund & Bengtsson, 2001). There are three major
routes of administration; water treatment, incorporation into feed, and injection (Cabello,
2006)

Unfortunately, due to few antimicrobials approved for aquaculture in Norway and
North America “off-label” usage can occur. “Off-label” refers to treatments at higher
doses than approved, by a route not approved, treatment for a non-approved disease, or
treatment of non-approved species (Burka et al., 1997). Furthermore, in Asian countries,
which account for the bulk of world aquaculture production, there are few if any
guidelines for OTC usage beyond residuals for exported products (A. Sapkota et al.,
2008)

Plant Agriculture. A minor but Significant use of tetracyclines is in the control
of plant pathogens. The potential of antimicrobials to treat plant disease was recognized
as early as the 19505 (McManus, 2000). OTC is registered in the US. for use on pear for
control of Erwinia amylovora which causes fire blight, and on peach and nectarine for
control ofXanthomonas arboricola, the causative agent of bacterial spot (McManus,
Stockwell, Sundin, & Jones, 2002). In Mexico, OTC is used for E. amylovora on apples
and in Latin America OTC is used on vegetable crops (McManus et al., 2002). OTC has
also been used to control disease caused by phytoplasmas (McManus, 2000). Application
rates of OTC are 3 gallons of 150 ppm solution per tree or 240 gallons per acre for
peaches and at 200 ppm are 50 — 100 gallons of solution per acre for pears (V idaver,
2002). The application rate for peaches can be increased for larger trees but is not to

exceed 500 gallons per acre per application (Vidaver, 2002).

In the US, total antibiotic usage on plants ranges from 20,000 to 65,000 kg
annually, which represents less than 0.5% of the total antibiotic production (McManus et
al., 2002). In 1999, the USDA estimated that 40% of pear acreage received 12,000 lbs of
OTC and apples received 3,000 lbs of OTC on 5% of the acreage (V idaver, 2002).
Throughout the 19903 OTC usage increased significantly, mainly due to increased
streptomycin (the other antibiotic commonly used in plant agriculture) resistance in E.
amylovora (Vidaver, 2002).

DEVELOPMENT OF RESISTANCE TO TETRACYCLINES

The development and spread of resistance to tetracycline includes the evolution of
antibiotic resistance traits and the subsequent horizontal transfer to diverse microbiota.
Below I provide an overview of the mechanisms of tetracycline resistance, followed by a
discussion of the evolutionary origins that have resulted in the diversity of resistance
genes today. This is followed by a discussion of horizontal transfer mechanisms that
have allowed for the dissemination of resistance genes throughout the bacterial domain
(Tables 1.1 to 1.5).

Mechanisms of Resistance. There are at least four types of resistances to
tetracycline; tetracycline efflux, ribosomal protection, enzymatic inactivation, and
ribosomal RNA mutation (Chopra & Roberts, 2001). For up to date information
regarding the TC resistance genes see Dr. Marilyn C. Roberts’ website:

http://faculty.washington.edu/marilynr/

The tetracycline efflux genes export tetracycline in an energy dependent manner
from the cytoplasm thereby detoxifying the cell by reducing the intracellular

concentration (Chopra & Roberts, 2001). Currently 23 tetracycline efflux genes have

been identified, the most recent of which was tet(41) found in an environmental strain of
Serratia marcescens (Thompson, Maani, Lindell, King, & McArthur, 2007) (Table 1.1).
Generally, the tetracycline efflux genes are found in Gram-negative bacteria, the
exceptions being tet(A), tet(L), tet(K), tet(3 3), tet(Z), tetA(P), and otr(B).

The tetracycline efflux genes are regulated by a repressor protein, TetR, which
controls expression of the efflux pump as well as its own expression (Chopra & Roberts,
2001). Upon binding a TC-Magnesium complex, TetR undergoes a conformational
change allowing release from two operators that overlap the divergently orientated
promoters for both the efflux pump, Tet(A), and the repressor protein, thereby allowing
mRNA transcription to proceed (Chopra & Roberts, 2001). This regulatory system is the
most sensitive, effector-inducible transcriptional regulation system described to date
(Chopra & Roberts, 2001 ).

Ribosomal protection proteins (RPPS) represent the second most diverse
mechanism of tetracycline resistance based on current data. RPPS mitigate tetracycline’s
effect on the bacterial cell by dislodging TC from the ribosome in a GTP dependent
manner (Connell, Tracz, Nierhaus, & Taylor, 2003). Currently there are 11 recognized
classes of RPPS. G+C% of sequence data suggests that the RPPS originated in Gram-
positive bacteria (Chopra & Roberts, 2001). The RPPS are well distributed throughout
both Gram-positive and Gram-negative organisms. Seven of the eleven RPPS (tet(M),
tet(O), tet(S), tet(W), tet(32), tet(Q), tet(36)) have been described in gram-negative
bacteria (Tables 1.1 — 1.5).

Regulation of the expression of RPPS is not well understood; however, it is known

to differ Significantly from that for the efflux pumps. It is speculated that RPPS are

regulated by translational (or transcriptional) attenuation of mRNA (Chopra & Roberts,
2001).

Enzymatic inactivation has not been as extensively characterized as the previous
two mechanisms of resistance. Currently only three enzymatic inactivation genes are
described, tet(X), tet(34), and tet(3 7). Interestingly, each of these genes appears to be
unique in their mechanism of resistance. The tet(34) and tet(37) genes are recently
discovered and hence, are not as well described. tet(34), originally isolated in Vibrio
Spp., encodes a protein that has homology to bacterial xanthine-guanine
phosphoribosyltransferases (N onaka & Suzuki, 2002). The tet(3 7) gene was cloned from
an oral metagenome, and is not homologous to tet(X), but appears to have a similar
mechanism of resistance (Dial-Torres et al., 2003).

The tet(X) gene was originally discovered in a Bacteroidesfragilis strain (Guiney,
Hasegawa, & Davis, 1984) but was inactive due to its requirement for oxygen to
function. When transferred into aerobically grown Escherichia coli, tet(X) was able to
detoxify tetracycline (Speer & Salyers, 1988). It was later shown that the Tet(X) protein
is a flavin dependent monooxygenase requiring FAD, NADPH, Magnesium, and Oxygen
to detoxify tetracyclines by regiospecifically hydroxylating carbon 11a which results in
this product breaking down inside of the cell (Speer, Bedzyk, & Salyers, 1991; W. Yang
et al., 2004).

The fourth major mechanism of tetracycline resistance is provided by bacteria acquiring
mutations in their ribosomal RNA (rrn) genes. In the gram-positive bacterium
Propionibacterium acnes, the suspected causative agent of acne, a G to C mutation at E.

coli equivalent base 1058 occurring in helix 34 of the rrn gene was shown to confer

resistance to tetracycline (Ross et al., 1998). The gram-negative bacterium Helicobacter
pylori has been shown to accumulate mutations conferring tetracycline resistance in the
rrn gene at base pairs 965 — 967 in helix 31 (Dailidiene et al., 2002), and at base pairs
926 — 928 (Gerrits, de Zoete, Arents, Kuipers, & Kusters, 2002). It is likely that these
mutations are Similar in that they all, either directly or indirectly, affect the binding of
tetracycline to the ribosome. This claim is supported by crystal structures of tetracycline
bound to the 303 ribosomal subunit showing interaction with both the helix 31 and 34
loops of the 16S rRNA (Brodersen et al., 2000).

Evolutionary Origins of Tetracycline Resistance Genes. Multiple biosynthetic
pathways for antibiotic synthesis have been estimated at being between 200 and 800
million years old (Wright, 2007). If antibiotics are ancient then it can be postulated that
resistances to those antibiotics are ancient as well. Combine this with the recent
knowledge that horizontal transfer is a major contributor in evolutionary processes
(Koonin, 2003) and it is not surprising that resistance to tetracycline was discovered in
Shigella dysenteriae only 5 years after the introduction of the antibiotic for human
therapy purposes (Chopra & Roberts, 2001). This raises an interesting but simplistic
question: Where did these genes come from?

It is generally thought that there are two main routes through which antibiotic
resistance has evolved: (a) evolution of resistance in antibiotic producers as self
protection and (b) in other microbes as protection against antibiotics produced by the
organisms described in (a) (Wright, 2007).

Most clinically relevant antibiotics are produced by soil dwelling actinomycetes

(D'Costa, McGrann, Hughes, & Wright, 2006), therefore, one may expect that these

 

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17

microbes represent a reservoir of antimicrobial resistance genes. In fact, this hypothesis
was first put forth by Walker & Walker in 1970 (Walker & Walker, 1970). D’Costa et a1.
(2006) recently tested this hypothesis. They screened a library of approximately 400
Streptomyces Spp., which produce over half of all known antibiotics, isolated from
various soil environments for their ability to evade 21 different antibiotics representing
natural, semi-synthetic, and fully synthetic antibiotics that act on eight major bacterial
targets. On average strains were resistant to between seven to eight drugs and two strains
were resistant to 15 of the 21 tested (D'Costa et al., 2006). Sixty percent were resistant to
tetracycline, 1% to minocycline, and 30% to tigecycline (D'Costa et al., 2006), indicating
that soil microbes represents a reservoir for resistance even to drugs that have been
recently introduced. Some of this resistance to tetracycline could indicate novel genes.
More evidence that soil microbes are a reservoir of tetracycline resistance genes was
provided by Dr. Handelsman’s group when they isolated a novel tetracycline efflux gene,
tcr, from a metagenomic library with DNA extracted and cloned from a pristine soil
sample (Riesenfeld, Goodman, & Handelsman, 2004).

The above studies demonstrate that Walker & Walkers’ postulation that antibiotic
producing organisms harbor antibiotic resistance genes is in fact true, however, they do
not provide an answer to the origins and mechanisms by which tetracycline resistance
genes developed. Multiple groups have addressed the question of describing the origins
of both the tetracycline efflux and the ribosomal protection genes.

The tetracycline efflux genes known today have 12 and 14 transmembrane
sequences (TMS) (Paulsen, Brown, & Skurray, 1996). Today’s efflux genes may be

ancestral to a 6-TMS gene that once had the physiological important function of

18

transporting sodium and potassium out of the cell (Guillaume, Ledent, Moens, & Collard,
2004). This hypothesis is based on two observations. First, the gram-positive
tetracycline resistance genes tet(K) and tet(L) confer low levels of resistance to
tetracycline and also can catalyze transport of sodium and potassium ions, and second,
the observation that a truncated Tet(K) protein can still transport potassium (Guillaume et
al., 2004). If present day gene variants are ancestors of ancient ion pumps, then at some
point, preference for tetracycline exclusion must have evolved. Monophyletic origin of
the tetracycline efflux pumps has been demonstrated and it has been suggested that the
specificity towards tetracycline appeared once, around the time they separated from the
multi-drug efflux sub-cluster, and was maintained despite considerable sequence
divergence resulting in present day gene variants (Aminov et al., 2002).

Considerably more work has been done regarding the evolution of the RPP genes.
Sanchez-Pescador and colleagues first showed that the tet(M) gene has significant
sequence identity, which implies homology, to the translation elongation factors EF-G
and EF-Tu (Sanchez-Pescador, Brown, Roberts, & Urdea, 1988). Since then, multiple
groups have worked to untangle the origins of these genes.

The observation has been made that, based on available sequences, there is no
evidence for recent transfer of RPPS from antibiotic producing strains to other bacteria
(Aminov, Garrigues-Jeanjean, & Mackie, 2001; Lau, Woo, To, Lau, & Yuen, 2004).
Indeed it appears that ancestral RPPS were present before divergence of the three
superkingdoms, and before the evolution of tetracycline production, and that EF-G and
the RPPS share common ancestry (Aminov et al., 2001; Kobayashi, Nonaka, Maruyama,

& Suzuki, 2007). Hence, the current theory is that the translational elongation factors

19

EF-G and EF-Tu shared a common ancestor with the present day RPPS. Over time
specificity towards tetracycline developed in the RPPs and was maintained. Further
evidence, besides considerable sequence identity in the N-terminal region of the two
genes, is that both the translational elongation factors and the RPPS hydrolyze GTP in a
ribosome dependent manner (Connell et al., 2003).

Recent recombination events between tet(O) and tet(W) genes in Megasphaera
elsdem'i and separately in the tet(M) gene variants has further driven the evolution of
RPPS (Oggioni, Dowson, Smith, Provvedi, & Pozzi, 1996; Stanton & Humphrey, 2003;
Stanton, McDowall, & Rasmussen, 2004). It is tempting to speculate that perhaps
recombination events such as this could provide for higher levels of resistance and for
resistance to newer tetracycline derivatives such as tigecycline which are active against
bacteria harboring traditional tetracycline resistance genes.

In summary, soil-dwelling antibiotic producers as well as native soil flora
represent reservoirs of tetracycline resistance genes. Both tetracycline efflux and RPP
genes have long evolutionary histories, with ancestry common to physiologically
important functions that likely predate the evolution of tetracycline biosynthesis. This is
a striking illustration of the function of what Gerard D. Wright has termed the “antibiotic
resistome”. The resistome contains all resistance genes, including those in pathogens and
non-pathogens, cryptic embedded resistance genes, such as tet(X) described above in
Bacteroides Sp. (Speer & Salyers, 1990), and precursor genes, as evidenced by genes
ancestral to present day tetracycline efflux genes and RPPS, that given sufficient time and
appropriate selective pressure could evolve resistance fimctions (Wright, 2007). This is

not a static system, but an ever-evolving reservoir which with sufficient time, and

20

through horizontal transfers, allows diverse phyla to become resistant to nearly any
antimicrobial compound.

Horizontal Transfer. Aminov et al. (2001) speculated that the rapid movement
of RPPS to taxonomically divergent bacteria is probably attributable to horizontal
transfer. They also suggested that specificity toward tetracycline in efflux pumps
appeared once and was maintained (Aminov et al., 2002). It follows from this
assumption that horizontal transfer is also responsible for the dissemination of
tetracycline efflux genes. This begs the question: How do tetracycline resistance genes
transfer to diverse bacteria?

There are three mechanisms that allow for horizontal transfer of DNA. These
include conjugation, transduction, and transformation. Conjugation is a process in which
two bacteria mate and DNA is transferred from one bacterium to another by the transfer
mechanisms of a self-transmissible DNA element (generally a plasmid) (Snyder &
Champness, 2003). Transduction refers to a process in which DNA is transferred
between two bacteria by a phage. There are two types of transduction, generalized and
specialized. In generalized transduction essentially any region of bacterial DNA can be
transferred from one bacterium to another (Snyder & Champness, 2003). Transfer via
specialized transduction is limited to genes located close to the attachment site of a
lysogenic phage in the bacterial chromosome (Snyder & Champness, 2003). Lastly,
transformation is the process in which bacterial cells take up DNA from the environment.

Horizontal dissemination of genes is further enhanced by mobile elements such as
transposons and integrons that can both act in a bacterial chromosome or inside a plasmid

framework. A transposon is a DNA sequence that can move from one location in a

21

bacterial chromosome to another, or to another DNA element present in the same cell
such as a plasmid (Snyder & Champness, 2003). A special type of transposon, a
conjugative transposon, contains genes that code for its own transfer (Snyder &
Champness, 2003) and can also mobilize other elements in cis or trans. Integrons are the
most recently described agent of genetic change. They are assembly platforms that can
incorporate exogenous circular DNA (gene cassettes; containing a single gene and a 3’
attachment site) through site-specific recombination and express them by their outward
orientated Pc promoter (Mazel, 2006). Integrons can be present in a chromosome (super-
integrons) or on mobile elements such as conjugative plasmids and transposons (Mazel,
2006).

The gram-negative tetracycline efflux genes are typically present on transposons
inserted into diverse plasmids (Roberts, 2003). With respect to tetracycline resistant
environmental isolates, multiple groups have found tet(A) located in the non-conjugative
transposon Tn1721 or Tn] 721-like transposons in diverse plasmids from bacterial
isolates from untreated hospital effluent (Rhodes et al., 2000), the normal flora of swine
(Sunde & Scrum, 2001), and Michigan apple orchards (Schnabel & Jones, 1999). Less
work has been done with regarding the Gram-positive TcR efflux genes in environmental
isolates, however, they are typically found on small plasmids (Chopra & Roberts, 2001).

Generally speaking, the RPPS tet(S) and tet(O) have been found on conjugative
plasmids or the chromosome whereas tet(M) and tet(Q) have been found on conjugative
transposons (Chopra & Roberts, 2001). The tet(M) gene is often associated with
Tn916/Tn1545-like conjugative transposons (Agerso, Pedersen, & Aarestrup, 2006;

Billington & J ost, 2006; De Leener, Martel, Decostere, & Haesebrouck, 2004; Wilcks,

22

Andersen, & Licht, 2005). The Tn916/Tn1545-like transposons are the most
promiscuous conjugative transposons described, and have a host range including both
Gram-positive and Gram-negative genera (Roberts, 2005). Dr. Marilyn Roberts has
hypothesized that the host-range of the mobile genetic element carrying a given tet gene
will ultimately determine its distribution throughout the bacterial superkingdom (Roberts,
2003). This hypothesis appears to have support as tet(M) is one of the more broadly
distributed resistance genes in bacterial isolates of environmental origin being found in
31 genera (Tables 1.1 — 1.5).

Unfortunately most work regarding horizontal transfer of these genes has been
done with isolates of clinical origins. Publications in which the authors have
characterized resistance genes as well as the mobile genetic elements on which they
reside in environmental isolates are conspicuously absent from public databases.
Especially absent is an understanding of the role of integrons in tetracycline resistance.
Work in this area represents a knowledge gap that is necessary to fill to have a
comprehensive view of the fate of tetracycline resistance genes in environmental
compartments. Understanding this dynamic will allow a better judgment of the risk
associated with the contamination of environments with tetracycline residues.

CROSS-RESISTANCE, CO-SELECTION, AND FITNESS
Multiple studies have shown that a bacterium carrying a plasmid with an
antibiotic resistance gene is at a fitness disadvantage to an isogenic progenitor without
the plasmid in the absence of antibiotic selective pressure (Nguyen, Phan, Duong,
Bertrand, & Lenski, 1989). However, it is also commonly observed that in multiple

environments, upon withdrawal of antibiotic usage resistance levels remain high (Ghosh

23

& LaPara, 2007; Langlois, Cromwell, Stahly, Dawson, & Hays, 1983). Clearly there are
non-obvious factors that help maintain tetracycline resistance in environmental settings
after it has developed.

Cross-resistance. Cross-resistance refers to the ability of one gene to encode
resistance to multiple compounds. Cross-resistance is problematic because genes that can
confer resistance to diverse compounds can be selected by the selective pressure of only
one compound. With reference to tetracycline the best examples are multidrug efflux
pumps. Multidrug efflux pumps (MDE) are membrane bound pumps which confer
resistance to a wide array of compounds. One such example is the acrAB operon in E.
coli. Expression of this operon increases with increasing concentrations of tetracyclines
(Viveiros et al., 2007). Indeed the acrAB MDE pump has the ability to confer resistance
to tetracycline as well as other compounds (Elkins & Nikaido, 2002). Pseudomonas
aeruginosa has an MDE pump, the mexXY-oprM system, that is induced and exports
tetracycline among other compounds (Poole, 2005). Shewanella oneidensis MR—l also
contains a similar pump that confers slight resistance to tetracycline (Groh, Luo, Ballard,
& Krumholz, 2007).

Co-selection. Co-selection can occur when two separate genes conferring
resistance to different compounds are resident on the same mobile genetic element.
Selective pressure provided by one compound can select for one resistance gene and also
maintain another co-resident, non-selected gene. Observance of this phenomenon has
been fairly common with TcR elements and examples in the literature are numerous. For
example, the conjugative plasmid R478 contains genes for both mercury and tetracycline

resistance (Gilmour, Thomson, Sanders, Parkhill, & Taylor, 2004). An IncF plasmid,

24

pRSB107, isolated by a culture independent approach from a sewage-treatment plant
encodes nine different antibiotic resistance genes (Szczepanowski et al., 2005). Schnabel
& Jones (1999) observed that 100% of the TcR phylloplane bacteria analyzed from a
Michigan apple orchard were linked with the streptomycin phosphotransferase genes
strB-strA. These three studies exemplify the increasing awareness of the role of co-
selection in tetracycline resistance gene dissemination.

Fitness Effects. As mentioned above, and as has been demonstrated with
tetracycline resistance on a plasmid, in the absence of antibiotic, carriage of a plasmid
results in reduced fitness (Nguyen et al., 1989). Given this situation, one might expect
that with antibiotic withdrawal and sufficient time the plasmid would be cured and
resistant bacteria would once again become susceptible. However, Dr. Richard Lenski’s
elegant work has shown that this may not always happen. Given sufficient generations,
compensatory mutations can occur in cells which negate the ill effects of plasmid
carriage. In fact, the tetracycline resistance gene (tet(B)) residing on the plasmid actually
provided a competitive advantage to host cells that had acquired compensatory
mutation(s) which allowed carriage of the plasmid without reduced fitness effects
(Lenski, Simpson, & Nguyen, 1994). Another study by Dr. Lenski’s group revealed that
due to the tight regulation of the tet(B) tetracycline efflux gene on the Tn] 0 transposon
by the TetR protein, carriage of Tn] 0 essentially poses no burden on the microbial cell
(Nguyen et al., 1989). It is important to keep the scale in perspective as this study
focused on the tetracycline gene and not the plasmid as a whole. The fitness
disadvantage of a plasmid must be the sum of all fitness effects of each gene resident on

that particular plasmid (Lenski et al., 1994).

25

Another mechanism by which bacterial fitness can be enhanced by carriage of
genes that have the ability to confer resistance to tetracycline is if that gene can provide a
physiological benefit to the host organism. As stated above in the evolution section, the
Gram-positive tetracycline efflux genes tet(L) and tet(K) can transport sodium and
potassium ions (Krulwich, Jin, Guffanti, & Bechhofer, 2001). Recently, Groh and
colleagues (2007) were able to show that (a) the mexF gene in S. oneidensis MR-l is
associated with increased fitness in sediment environments and (b) this gene is required
for resistance to tetracycline. Both of these examples demonstrate the recent realization
that other factors are contributing to the maintenance of resistance in natural
environments.

DISSEMINATION OF RESISTANCE IN THE ENVIRONMENT

The evolution of tetracycline resistance genes and mechanisms to horizontally
transfer them between diverse phyla, along with the anthropogenic use of tetracyclines,
and non-antibiotic selective pressures has likely contributed to the environmental
dissemination of tetracycline resistant bacteria. How much impact humans have had on
this process is hotly debated. The goal here is not to debate this issue, but to provide an
overview of the major pathways through which this dissemination occurs.

The various uses of tetracycline antibiotics including human therapy, animal
husbandry, aquaculture, and plant agriculture were described above. Inevitably
tetracycline residues and resistant microbes make their way to the environment. Figure
1.1 shows the anthropogenic use of tetracyclines including the primary and secondary
reservoirs of tetracyclines, resistance genes, resistant bacteria, and the major paths in

which they can be transferred from one environment into another. Primary reservoirs

26

refer to environments that receive direct input of tetracyclines through human activity
whereas secondary reservoirs refer to environments in which accumulation of
tetracyclines occurs due to transfer from a primary reservoir. Figure 1.1 illustrates a
cycle through which the development, maintenance, and transfer of tetracycline
resistance genes could potentially be enhanced.

Evidence for tetracycline resistant microbes residing in different environmental
compartments is provided by Tables 1.1 — 1.5 which shows results of a comprehensive
literature review of genera that have been isolated from specific environments and which
tetracycline resistance genes they harbor. Currently, empirical evidence may be lacking
for some of the pathways presented in Figure 1.1. It is not intended to be a concrete
presentation of every pathway, but rather, a guide in thinking about alternative pathways
that may exist and have yet to be researched. Presented below is a discussion of relevant
research in the major environmental reservoirs and pathways exhibited in Figure 1.1. The
intention with this discussion was not to be comprehensive, but to illustrate the
representative studies that have identified these pathways as contributors to spread of
tetracycline resistance.

Air Transport. The major input of tetracycline residues and resistant microbes
into the air is likely through transfer from feces of animals that have been treated with
TCs, especially air indoor concentrated animal feeding operations (CAF Os). A recent
empirical investigation of airborne particulate matter sampled for a 20 year period from a
swine CAFO found that TCs were present in samples 12 out of 20 years (Hamscher,
Pawelzick, Sczesny, Nau, & Hartung, 2003). Concentrations of TCs in particulate matter

ranged from 0.2 to 5.2 mg/kg with a mean of 0.81 mg/kg. Maximum concentrations were

27

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28

found to be; 1.1 mg OTC/kg, 5.18 mg TC/kg, and 2.12 mg CTC/kg (Hamscher et al.,
2003). The author’s speculate that the drug residues originate from two sources, dried
liquid manure particles from animal waste and dry powder animal feed supplemented
with antibiotics.

Numerous studies have implied that TC resistant bacteria can become airborne
through manure at CAF Os. To the best of the author’s knowledge only one study has
identified tetracycline resistance genes in such samples. The tetracycline resistance genes
tet(K), tet(L), and tet(M) were found in various combinations in Enterococcus spp. and
Streptococcus spp. isolates from air inside a swine CAFO (Table 1.3). Chapin et al.
(2005) isolated Enterococcus spp., coagulase negative staphylococci, and viridians group
streptococci from air inside a CAF O facility, 90% of which were resistant to tetracycline.
Almost all of the tetracycline resistant bacteria also had evidence of cross-resistance to
one or more antibiotics including erythromycin, clindamycin, and virginiamycin (Chapin,
Rule, Gibson, Buckley, & Schwab, 2005). Another study by Gibbs et a1. (2004)
investigated external air both upwind and downwind of a swine CAFO that administered
oxytetracycline via feed. They found that microbes isolated inside or downwind of the
facility showed resistance to OTC whereas microbes isolated upwind never exhibited
resistance. Furthermore, at one facility while swine were present in the barns 86%
(50/56) were OTC resistant. When air was sampled downwind of this same barn after it
was emptied, cleaned, and disinfected only 9% (5/57) of isolates were OTC resistant
(Gibbs, Green, Tarwater, & Scarpino, 2004). In a follow up study Gibbs et a1. (2006)
showed once again that tetracycline resistant bacteria were significantly more dense in air

up to 150 m downwind of a CAF O than upwind. Staphylococcus aureus, an important

29

human pathogen, represented 76% of the total bacteria isolated including samples from
both inside and outside of the facility (Gibbs et al., 2006).

With sufficient wind carriage airborne TC resistant bacteria or particulate matter
sorbed with TCs could easily be transferred to human and animal hosts, as well as water,
plant, and soil environments (Figure 1.1). Deposition onto water, plants, and soil, or
causation of animal infection by airborne tetracycline resistant bacteria from a CAF O
would likely be difficult to determine. Not surprisingly there is a lack of published
studies that have attempted such experiments. Separating natural background resistance
from acquired resistance would likely prove to be especially problematic. However,
Gibbs and co-workers finding that statistically similar amounts of TC and OTC resistant
bacteria were found both inside and 150 m downwind of a swine CAFO strongly implies
that aerial deposition onto the above mentioned environments is inevitable (Gibbs et al.,
2006)

Many groups have attempted to establish a link between exposure to food
production animals receiving antibiotics and antibiotic resistance in the human flora. It is
difficult to establish a cause and effect relationship due to uncontrollable variables;
however, such studies do provide compelling evidence that transfer of airborne resistant
bacteria to farm workers is likely taking place. One study examined the resistance of
bacteria isolated from 113 farm workers versus 113 non-farm workers and found that
tetracycline resistant enterobacteria and Escherichia coli were isolated more frequently in
pig farmers (Aubry-Damon et al., 2004). Another study found that slaughter plant
workers are at higher risk of exhibiting multidrug resistant Escherichia coli than non-

swine workers (Alali et al., 2008). Abigail Saylers’ group found that the ribosomal

3O

protection protein gene tet(Q) has transferred between animal commensal Prevotella spp.,
human colonic Bacteroides spp., and human oral Prevotella spp. strains (N ikolich, Hong,
Shoemaker, & Salyers, 1994). Despite not being able to establish a direction of transfer,
they were able to show a recent transfer of tet(Q) between a human oral strain of
Prevotella spp. and a human colonic strain of Bacteroidesfragilis (N ikolich et al., 1994).
This suggests a link for how tet(Q) could transfer between animal ruminant Prevotella
spp. and Bacteroides spp. resident in the human colon.

Water transport

Aquaculture. The most commonly recognized way in which tetracycline residues
and resistant organisms can enter water environments is via aquaculture. As was
mentioned above in the tetracycline usage section, OTC is used for therapeutic purposes
in aquaculture and is the most commonly used antibiotic in this industry (A. Sapkota et
al., 2008). Often the antibiotic is mixed directly into the water phase with feed at
concentrations ranging from 50 to 2,000 ppm (Shao, 2001). Another, perhaps more
intensive, way in which tetracyclines can reach aquaculture environments is through
integrated fish farming, a practice in which manure from livestock production is used as
feed in fish ponds (Petersen, Andersen, Kaewmak, Somsiri, & Dalsgaard, 2002). Asian
countries, which account for 94% of global aquaculture production, have a history of
administering wastewater, animal waste, and human waste to fish ponds (A. Sapkota et
al., 2008). Any co-resident TCs with the waste are transferred to the aquaculture
environment. These residues can bioaccumulate in cultured species, be deposited to
sediments (which can be as high as 40% of medicated feed (Capone, Weston, Miller, &

Shoemaker, 1996)), excreted via feces (as high as 90% can be excreted (Cravedi,

31

Chouber, & Delous, 1987)), and move from sediment back into the water column through
dissolution (Graslund & Bengtsson, 2001).

An abundance of studies have traced OTC residues in cultured species and have
found maximum muscle concentrations after OTC medication of between 4 and 20 ppm
(Gomez-Jimenez, Espinosa-Plascencia, Valenzuela-Villa, & del Carmen Bermudez-
Ahnada, 2008; Namdari, Abedini, & Law, 1996; Nogueira-Lima, Gesteira, & Mafezoli,
2006; Uno, Aoki, Kleechaya, Tanasomwang, & Ruangpan, 2006) depending on the
species, water environment conditions, and amount of antimicrobial administered. One
study revealed concentrations reaching as high as 10,050 ppm in hemolymph tissue of
cultured shrimp (Gomez-Jimenez et al., 2008). In the United States, Japan, and European
Union, regulatory agencies have set maximum residue limits of 0.2 ppm, 0.2 ppm, and
0.1 ppm, respectively, (Gomez-Jimenez et al., 2008) for OTC residues in edible food
tissues brought to market. Generally farmed fish are given adequate withdrawal time to
allow clearance of OTC from the animal. However, these three regions represent only
4% of world aquaculture production (A. Sapkota et al., 2008) and guidelines for
antibiotic usage in developing countries, which account for the bulk of world aquaculture
production, are poorly established. Due to this lack of information, combined with
globalization, it is unclear to what extent contaminated aquaculture products are reaching
the table in many countries. For example, in 1991 Japanese health authorities found
unacceptable levels of OTC in farm-raised shrimp imported from Thailand (Graslund &
Bengtsson, 2001). Similarly, in 1993 the Thai Medical Sciences Department found that

24% of shrimps exported contained OTC residues (Graslund & Bengtsson, 2001).

32

Ultimately some OTC residues accumulate in sediments below aquaculture cages.
This process is dependent on four dominant factors: 1) the total amount of OTC added 2)
the percentage of OTC that reaches the sediment 3) the area of sediment over which OTC
is deposited and 4) the depth of the sediment through which OTC is distributed (Coyne et
al., 1994). Concentrations found in marine sediments have ranged from 1 to 300 ppm
and likely depend upon the characteristics of the local environment. Kerry et al. (1994)
found peak OTC concentrations of 9.9 ppm with a half-life of 16 days in sediments below
fish cages. At a depth of 8 cm OTC was still detectable by HPLC 19 days after cessation
of medication, however, 33 days afterwards it was undetectable (Kerry et al., 1994).
Samuelsen et a1. (1992) found much higher OTC concentrations in sediments. After 10
days of OTC medication sediment concentrations ranged from 25 ppm to 300 ppm and
stayed stable for 75 to 200 days at these concentrations. Residues were detectable even
after as long as 550 days and persisted in sediment depths of 2 — 8 cm for at least 245
days at concentrations of approximately 30 ppm (Samuelsen, Torsvik, & Ervik, 1992).
Furthermore, immediately after 10 days of feeding, 100% of culturable bacteria were
OTC resistant. This percentage decreased during the first 75 days post medication and
stabilized at a level of 10 to 50% for at least 550 days. Surprisingly few studies have
recently been conducted on the persistence of OTC in aquaculture sediments. This is an
area open to investigation. Similarly, no studies were found in which investigators
measured soluble water concentrations of OTC in aquaculture cages. These could be
appreciable considering that modeling studies estimated that 10 — 15% of OTC would be
released as a pulse to receiving waters during medication and the first 5 days after (Rose

& Pedersen, 2005).

33

Multiple studies have shown that OTC present in the upper layers of sediment can
dissolve back into the water column (Capone etal., 1996; Hektoen, Berge, Honnazabal,
& Yndestad, 1995). In one study boxes filled with sediment spiked with 200 ppm OTC
were placed at a depth of 15 m in seawater (Hektoen et al., 1995). It was found that the
OTC concentration decreased in the upper 2 cm from 200 ppm to approximately 30 ppm
over the course of more than 200 days, however, OTC in the 6 to 7 cm range stayed at
relatively high levels being measured at approximately150 ppm after more than 200 days
(Hektoen et al., 1995). The half-life of OTC in 0 to 1 cm sediments was 151 days
compared to more than 300 days in the 5 to 7 cm depth (Hektoen et al., 1995). Smith and
Samuelsen (1996) used a modeling approach to predict that OTC concentrations in the
bottom 1 cm of the water column would range between 0.016 ug/mL and 0.11 ug/mL
depending on the sediment concentration. They further assumed that since previously
approximately 10% of total OTC in seawater is bioavailable, the predicted bioavailable
concentrations would range from 0.0016 ug/mL to 0.011 ug/mL (Smith & Samuelsen,
1996)

Tetracycline resistance in water environments is currently best characterized in
aquaculture environments. Out of 115 unique isolate/ gene combinations present in the
literature from water environments 71 (62%) were isolated from aquaculture sources
(Table 1.1). The two genera isolated harboring the most tet genes are Aeromonas spp.
and Vibrio spp. likely because both are important fish pathogens. Other genera identified
to contain more than three different tet genes include Escherichia spp. (11), Citrobacter
spp. (7), Pseudomonas spp. (7), Acinetobacter spp. (4), Photobacterium spp. (4), and

Plesiomonas spp (4). The bulk of resistance genes found in aquaculture environments to

34

date are efflux genes (Table 1.1). It is unknown whether this bias is due to the type of
bacteria typically present in aquaculture environments, since efflux genes are historically
more commonly associated with Gram-negative bacteria, or if the bias is due to lack of
screening for RPPS. Nonetheless, aquaculture isolates continue to be a significant
reservoir for tetracycline resistance genes exemplified by the discovery of new
tetracycline gene variants. Three more recently discovered genes, tet(31), tet(34), and
tet(39), discovered in 2000, 2002, and 2005, respectively, were isolated from Aeromonas
salmonidicz'a, Vibrio spp., and Acinetobacter spp. from aquaculture sources (Agerso &
Guardabassi, 2005; L'Abee-Lund & Sorum, 2000; Nonaka & Suzuki, 2002).

Few studies have specifically dealt with temporal and spatial dynamics of OTC
resistance in aquaculture environments. As mentioned above, in some countries
guidelines are established requiring a certain period of drug free rearing to keep OTC
residues out of edible fish tissues. However, antibiotic resistant bacteria may persist in
aquaculture environments after the cessation of medicated feeding (Samuelsen et al.,
1992). This persistence creates an environment that could allow for the transfer of
resistance between bacteria or the clonal expansion and dissemination of already resistant
bacteria. In fact, resistance to CTC and OTC appears to be a worldwide phenomenon in
aquaculture environments, as it has been reported in China, India, Japan, Philippines,
Indonesia, South Korea, Bangladesh, Thailand, Chile, Norway, the United States, and
Taiwan (A. Sapkota et al., 2008).

Kim et a1. (2004) took a closer look at a regional examination of OTC resistance
in aquaculture environments in Japan and Korea. Interestingly, they were able to see a

contrasting distribution in Vibrio spp. isolated from healthy fish at the same location and

35

sampling time. In one set of healthy fish Vibrio spp. isolates were positive for the tet(M)
gene and had a specific 16S rDNA RFLP pattern (Kim, Nonaka, & Suzuki, 2004).
Another set of healthy fish isolated at the same time and location harbored Vibrio spp.
isolates that were negative for tet(M) and had a different 16S rDNA RF LP profile (Kim,
Nonaka, & Suzuki, 2004). This indicates that within the same farm differences can exist
in the diversity of TC resistance genes on a microenvironment scale. Also, the
observation was made that Vibrio spp. harboring tet(M) genes that shared the same 16S
rDNA RFLP pattern while also lacking the tet(S) gene and a marker for Tn1545-Tn916-
like transposons were isolated from healthy fish and seawater at two separate locations in
Korea as well as in diseased fish in Japan (Kim et al., 2004). It is possible that there was
a clonal expansion and dissemination of this Vibrio spp. strain throughout the region.

Resistance can also be prevalent in sediment environments below aquaculture
sites. Petersen et al. (2002) investigated the temporal occurrence of OTC resistance in
Acinetobacter spp. and Enterococcus spp. in an integrated fish farming environment.
They found that there was a significant development of resistance during the first 2
months after fish production was initiated. Resistance to OTC in Acinetobacter spp. rose
to 100% of cultured isolates, which is interesting because OTC was not fed to the broilers
whose manure was used in the fish farm, indicating the probable co-selection of OTC
resistance genes (Petersen etal., 2002). In contrast, Enterococcus spp. showed no
significant change in resistance (Petersen et al., 2002).

The persistence of OTC resistant bacteria in the environment raises the concern
that resistance could transfer to clinically relevant pathogens. Furushita et a1. (2003)

found that sequences of tet(C), tet(D), and tet(Y) from aquaculture isolates shared 100%

36

sequence identity to those of clinical strains. Furthermore strains of Photobact‘erium
spp., Vibrio spp., Alteromonas spp., and Pseudomonas spp. could transfer their resistance
determinants to E. coli via conjugation (F urushita et al., 2003) implying a potential
pathway between environmental and human commensal bacteria. Similarly, it has been
demonstrated that IncU R-plasmids previously only associated with aquaculture
environments were also associated with human isolates and that this dissemination
occurred in four separate countries (Norway, Scotland, England, and Germany) (Rhodes
et al., 2000). In 2005 a new tetracycline efflux pump, tet(3 9), was discovered in isolates
from fish farms as well as a clinical specimen from human urine collected in the
Netherlands in 1986 (Agerso & Guardabassi, 2005). Although none of the above
examples provide definitive proof that horizontal transfer is occurring they are consistent
with this hypothesis.

Surface Water. Surface waters are prone to TC residue and resistant microbe
contamination through wastewater treatment plant (WWTP) effluent discharges, direct
discharges of sewage from households or hospitals, aquaculture sites, and runoff from
soils with TC contaminated manure.

The presence and concentration of tetracyclines has been measured in surface
waters by multiple investigators. Kolpin et a1. (2002) tested 84 samples in a USGS
national reconnaissance study. Even though their study was purposefully biased towards
streams likely to be impacted by anthropogenic contamination they infrequently detected
tetracyclines. CTC was detected in only 2 of 84 samples (median concentration of 0.42
ug/L) (Kolpin et al., 2002). Similarly, OTC and TC were detected at only one site (0.34

ug/L and 0.11 ug/mL, respectively) (Kolpin et al., 2002). In 2008 this same group

37

published another study in which they sampled surface waters that are sources for
drinking water supplies. Of the 47 surface waters analyzed tetracyclines were never
detected (F ocazio et al., 2008). Similarly Batt and colleagues were unable to detect
tetracyclines in river waters, although they were able to in WWTP outfalls (Batt, Bruce,
& Aga, 2006).

However, TCs have been detected frequently in other surface waters. This is
likely attributable to different site characteristics (e. g. organic C loads, flow rate etc.) and
susceptibilities to TC contamination. It has been shown experimentally that TCs can be
transported to surface waters through overland flow and field drainage after manure
application (Davis, Truman, Kim, Ascough, & Carlson, 2006; Kay, Blackwell, & Boxall,
2004, 2005). Choi and colleagues detected OTC, minocycline (MCLN), doxycycline
(DXC), meclocycline (MECN), democlocycline (DMC), and TC in river water at
concentrations ranging from 0.01 to 0.2 ug/L (Choi, Kim, Kim, & Kim, 2007). Yang et
a1. (2003) also detected CTC, OTC, DMC, TC, and DXC in the Poudre River in Northern
Colorado at concentrations ranging from 0.05 to 1.14 ug/L.

In fact, Dr. Kenneth Carlson’s group at the Colorado State University has
conducted the most extensive and comprehensive study of TC residues, resistant bacteria,
and resistance genes in surface waters to date. They’ve studied the Poudre River in
Northern Colorado temporally and spatially. Sampling at five separate sites revealed that
TCs contamination in surface waters likely originated from anthropogenic sources
including urban and agricultural impacts (Yang & Carlson, 2003). A pristine site located
in the mountains upstream of any cities revealed no detectable tetracyclines (Yang &

Carlson, 2003). As the sampling sites became more impacted by human activity, more

38

tetracyclines were detected. At a site directly downstream of a WWTP they detected a
large spike in DMC (0.33 ug/L) (Yang & Carlson, 2003). Their data also show the
impact of OTC and CTC, two commonly used antibiotics in animal husbandry, on the
river at sites likely to be impacted by agricultural runoff. At the site directly downstream
of the WWTP they only detected TC and DMC. OTC and CTC were also absent from
the effluent coming from the WWTP. However, further downstream after the river
flowed through areas where the land use is predominantly agricultural OTC and CTC
were first detected (0.07 and 0.15 ug/L OTC and 0.19 ug/L CTC at site S) (Yang &
Carlson, 2003). In a follow up study they sampled the same five sites over a period of
seven months and showed results consistent with the previous study indicating that this
was not a one time event (Yang, Cha, & Carlson, 2004).

Further studies were conducted by Dr. Carlson’s group that measured the
concentrations of tetracyclines in sediments, the presence and quantity of specific
tetracycline resistance genes, and of resistant bacteria. Sediment concentrations of TC
residues were significantly higher than in surface waters (maximum total concentration of
tetracyclines of 100.9 and 399.1 ppb for high and low flow events, respectively) (Pei,
Kim, Carlson, & Pruden, 2006). This is not surprising considering the strong sorption of
tetracyclines to soils (see the terrestrial section of this review). The tet(O) gene was the
only gene detected at the pristine site, whereas tetB(P), tet(S), tet(W), and tet(O) were all
detected at the impacted sites (Pei et al., 2006). The authors quantified tet(W) and tet(O)
via RT-PCR and found that tet(O) concentrations were about an order of magnitude
higher at impacted sites compared to pristine sites (Pei et al., 2006). They returned to this

site for another study, this time more extensively analyzing tet(O) and tet(W) gene

39

concentrations by RT-PCR (Pruden, Pei, Storteboom, & Carlson, 2006) and also
investigating surface runoff as sources of resistance genes. Gene concentrations followed
the pattern (highest to lowest) of dairy lagoon water > irrigation ditch water >

urban/agriculturally impacted sediment (p < 0.0001) (Pruden et al., 2006). Gene

concentrations generally were between 10.7 to 10.5 gene copies/16$ rrn copy. Although

concentrations of TcR genes did not differ significantly from any of the sites along the
river when normalized to 16S rRNA gene copy number, lowly impacted sites were
significantly different from sites heavily impacted by urban and agricultural runoff
without this normalization (Pruden et al., 2006). This may in fact be a better measure as
it is known that a single bacterium can harbor one to fifteen copies of rrn genes.
Ribosomal operon gene copy number varies with growth strategy as copiotrophs typically
have higher copy numbers than oligotrophs (Klappenbach, Dunbar, & Schmidt, 2000).
An earlier study indicated that the sites subject to human impact had higher organic C
amounts indicative of significant surface runoff. It is possible that bacteria originating
from a high nutrient environment would have higher rrn copies per bacterium which if
transfered to surface water, could skew the data when TcR gene copy number is
normalized per rm copy.

Other investigators have examined the presence of tetracycline resistant bacteria
and their genes in aquatic environments likely to be impacted by agricultural practices
including the J iazhou Bay in China (Dang, Ren, Song, Sun, & An, 2008) and the Mekong
River in Southeast Asia (Kobayashi, Suehiro, Cach Tuyen, & Suzuki, 2007). Sapkota et
al. (2007) examined surface waters impacted by a swine CAF O. They found that

Enterococcus spp., E. coli, and fecal coliforms were significantly higher at surface water

40

sites downstream of the CAF O than upstream. Although only marginally significant (p =
0.06), bacteria downstream had a higher percentage resistance to TC supporting the
possibility that the CAFO is the source of resistance (A. R. Sapkota, Curriero, Gibson, &
Schwab, 2007).

Ground Water. Tetracyclines are rarely detected in groundwater samples. In a
recent national reconnaissance study performed by the USGS, 47 groundwater sites were
examined for a multitude of pharmaceuticals and personal health care products (Barnes et
al., 2008). Tetracyclines were never detected at any of the 47 groundwater sites. A
concurrent study sampled groundwater sources used for drinking water and again, no TCs
were detected (Focazio et al., 2008). Hirsch and colleagues sampled groundwater wells
in Germany and similarly did not detect any TCs (Hirsch, Temes, Haberer, & Kratz,
1999). In a similar study, Campagnolo and colleagues only detected CTC at a
concentration of 2 ug/L in a field well near a poultry farm that had used tetracyclines
(Campagnolo et al., 2002). Mackie et al. (2006) sampled groundwater monitoring wells
adjacent to swine manure lagoons at two CAFO sites in Illinois that had a history of TC
usage. They were only able to detect OTC twice out of 45 samples at concentrations of
0.08 and 0.13 ug/L, TC once at a concentration of 0.4 ug/L, and the TC degradation
products anhydrotetracycline, B-apooxytetracycline, and anhydrochlortetracycline three
times at concentrations ranging from 0.1 ug/L to 0.3 ug/L (Mackie et al., 2006). Rare
detection of TC residues in groundwater is not surprising considering that tetracyclines
sorb strongly to soils and are rarely detected below depths of 30 cm (Table 1.6) (Pils &

Laird, 2007; Sarmah et al., 2006).

41

Although TC residues are rarely detected in groundwater, TC resistant bacteria
and genes have been commonly detected in groundwater impacted from CAFOs (Table
1.3) (Anderson & Sobsey, 2006; A. R. Sapkota et al., 2007). University of Illinois
researchers have spent years characterizing the presence, persistence, and mobility of tet
genes in aquifers located below swine manure lagoons at CAFOs with a history of TC
use. In two separate studies, they were able to detect numerous RPPS and efflux genes in
groundwater monitoring wells proximal to the swine lagoons (Aminov et al., 2002; Chee-
Sanford, Aminov, Krapac, Garrigues-Jeanjean, & Mackie, 2001). Monitoring wells
located behind the swine lagoons and upstream of the groundwater flow pattern did not
contain tet genes. The tet genes may be mobile throughout the aquifer as they detected
tet(B), tet(H), tet(Z), and tet(Q) 250 m downgradient from the swine lagoons (Aminov et
al., 2002; Chee-Sanford et al., 2001). They also were able to culture isolates of
Enterococcus spp., Staphylococcus spp., and Lactobacillus spp. containing tet(M) from
groundwater samples (Chee-Sanford et al., 2001).

The authors subsequently conducted a long-term 3 year sampling of these sites to
determine any patterns in resistant gene flow. The presence of RPPs tet(M), tet(O),
tet(Q), and tet(W), and efflux genes tet(C), tet(H), and tet(Z) were monitored over six
sampling dates (Koike et al., 2007). Two significant conclusions were made. First, a
principle component analysis (PCA) determined how the monitoring wells grouped based
on well tet gene profile compared to the let gene profile of the swine lagoons. At one site
five monitoring wells that were all located close and downgradient from the swine lagoon
grouped with the lagoon in PCA (Koike et al., 2007). This result indicates that the swine

lagoon is indeed the source of the tet gene contamination. This finding was corroborated

42

when they analyzed 100 sequences of tet(W) genes isolated from the various wells and
lagoons. The tet(W) genes present in the groundwater monitoring wells shared over
99.8% identity with sequences from lagoon samples, whereas tet(W) genes isolated from
control wells exhibited considerably less sequence identity (Koike et al., 2007).
Although these studies confirmed that bacteria harboring tet resistance genes can
contaminate groundwater samples via leeching from swine lagoons, they cannot
determine if this is due to horizontal transfer of genes from lagoon bacteria to natural
bacteria or if it is due to movement of lagoon bacteria into the groundwater phase.

Wastewater Treatment Plants. Wastewater treatment plants (WWTPs) are
considered to be areas prone to antibiotic contamination and harboring resistance traits
since they receive wastewater from households and hospitals that may be contaminated
with these agents (Kummerer & Henninger, 2003; Rhodes et al., 2000; Thomas, Dye,
Schlabach, & Langford, 2007).

Typically antibiotic concentrations in WWTP influent water are in the ppb range
from less than 1 to 50 ppb whereas effluent concentrations typically are between 0.5 and
10 ppb if detectable. Karthikeyen et a1. (2006) surveyed WWTPs in Wisconsin and
found maximum influent concentrations of 48 ppb (TC), 0.31 ppb (CTC), 47 ppb (OTC),
and 10 ppb (DXC) compared to maximum effluent concentrations of 3.6 ppb (TC), 0.42
ppb (CTC), 0.42 ppb (OTC), and 10.9 ppb (DXC). They measured soluble TC maximum
influent concentrations of 1.2 ppb and maximum effluent concentrations of 0.85 ppb
(Karthikeyan & Meyer, 2006). Yang and colleagues surveyed a WWTP in Fort Collins,
CO and found maximum influent concentrations ranging from 0.1 to 0.3 ppb for TC,

DMC, CTC, and DXC compared to maximum effluent concentrations of 0.06 ppb (CTC)

43

and 0.07 ppb (DXC) (Yang, Cha, & Carlson, 2005). TC and DMC were not detected in
effluent. In China influent concentrations have been measured for TC at 1.3 ppb
compared to 0.18 ppb in effluent (Gulkowska et al., 2008). Similarly, a WWTP in
Canada was shown to have a maximum effluent concentration of 0.98 ppb during the
study period (Miao, Bishay, Chen, & Metcalfe, 2004).

Given that TC concentrations in municipal WWTPs seem to be similar regardless
of geographical location it is of interest to know if these environments are selecting or
enriching TC resistant bacteria that could disseminate to the environment on a global
scale. Ferreira da Silva et a1. (2007) studied a WWTP in Portugal and they found that the
percentage of tetracycline resistant Escherichia spp., Shigella spp., and Klebsiella spp.
increased in effluent water compared to raw influent water. This could indicate either (a)
the transfer of TcR determinants in the WWTP environment or (b) the enrichment of
resistant bacteria. Furthermore, TC resistance had a positive correlation with ampicillin,
ciprofloxacin, and mercury resistances indicating the potential for co-selection of TcR
resistant determinants (Ferreira da Silva, Vaz-Moreira, Gonzalez-Pajuelo, Nunes, &
Manaia, 2007). This is problematic because resistant bacteria could be discharged in the
effluent water to surface waters. Support of this interpretation is provided by Goni-
Urriza and colleagues study that sampled Enterobacteriaceae spp. and Aeromonas spp.
upstream and downstream of a WWTP in Spain. Levels of resistance increased from
12.5% and 0% upstream to maximum values of 24.3% and 27.5% downstream for
Enterobacteriaceae spp. and Aeromonas spp., respectively (Goni—Urriza et al., 2000).

As is commonly known to microbiologists, less than 1% of known bacteria are

currently cultured (Torsvik, Goksoyr, & Daae, 1990; Torsvik & Ovreas, 2002). Hence,

44

studies using only cultured indicator organisms are used could drastically underestimate
the extent of TcR in the environment. To avoid this bias a few research groups have
tested by non-culture based methods for the presence and quantity of TcR genes in
WWTP environments (Auerbach, Seyfried, & McMahon, 2007; Pruden et al., 2006).
Pruden et a1. (2006) tested for the presence of tet(O) and tet(W) by a PCR
presence/absence assay throughout multiple stages in the WWTP process and detected
both genes throughout all stages including treated drinking water (Pruden et al., 2006).
Auerbach et a1. (2007) tested for the presence of 10 tet genes encompassing both efflux
and RPP determinants in WWTPs in Wisconsin. They consistently found a high diversity
of tet genes and in multiple cases found all 10 genes to be present in WWTP
environments (Auerbach et al., 2007) whereas in two control lake samples they only

found tet(A). Concentrations of tet(G) and tet(Q), measured by RT-PCR, ranged from 1

x 107 — l x 109 copies per mL in influent, activated sludge, and biosolids (Auerbach et

al., 2007). Effluent waters had concentrations ranging from 1 x 104 to l x 106 per mL,

about three orders of magnitude lower (Auerbach et al., 2007). It is possible that this
reduction in tet gene copy number is due to one or a combination of the following: cell
death in the biosolid phase, absence of selective pressure due to decreased antibiotic
bioavailability, or simply retention by the biosolid phase.

Terrestrial Transport

Soil. As mentioned above, tetracyclines are administered to animals for
prophylactic, therapeutic, and growth promotion purposes. Multiple studies have
demonstrated the development, selection, and maintainence of TC resistance traits in the

animals (Table 1.5). Significant amounts of tetracyclines are excreted unaltered in urine

45

and feces (Sarmah et al., 2006). Concentrations of tetracyclines have been found up to 40
ppm in manure (Martinez-Carballo, Gonzalez-Barreiro, Scharf, & Gans, 2007). Not
surprisingly, the presence of TC resistant microbes has been demonstrated in manure at
all stages in the storage process (Stine et al., 2007). In the United States agricultural
animals produce approximately 128 billion lbs of manure annually (Sarmah et al., 2006),
most of which is recycled to fields as fertilizer. In recent years concern has developed
that manure management practices may be enhancing the spread of TcR. TC residues
and TcR bacteria may leach from manure storage areas into groundwater systems, runoff
to surface waters after rainfall, or accumulate through land application of manure. In
regards to land application, TcR enhancement could occur through TC residue transfer to
soil and subsequent selection for resistant indigenous populations, soil deposition and
enrichment of resistant microbes originating from animal sources, or horizontal transfer
of TcR genes from animal commensal bacteria to indigenous soil bacteria.

Soils fertilized with TC contaminated manure typically contain concentrations up
to 300 ppb (Table 1.6). Tetracyclines are known to have a strong capacity for sorption to
soil that is dependent on humic material, pH, and cation exchange capacity (Gu,
Karthikeyan, Sibley, & Pedersen, 2007; Pils & Laird, 2007; Sassman & Lee, 2005; Tolls,
2001). Correspondingly, tetracyclines detected in manured fields have been shown to
accumulate in the top 30 cm and are infrequently detected below this depth (Hamscher,
Pawelzick, Hoper, & Nau, 2005; Hamscher, Sczesny, Hoper, & Nau, 2002; Kay,
Blackwell, & Boxall, 2004). Furthermore, these residues can persist in top-soil for years

and accumulate with repeated manure applications (Hamscher et al., 2005).

46

Few studies have determined the tet gene variants in TC resistant cultured isolates
originating fi'om manured soils. In fact only six studies have been published to date (Fig.
1.2). Ghosh and LaPara (2007) have performed the most comprehensive study to date in
which they cultured soil bacteria from swine farms using sub-therapeutic tetracyclines,
dairy farms using TCs as therapeutics, and non-agricultural soils. They used both
nutrient rich and nutrient poor media to culture a wider variety of soil bacteria than is
typically isolated in soil studies. This study alone represents 50% of the unique
isolate/TcR gene combinations from soil environments (Table 1.2). One of the swine
farms sampled ceased operation during the study period and it was found that resistance
levels remained high for at least 18 months (Ghosh & LaPara, 2007). Bacteria were
sampled at various distances (5, 20, and 100 m) from a pen in which swine manure had
been previously stored. Interestingly, there was considerable overlap in the types of
resistant bacteria at 5 and 20 m from the pen, however, at 100 m Streptomyces spp. were
most prevalent, representing approximately 50% of the isolates (Ghosh & LaPara, 2007).
This indicates a shift from bacteria originating from the animal manure to soil indigenous
bacteria as Streptomyces spp. are dominant members of soil communities and antibiotic
producers likely to harbour resistance genes. Ultimately, a conclusion regarding the
safety of the application of TC contaminated manure cannot be drawn from this study.
However, the fact that this study alone represents 50% of unique gene/isolate
combinations known in soil environments demonstrates that (a) soil is a significant
reservoir of TcR and (b) the extent of horizontal dissemination of TcR genes is likely to

be vastly underestimated.

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No study to date has provided conclusive evidence that transfer of TC residues
and resistant microbes into soil environments enhances the transfer of resistance to
clinical pathogens. However, this is not to say that this process is not occurring. In fact,
it is possible that a lack of evidence is due to a lack of appropriately-controlled, long-term
studies. Field scale studies typically involve sites which have been subject to agricultural
practices for many years. Adequate data regarding antimicrobial usage is rarely available
to researchers and this makes untangling introduced resistance from background
resistance a nearly impossible task. Furthermore, few studies have combined culture-
independent molecular methods with culture methods. Both have inherent bias that could
vastly underestimate the true extent of resistance. The fact that less than 1% of known
bacteria have been cultured limits our understanding of the extent of antibiotic resistance.
Culture-independent methods can be expensive and time-consuming. For example,
examining the entire extent of TcR genes in any sample requires detection of 38 tet gene
variants and this does not include genes yet to be discovered. Even when tet genes are
identified by molecular methods, few studies adequately investigate the mechanisms of
horizontal transfer that will allow judgment of the capability for further dissemination of
resistance. Clearly TcR resistance in soil environments and the impact of animal
agriculture on such warrants further investigation.

Plant. Plant agriculture represents the least researched terrestrial system with
regards to tetracycline resistance. OTC is sprayed in orchards at concentrations of ~ 300
ppm to control various plant diseases (McManus et al., 2002). Ironically, this system

represents the most direct application of OTC (OTC is sprayed directly onto plant

49

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50

surfaces after dissolution into water), yet is the least well characterized.

Currently only one study has investigated the cultivatable TcR fraction from
phylloplane bacteria associated with OTC treated plants. A survey of resistance in
Michigan apple orchards revealed the presence of tet(A), tet(B), tet(C), and tet(G) in
isolates mostly of Pantoea agglomerans or fluorescent and nonfluorescent Pseudomonas
spp. (Schnabel & Jones, 1999). All TcR elements were located on plasmids. In
particular tet(A) and tet(B) were exclusively associated with TnI 721-like and Tn10
transposons, respectively (Schnabel & Jones, 1999). The author’s go on to speculate that
development of TcR in the major plant pathogen Erwinia amylovora (the causative agent
of fire blight) will not be a short term consequence of OTC application in apple orchards
(Schnabel & Jones, 1999). This conclusion was based on a number of astute
observations; however, little is known regarding the transfer of TcR to other genera that
may be of consequence to human health. Given the lack of studies on OTC usage and
bacterial resistance that could ultimately end up in retail food it certainly represents an
area that deserves future research consideration.

SIGNIFICANCE OF RESEARCH

It is my belief that in a world of limited time and resources, scientists should be
able to justify the significance of their research to the community that a) provides funding
and b) will reap the benefits from it. Considerable time has been spent describing the
broader antibiotic resistance problem in the context of tetracycline resistance. Now the
discussion narrows and shifts towards the relevancy of the topic of research, that is,

tetracycline resistance in the soil agricultural environment.

51

Economic Impacts. As has been eluded to, there is heated debate as to whether
or not antimicrobials should be eliminated for grth promotion purposes (Hileman,
2001). The best justification for researching antimicrobial resistance in the agricultural
environment can be provided by an analysis of the potential economic impact of either
withdrawal of antibiotics for growth promotion, or in continued use, which could
potentially enhance the problem of treating antibiotic resistant pathogens.

The economic justification for the continued use of antimicrobials in farming
practices (aquaculture, animal husbandry, and plant agriculture) is that usage will
decrease costly bacterial infections that could decimate crops or animals. The benefits of
therapeutic/prophylactic usage are obvious and their purpose is rarely debated. It is sub-
therapeutic usage that takes the most scrutiny. Farmer’s argue that sub-therapeutic
antimicrobial usage allows them to use less feed that in turn helps keep food prices low.
It has been estimated that there is an additional net return of $2.99 per pig if
antimicrobials are used from weaning to market for each $0.7 invested in the antibiotic
(Cromwell, 2002). The economic return if used in the breeding diet is $7.12 net return
per litter and $2.63 if used in lactation feed per litter (Cromwell, 2002). On a broader
scale it has been estimated that the complete withdrawal of antimicrobials for growth
promotion in food production in the US would cost consumers $5 — 40 per capita per
annum (Phillips et al., 2004). Another, often overlooked, factor is the added cropland
that would need to be used because of the decrease in feed efficiency. This has been
estimated to be an additional 2 million acres of cropland in the US alone (Phillips et al.,
2004). Transition of more land to cropland for feed production would also have a

significant environmental impact.

52

Concern has been raised that antimicrobial residues that ultimately end up being
applied to land through contaminated manure can aid in selection of resistance
determinants that may ultimately transfer to pathogens seen in the clinic. Potential for
resistance transfer and enrichment has been demonstrated via consumption of TcR and
TC contaminated food, water, and air (Chapin, Rule, Gibson, Buckley, & Schwab, 2005;
Koksal, Oguzkurt, Samasti, & Altas, 2007; Zhang et al., 2007). If this occurrs a
disastrous economic consequence is that infectious diseases will become more difficult
and costly to treat. In 1992 it was estimated that approximately $120 billion (15%) of all
healthcare expenditures were related to treating infectious disease and that antibiotics
were the most commonly prescribed drugs (Cassell, 1997). Various sources have
estimated that the annual cost of treating drug resistant infections in the US ranges from
$1.5 to $ 4 billion (Cassell, 1997; Twomey, 2000).

What we have come to is a difficult tug of war between two different vested
interests, with economic and ethical consequences on both sides. On the one hand the
world’s population is exponentially increasing and maintaining efficient and high levels
of food production is essential to provide sufficient nourishment to the masses. Usage of
antimicrobials for sub-therapeutic purposes may help this high level be maintained. On
the other hand this usage could have consequences to infectious disease treatment, and
this can become a quality of life issue. Therefore it becomes researcher’s responsibility
to find and fill the knowledge gaps to determine how much of a risk the practice of sub-
therapeutic antimicrobial grth promotion presents.

Knowledge Gaps. Despite 60 years worth of research on tetracyclines,

significant gaps in understanding regarding the environmental dissemination of

53

tetracycline resistance genes still exist. Two particularly relevant gaps are the molecular
identification of tetracycline resistance genes in soil environment and the effect that
tetracycline chemistry has on the bioavailability of the antibiotic in environmental
matrices.

A comprehensive literature review was conducted to investigate the extent to
which molecular investigation of TcR determinants has been researched. This survey
revealed that 111 studies have characterized tetracycline resistance genes in cultured
isolates originating from environmental, food, and animals by molecular methods (Fig.
1.2). Bacteria isolated from human clinical sources were omitted in this survey because
this abundant data would obscure the environmental information. There has been a
significant body of work completed since 2000. Eighty three percent (92/1 11) of the total
studies have been published in the last eight years. Of all studies only 6/111 (6 %) are
from soil environments. Despite this low number of studies genera/ gene combinations
unique to soil represent 17% of the total unique genera/ gene combinations. Of these six
studies 84% of the unique genera/ gene combinations only come from two studies, one
published in 2007 and the other in 2008 (Ghosh & LaPara, 2007; Srinivasan et al., 2008).
This review demonstrates the strong likelihood that there is a significant amount of
unexplored molecular diversity in present agricultural soil environments, especially those
that have been impacted by manure contaminated with tetracycline residues.

A second major area that has been ignored is the effect of tetracyclines’ chemistry
on the bioavailability of the molecule and how this controls the expression of tetracycline

resistance genes in environmental matrices. Bioavailability is defined as the ability of

54

tetracyclines to leave the environmental matrix, penetrate the microbial cell’s membrane,
and act on their targets.

There have been multiple investigators that have identified the gap in
understanding of bioavailability in a natural context (Chopra & Roberts, 2001; D'Costa,
Griffiths, & Wright, 2007; Kummerer, 2004). Despite public knowledge of this gap, few
studies have attempted to address the topic of tetracycline bioavailability to microbial
cells in natural environments (Chander, Kumar, Goyal, & Gupta, 2005; Verma, Robarts,
& Headley, 2007). Furthermore, these two studies have only implicitly addressed the
topic of tetracycline bioavailability in natural environments. To the best of the author’s
knowledge, no studies exist in which mechanistic examination of bioavailability and how
it controls expression of the tetracycline efflux genes in the context of the relevant
environmental tetracycline chemistry have been conducted.

Objectives —

Presented herein are the results of research on three specific objectives:

1) To determine the full-coding sequence of potentially novel RPP genes
detected in an agricultural soil environment impacted with manure
contaminated with tetracycline residues.

2) To examine the diversity of soil genera harboring tetracycline efflux pumps in
the environment mentioned above.

3) To conduct mechanistic research into factors that would control
bioavailability of tetracycline in environmental matrices, and hence,
expression of tetracycline efflux genes through modulation of the TetR

repressor protein.

55

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CHAPTER II
CHARACTERIZATION OF TETRACYCLINE EFFLUX PUMPS IN A
MAN URED AGRICULTURAL SOIL BY BOTH CULTURE DEPENDENT AND
INDEPENDENT METHODS
INTRODUCTION
In the United States tetracycline (TC), oxytetraycline (OTC), and
chlortetracycline (CTC) are approved for and commonly given as grth promoting
agents to swine (Sarmah, Meyer, & Boxall, 2006). Growth promotion refers to the sub-
therapeutic use of an antibiotic, typically mixed in with feed, to improve pig weight gain
and feed efficiency (Gaskins, Collier, & Anderson, 2002). It is estimated that about 24.6
million lbs of antimicrobials are given annually to animals for non-therapeutic purposes,
whereas 3 million are given to humans (Gorbach, 2001). As much as 75% of a single
dose can be excreted un-metabolized in urine or feces (Sarmah et al., 2006). Hence a
significant percentage of the antibiotic makes its way into the environment. In the United
States confined animal-feeding operations (CAFOs) generate 128 billion pounds of
manure annually (Sarmah et al., 2006). This manure is stored for some time and
ultimately spread onto fields. Multiple investigators have found manured soil
concentrations of tetracyclines in the range of 200 ppb (Table 1.6). Correspondingly,
tetracycline resistance has also been observed in soil environments (Table 1.2).
Few investigators have analyzed genes conferring tetracycline resistance in soil

systems. Only six studies have cultured antibiotic resistant bacteria from soils and
examined their tetracycline resistance elements by molecular methods (Fig. 1.2). Out of

those six studies, four focused on agricultural soil systems (Agerso & Sandvang, 2005;

Ghosh & LaPara, 2007; Srinivasan et al., 2008; Stine et al., 2007) and of these four

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studies, 93% of the unique gene/isolate combinations come from two very recent studies
(Ghosh & LaPara, 2007; Srinivasan et al., 2008). In other words, agricultural soils
fertilized with TC contaminated manure are under-studied with regards to molecular
tetracycline resistance. This is particularly surprising given that tetracycline has been
studied for 60 years and soil has been hypothesized as a reservoir for antimicrobial
resistance genes for at least 38 years (Walker & Walker, 1970).

Previously, Dr. Carlos Rodriguez-Minguela examined the extent of tetracycline
resistance in an agricultural soil (Table 2.1) that was sampled 1 week after tetracycline
contaminated manure was applied (Rodriguez-Minguela, 2005). His work focused on
characterizing ribosomal protection proteins (RPPS), one of the four known mechanisms
of tetracycline resistance, that confer resistance to tetracycline by dislodging it from the
ribosome in a GTP-dependent manner (Connell, Tracz, Nierhaus, & Taylor, 2003). By
utilizing degenerate RPP primers he found that tet(M) and tet(O/W) variants were most
predominant 1 week post-application (Rodriguez-Minguela, 2005). Other genes detected
included tet(O), tet(Q), tet(3 6) and two putative novel RPPS referred to herein as clone
397 and clone 492 (Rodriguez-Minguela, 2005). The partial sequence of clone 397 has
71% amino acid identity with its closest relative, Tet(32), and potentially represents a
new RPP gene based on the definition of S 80% amino acid identity to the closest
characterized tetracycline resistance gene (Levy et al., 1999; Rodriguez-Minguela, 2005).

Table 2.1. Site # 21 Soil (Michigan Farm)

 

 

Source Manure Application Antibiotic Used Soil Type
Wheat field 1 week before Cl-tetracycline Capac loam
sampling

 

El-tetracycline was routinely used as swine feed supplement (growth promoting agent)

 

79

Clone 492 shared 85% amino acid identity to Tet(O/W), however, the last 135 amino
acids were 98% identical to Tet(32) (Rodriguez-Minguela, 2005). Unfortunately the
culture approaches used by Rodriguez-Minguela failed to yield a bacterial isolate
harboring clone 397 or 492, and so definitive proof of functional tetracycline resistance
remaines unknown.

The advent of culture independent methods, termed metagenomics, represents
another approach to recover the full-coding sequences of clones 397 and 492.
Metagenomics refers to any culture-independent sequence or expression based analysis of
a microbial community (Schloss & Handelsman, 2005). Less than 1% of known bacterial
species are currently cultivatable (Torsvik, Goksoyr, & Daae, 1990). Construction of
metagenomic insert libraries (e. g. fosmid, cosmid, or bacterial artificial chromosome) of
random community DNA can circumvent the need for cultured isolates to study the
genomes of these bacteria. Three studies have isolated tetracycline resistance genes
through construction of various types of metagenomic libraries (Diaz-Torres et al., 2003;
Diaz-Torres et al., 2006; Riesenfeld, Goodman, & Handelsman, 2004). In two of these
studies the use of metagenomics allowed the discovery of two novel tetracycline
resistance genes, tcr, a tetracycline efflux gene, from a pristine soil (Riesenfeld et al.,
2004) and tet(3 7), a tetracycline inactivating enzyme, from the human oral metagenome
(Diaz-Torres et al., 2003). Given the success of previous investigators in isolating novel
tetracycline resistance determinants and the problem of recovering full-coding sequences
of clones 397 and 492, metagenomics was an appropriate method to pursue.

This metagenomic approach was supplemented with an analysis of the

cultivatable fraction of bacterial isolates harboring tetracycline efflux genes in the same

80

soil sample previously used by Dr. Rodriguez-Minguela. He recovered tet(M) in
Paenibacillus spp. and Enterococcus spp. and tet(O) in Corynebacterium spp.
(Rodriguez-Minguela, 2005). Given the lack of studies examining molecular tetracycline
resistance in agricultural soil environments it is also of interest to characterize the
fraction resistant to tetracycline due to the other major mechanism, tetracycline efflux.
The tetracycline efflux pumps are membrane bound proteins that exchange a tetracycline-
magnesium complex in an energy dependent manner for a proton thereby reducing the
intracellular concentration and detoxifying the cell (Chopra & Roberts, 2001).

The goals of my study were to:

1) To recover the full-coding sequences of clones 397 and 492 via metagenomic

library construction and demonstrate functional tetracycline resistance

2) To analyze the extent of resistance to tetracycline via efflux pumps in the

cultivable and non-cultivable (via metagenomics) bacterial fraction.
MATERIALS AND METHODS

Sample. Soil used for this study was a Capac loam sampled from a wheat field
on a Michigan farm one week after application with manure from animals that received
Cl-tetracycline routinely as a growth promoting agent (Table 2.1) (Rodriguez-Minguela,
2005)

Soil Microcosms. Samples of site 21 soil (5 g) were transferred into serum
bottles. These samples were supplemented with 1 mL of a 40 ug/mL tetracycline
(Sigma-Aldrich Co., St. Louis, MO) + 200 ug/mL cyclohexamide (Sigma-Aldrich Co.,
St. Louis, MO) 10 ug/mL tetracycline + 200 ug/mL cyclohexamide, or water + 200

ug/mL cyclohexamide solution. Serum bottles were sealed and incubated aerobically for

81

10 days. Every other day bottles were opened in a laminar flow hood for 10 minutes to
allow atmosphere replacement.

DNA Extraction, Purification, Sizing, and Cloning. After 10 days incubation,
DNA was extracted from 2.5 g samples from soil microcosms by a method modified
from (Zhou, Bruns, & Tiedje, 1996). After this extraction step the soil DNA was
amenable to PCR, however, attempts at library construction failed indicating that the
DNA was not of sufficient quality for cloning purposes. Subsequently, a number of
additional steps were taken to further purify the DNA. DNA was concentrated with YM-
10 Microcon filters (Millipore Co., Billerica, MA) for about 30 min at a speed of 500 rcf
in a table top centrifuge. After sufficient concentration the DNA was loaded onto a 1%
pulse field certified (PFC) agarose (Bio-Rad, Hercules, CA) pulse field gel
electrophoresis (PF GE) gel with the 2 cm of gel below the wells cut out and refilled with
1% PFC agarose + 2 % polyvinylpyrrolidone (PVP; Sigma-Aldrich Co., St. Louis, MO)
in 1X tris-acetate-EDTA (TAE) buffer. The gel was run with the auto algorithm on a
Bio-Rad CHEF MapperTM PFGE unit (Bio-Rad, Hercules, CA) to separate DNA in a size
range from 10 — 100 Kb. After the PFGE nm, the gel regions corresponding to the Low
Range and Mid Range I PFG Markers (New England Biolabs Inc., Ipswich, MA)
flanking the DNA was cut from the gel and stained in a bath with 100 mL 1X TAE and
15 uL SYBR® Safe (Invitrogen, Carlsbad, CA). Cut gel fragments containing PFG
markers were visualized with an Eagle Eye 11 gel imaging system (Stratagene, La Jolla,
CA) and cover slips were used to make small cuts in the gel corresponding to DNA sizes
of 35 — 60 Kb. The gel slices were re-aligned to re-fonn the original gel and slices

corresponding to soil DNA 35 — 60 Kb in size were cut. DNA was extracted from these

82

slices by electroelution in dialysis tubing (Size MC-l 8, 12,000 — 16,000 MWCO Daltons,
25 mm flat width; Sargent-Welch, Buffalo, NY) as described in (Sambrook & Russell,
2001). Bags containing DNA were dialyzed in 1 L sterilized water at 4°C for 20 minutes.
After dialysis liquid containing the DNA was removed from the bags, concentrated with
YM-100 Microcon filters, and finally subject to end-repaired and cloned as described in
the CopyControlTM Fosmid Library Production Kit (EPICENTRE® Biotechnologies ,
Madison, WI).

Restriction Analysis and Library Storage. The FosmidMAXTM Kit
(EPICENTRE® Biotechnologies , Madison, WI) was used according to the
manufacturer’s protocol to extract fosmid DNA. Two micrograms of fosmid DNA from
fourteen randomly chosen clones were digested with EcoRI (New England Biolabs Inc.,
Ipswich, MA) according to the manufacturer’s protocol and run on a PF GE set to
separate DNA in the l to 20 kb size range.

The library was amplified and stored as liquid gel pools as described in (Hrvatin
& Piel, 2007).

Construction of Control Strain EPI300TM-397. Before PCR based screening
for the presence of clone 397 a control strain (referred to as EPI300TM-3 97) was
constructed to test the sensitivity of the PCR screen. The 1.3 kb fragment of clone 397
was PCR amplified from pCR®4-TOPO® vector (Invitrogen, Carlsbad, CA) containing
the clone 397 insert (Rodriguez-Minguela, 2005). PCR was conducted with primer set 1
(Table 2.2) as described in Rodriguez Minguela (2005). After amplification the PCR
mixture containing the amplified fragment was cleaned using ExoSAP-IT® (U SB Co.,

Cleveland, OH) according to the manufacturer’s protocol. This purified 1.3 kb fragment

83

was then end-repaired and cloned into pCClFOSTM (EPICENTRE® Biotechnologies,
Madison, WI) according to the manufacturer’s protocol. Vector containing insert DNA
was electroporated into electrocompetent EPI300TM cells prepared as described in
Sambrook & Russell (2001). Transforrnants were plated on LB + 12.5 ug/mL
chloramphenicol (Sigma-Aldrich Co., St. Louis, MO). Ten randomly chosen clones were
further analyzed by PCR for the presence of clone 397 with primer set 15 (Table 2.2).
The PCR master mix contained 5 uL 5X GoTaq® Buffer (Promega, Madison, WI), 1.5
uL MgC12 (25 mM stock; Promega, Madison, WI), 0.5 uL dNTPs (100 uM Mix;
Promega, Madison, WI), 0.375 uL 397(FWD) (10 uM stock), 0.375 uL 397(REV) (10
uM stock), 1 uL 100X bovine serum albumin (BSA; New England Biolabs, Ipswich,
MA), 0.25 uL GoTaq® DNA Polymerase (Promega, Madison, WI), and 15 uL sterilized
water per reaction giving a total volume of 24 uL. To this 1 uL of target DNA (100 ng)
was added. Reactions were cycled on a Gene Amp PCR System 9700 (Applied
Biosystems, Foster City, CA) thermocycler. Cycling conditions were as follows: initial
denaturation at 98°C for 2 min, followed by 25 cycles of denaturation at 95°C for 40 s,
annealing at 60°C for 40 s, and extension at 60°C for 25 s. This was followed by a final
extension of 1 min at 60°C. Presence of arnplicons were verified on 35 mL 3% MetaPhor
Agarose (Carnbrex Co., East Rutherford, NJ) gels in 1 X TAE supplemented with 1 uL
SYBR® Safe (Invitrogen, Carlsbad, CA) run at 100 volts for 45 mins.

PCR Screen for Clone 397. Before conducting the PCR based screen for clone
397, the method was tested for its sensitivity. This was done by diluting the EPI300TM-

397 control strain in various amounts and adding it to a clone pool of 99 randomly picked

soil DNA fosmid clones constructed with the same vector (courtesy of Dr. Thomas

84

Schmidt, Michigan State University). Ratios tested included one positive in 100 clones,
one positive in 1,000 clones, and one positive in 10,000 clones (Fig. 2.1). Dilutions of
pools were also tested for inhibitory effects of an overabundance of cell material. PCR
was done on cell lysate after lysis in a thermocycler at 95°C for 10 mins. The PCR
protocol used was as described above for primer set 15 with the exception that 50 cycles
were used to increase the sensitivity of the assay. This test verified that the method is
sensitive enough to detect one positive in 10,000 clones.

To screen the library liquid gel pools were used as inoculum for forty-one 1 mL
overnight cultures of LB + 12.5 ug/mL chloroamphenicol. After growth, aliquots of cells
were lysed at 95°C for 10 min in a thermocycler and subject to PCR with primer set 15 as
described above.

Functional Screen of Soil Fosmid Library and Reduction of Redundancy.
The soil fosmid library was screened by activity for the presence of expressed
tetracycline resistance genes. Liquid gel pools (Hrvatin & Piel, 2007) were used as
inoculum for forty-one 1 mL overnight cultures (37°C, 150 rpm) in LB + 12.5 ug/mL
chloramphenicol. After growth, 41 individual LB agar plates containing 12.5 ug/mL
chloramphenicol + 5 ug/mL tetracycline were plated with 15 uL fi'om each overnight
culture and grown at 37°C overnight. Colonies (262) were picked into 96 well plates
with LB + 12.5 ug/mL chloramphenicol + 5 ug/mL tetracycline and grown at 37°C
overnight with shaking at 150 rpm. It was expected that using liquid gel pools as
inoculum for overnight cultures would introduce redundancy due to clonal growth of
fosmid clones. As such, it was necessary to reduce this redundancy before screening the

262 isolated colonies for the presence of tetracycline resistance genes. This was done by

85

 

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87

pooling 100 uL of culture from rows of 96 well plates (for example A1 to A12) into a
total volume of 1.2 mL, extracting fosmid DNA (as described above), and subjecting this
pool to DNA restriction analysis with EcoRI (New England Biolabs Inc., Ipswich, MA)
according to manufacturer’s protocols. This pooled restriction digest was then mm on a
125 mL 1% UltrapureTM agarose (Invitrogen, Carlsbad, CA) gel with 9 uL SYBR® Safe
(Invitrogen, Carlsbad, CA) at 4°C for 4 h at 75 volts. The pooled digest patterns were
compared to the digest pattern of the fosmid clones on the end of the rows (for example
A1 and A12, each digested individually). If overlapping patterns were detected in the
pooled digest each clone in an entire row was analyzed individually to identify unique
clones. This method allowed the identification of seven (referred to as TCl — TC7)
fosmid clones with unique digest patterns selected for further study (Fig. 2.5).
Sub-Cloning of Fosmids TCl, TC3, TCS, and TC6. Fosmids TCl, TC3, TC5,
and TC6 were selected for sub-cloning to identify the tetracycline resistance gene
resident on insert DNA. Fosmid DNA to be sub-cloned was extracted with Qiagen’s
Plasmid Midi Kit (Valencia, CA) according to the manufacturer’s protocol. DNA was
randomly sheared by sonication (done in a bath with the following conditions:
continuous, 50% duty cycle, output level = 7; W-3 85 Heat Systems-Ultrasonics Inc.,
Plainsview, NY) for 15 s to a size range of 2 to 4 kb. This DNA was then used for
cloning into the pCC 1 FOSTM vector as described in the manufacturer’s protocol with the
exception that electroporation was used instead of transfection to transfer the ligated
vectors into EPI3OOTM host cells. Preparation of electrocompetent cells and
electroporation was performed as described in Sambrook & Russell (2001). Transformed

cells were plated on LB containing 12.5 ug/mL chloramphenicol and 5 ug/mL

88

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Figure 2.1. Test of PCR Screening Sensitivity for Clone 397. EPI300TM host cells
containing the pCClFOSTM vector with a fragment of clone 397 were diluted with 99
randomly picked soil fosmid library clones to give positive to background ratios as
described. (a) 1 positive cell to 100 total cells. (b) 1/10th dilution of (a). (c) 1 positive to
1000 total cells. (d) 1/10th dilution of (c). (e) 1 positive to 10,000 total cells. (f) 1/10th

dilution of (e). (g) negative control including the 99 cells used as background. (h) 1/10th
dilution of (g). + cells = EPI300TM-397 cell lysate. + pCCFOSlTM-397 = vector DNA.
+ pCR4®-397 = original source of clone 397. -— = negative PCR control. The ladder is
Invitrogen’s 100 bp ladder.

 

tetracycline to select clones with tetracycline resistance genes inserted. For each fosmid
ten clones were randomly selected and digested with EcoRI to find the smallest insert
conferring tetracycline resistance. The ends of this clone were then sequenced as
described below.

Cultivation of Tetracycline Resistant Soil Isolates. Soil samples (5 g) prepared
as described above were added to 20 mL of phosphate saline buffer (pH 7.5)
supplemented with sodium pyrophosphate (Sigma-Aldrich Co., St. Louis, MO) and
dithiothreitol (DTT; Sigma-Aldrich Co., St. Louis, MO) and diluted for plating as

previously described (Stevenson, Eichorst, Wertz, Schmidt, & Breznak, 2004). Media

used included LB agar (Becton Dickinson and Company, Franklin Lakes, NJ), 1/10th

89

strength R2A (Becton Dickinson and Company, Franklin Lakes, NJ) supplemented with
15 g Bacto Agar (Becton Dickinson and Company, Franklin Lakes, NJ) per mL, and Soil
Media (SM = 25 mL of soil extract, 8 g of phytagel, and 0.6 mmol CaC12 per liter of
media). Plates were incubated at 25°C for 9 days before colonies were randomly selected
and transferred to liquid culture of the same medium. DNA from liquid cultures was
extracted by the Joint Genome Institute (J GI) standard operation procedure available at
the following web address:
http://my.igi.doegmr/general/protocols/DNA Isolation Bacterial_CTAB_Protocol.doc

PCR of Tetracycline Resistance and 16S rRNA Genes. The general master
mix for PCR of tetracycline efflux genes tet(A) — tet(30) (primer sets 3 — 13) included 5
uL 5X GoTaq® Buffer (Promega, Madison, WI), 2.5 uL 1 mM dNTPs (Promega,
Madison, WI), 0.8 uL 25 mM Magnesium Chloride (Promega, Madison, WI), 1 uL
GoTaq® DNA Polymerase (Promega, Madison, WI), and 9.7 uL sterilized water. To this
2.5 uL of each primer was added giving a final volume of 24 uL. Thermocycling
conditions were performed as previously described (Aminov et al., 2002). Clones 397
and 492 were amplified as described for clone 397 above. The tet(31) and tet(X) genes
were amplified as previously described (Agerso & Sandvang, 2005; Ghosh & LaPara,
2007; Ng et al., 2001). Amplification of 16S rRNA genes was performed as previously
described (Eichorst, 2007).

Sequencing of Fosmid Ends and 16S rRNA Genes. All sequencing was
conducted by staff at the Michigan State University Research Technology Support
Facility — Genomics Core (East Lansing, MI). Sequencing was performed on an ABI

PRISM® 3730 Genetic Analyzer (Applied Biosystems, Foster City, CA). Primers used

90

for sequencing included T7, 8F, and pEpiFOS-RSP (Table 2.2). Sequences were
automatically quality trimmed based on a Q20 value. Basic Local Alignment Search
Tool (BLAST) searching was done with both BLASTN and BLASTP algorithms against
the non-redundant database (Altschul et al., 1997). Nucleotide sequences were translated

to protein at the following website: http://www.expasv.ch/tools/dna.html

 

RESULTS AND DISCUSSION

F osmid Library Construction. A fosmid library was constructed with DNA
extracted from site # 21 soil (Table 2.1) as described in materials and methods. Prior to
cloning, PCR analyses of the final DNA preparation verified the presence of clone 397
but not clone 492 in purified DNA (Fig. 2.3). The absence of clone 492 may be due to it
being located on a plasmid (or other DNA) less than 30 Kb in size. EcoRI restriction
analysis of 14 randomly selected fosmid clones indicated an average insert size of
approximately 30 kb (Fig. 2.4) and based on an estimated 27,000 to 30,000 clones, this
library represents approximately 810 — 900 Mb of soil DNA; 135 to 150 genome
equivalents assuming an average genome size of 6 Mb.

Sequence and Functional Based Screening for Cloned Tetracycline
Resistance Genes. Unfortunately, no positive gel pools were identified for clone 397 by
PCR based screens (Materials and Methods). Absence of a positive hit for clone 397 was
not due to detection limit problems as tests for the sensitivity of the method revealed that
positive detection was still obtainable with one positive clone in 10,000 (Fig. 2.1). It
appears that the inability to obtain a fosmid containing clone 397 was due to insufficient
enrichment of the target gene. Based on previous results, the highest copy number per

gram of soil achieved for clone 397 was 10,000 (Rodriguez-Minguela, 2005). It is

91

commonly assumed that 1 g of soil contains 1 billion bacteria. This would correspond to
one in 100,000 bacteria harboring clone 397 if a single gene was present on the
chromosome. Given that the library represents 135 to 150 genome equivalents it appears
that recovery of the full coding sequence of clone 397 will remain elusive until either
better enrichment can be achieved, or an alternative, more appropriate method is used.

To complement the sequence based screening, a functional based screen of the
fosmid library was also conducted. This involved growing overnight cultures of the
clone pools and plating on LB plates with 5 ug/mL TC and 12.5 ug/mL CAM. It was
expected that the overnight “pre-growth” step prior to plating would introduce a
considerable amount of redundant TC resistant clones. This redundancy was eliminated
as described in the materials and methods. Restriction analysis with EcoRI (Fig. 2.5)
revealed seven TC resistant fosmid clones (TCl —- TC7) with unique restriction patterns
and varying relative minimum inhibitory concentrations (MICs). It is important to note
that I cannot rule out the possibility that two clones could contain fragments of the same
DNA molecule cloned in different locations giving rise to different restriction digest
patterns.

Tetracycline Resistance Genes Present on Fosmids. The seven TC resistant
fosmid clones were then subject to either sub-cloning or PCR to determine the
tetracycline resistance gene(s) present. Figure 2.6 summarizes the results. Three clones
contain tet(Y) (TC4, TC6, and TC7), two clones contain tet(A) (TCl and TC5), one clone
contains tet(31) (TC3) and the last clone contains tet(C) (TC2). In addition to containing
tet(A), clone TCl also contains tet(30). Interestingly, all of these genes are tetracycline

efflux pumps. This could be due to the fact that ribosomal protection proteins are

92

 

 

 

 

Figure 2.2. PCR Verification of Presence of Clones 397 and 492 in Soil DNA. 3%
Metaphor Agarose gel in 1X TAB stained with SYBR Safe. Ladder is Invitrogen’s 100
bp ladder. 1, 2, 3, & 4 refer to 4 separate soil DNA samples, + and — are positive and
negative controls respectively.

  

|"“l
C‘ 0‘

M V

- .- -

Figure 2.3. Verification of Presence of Clones 397 and 492 in Purified Soil DNA to
be Cloned. 3% Metaphor Agarose gel in 1X TAE stained with SYBR Safe. Ladder is

Invitrogen’s 100 bp ladder. 397 and 492 refer to sample DNA. + and — are positive and
negative controls respectively.

    

93

23.1 Kb

9.42 Kb

4.36 Kb

1.02 Kb

 

Figure 2.4. Average Insert Size of Soil Fosmid Library. Gel showing the EcoRI
restriction patterns of fourteen randomly chosen fosmid clones. The outermost ladder is
Invitrogen’s J. phage digest ladder, the middle ladder is NEB’s Low Range PFG ladder
and the innermost ladder is Invitrogen’s 1 Kb DNA ladder. Average insert size equals
29.94 Kb. Library size was between 27,000 and 30,000 clones.

94

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95

generally found in Gram-positive organisms (Chopra & Roberts, 2001) and may not be
efficiently expressed in a Gram-negative host such as E. coli. Heterologous gene
expression is one of the known limitations of functional metagenomics (Handelsman,
2004) and usage of a Gram-positive host cell strain may permit the isolation of more
RPPS. To date, only one study has isolated RPPS via a metagenomic method (Diaz-
Torres et al., 2006).

The finding of tet(A) and tet(30) co-resident on the insert present in fosmid TC1
is of particular interest. Previously tet(3 0) has only been found in Agrobacterium
tumefaciens C58 (Luo & Farrand, 1999) and detected by DNAzDNA hybridization in
various garden and farm soils (Patterson, Colangeli, Spigaglia, & Scott, 2007).
Resistance to tetracycline in Agrobacterium tumefaciens C58 was only identified after
insertion of 18426 into the tetR gene. Luo & Farrand (1999) went on to show that this
tetR variant could bind the Ptet promoter but was unable to be de-repressed by
tetracycline, explaining why resistance was only detected after mutation of the tetR gene.
This non-inducible TetR protein could also interact with promoters from other tet gene
variants and was partially dominant when two tet variants were present in the same cell
resulting in low level TC resistance (Luo & F arrand, 1999). Interestingly, relative MIC
tests with TC1 (Fig. 2.5) indicated that carriage of this fosmid conferred high levels of
tetracycline resistance. There are three possible scenarios explaining how TC1 can carry
tet(30) and another tetracycline efflux gene (tet(A)) and still exhibit high levels of
resistance. First, this could be the first observation of a fully functional tetR variant for
tet(30). Second, perhaps tet(30) is not associated with a tetR in this fosmid capable of

operator binding, or alternatively no tetR gene at all. And third, it is also possible that

96

only partial sequence of the tet(30) and/or tetR gene(s) were cloned, and hence are non-
functioning. However, the third scenario is not likely since sequencing revealed a tnpA
gene and an area with no significant homology flanking the ends of the pCClFOSTM
vector (Fig. 2.6). The observation of co-carriage of tet(A) and tet(30) warrants further
investigation as it may be the first discovery of a functional TetR(3 0) variant.

The tet(Y) variant was the most frequently isolated efflux gene (Fig. 2.6, Table
2.3). This efflux gene was first discovered from the exogenous isolation of plasmids via
biparental matings with bacteria from piggery manure as donors and Escherichia coli
CV601 and Pseudomonas putida UWCl as recipients (Smalla et al., 2000). Aeromonas
spp. and Photobacterium spp., both from water environments, have been found to harbor
tet(Y) (Table 1.1) (Furushita et al., 2003; Gordon eta1., 2008b). Sub-cloning of TC6
revealed a sequence with 100% amino acid identity to the TetR(Y) repressor protein
variant from plasmid pAB5 S9 isolated from Aeromonas bestarium from a river in
Brittany, France (Gordon et al., 2008a). Interestingly, Gordon et a1. (2008) recently
reported that this was, to their knowledge, the first discovery of a functional repressor
protein for the tet(Y) gene. Therefore, the discovery of tetR(Y) in fosmid TC6 may
represent the second report of a functional tet(Y) repressor protein. Similar to pABS S9
the tet(Y) variant in fosmid TC6 was linked with the streptomycin resistance gene srrB.
However, it is unlikely that the insert in fosmid TC6 is from plasmid pAB5 S9 since end
sequencing of TC6 revealed merA and merP genes involved in mercury resistance
(discussed below) that were not present on pABS S9 (Gordon et al., 2008a). It is

unknown if the tet(Y) genes present in fosmids TC4 and TC7 also contain functional tetR

genes because the presence of tet(Y) was confirmed by PCR instead of sub-cloning as

97

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was done for TC6. The finding of tet(Y) in fosmids TC4, TC6, and TC7 represents only
the second report of this gene being isolated from a soil environment (Schmitt, Stoob,
Hamscher, Smit, & Seinen, 2006).

The discovery of tet(3 1) on fosmid TC3 represents only the second time this gene
has been detected in any environment and the first identification in soil. This is likely not
due to the rarity of this sequence in the environment, but the lack of studies that
exhaustively search for tetracycline resistance gene variants. Out of the five studies that
analyzed tetracycline resistance by culture dependent methods and PCR or DNA
hybridization only one (Agerso & Sandvang, 2005) screened for the tet(31) gene, and this
was only after isolates first lacked tet(A), tet(B), or tet(C) meaning that isolates carrying
the A, B, or C variants and 31 would have been missed.

Currently little is known regarding the tet(31) variant. It was originally discovered on
plasmid pRASZ isolated from the fish pathogen Aeromonas salmonicida subspecies
salmonicida in Norway (L'Abee-Lund & Sorurn, 2000) and hence, the finding of tet(3 1)
on fosmid TC3 is quite interesting. When sub-clone end sequences of TC3 were matched
against NCBI’s non-redundant nucleotide database with BLAST (Altschul et al., 1997),
both ends matched a sequenced fragment of pRAS2 Surprisingly, homology was not
only over tet(31) and tetR(31) but also over ~ 600 bp of flanking sequence. The sequence
corresponding to base-pairs 350 — 950 in pRAS2 (accession number AJ250203) was not
annotated and so the sequence was translated and subject to protein BLAST analysis.
This revealed the presence of what looks to be gene fragments possibly formerly
involved in phenazine biosynthesis (Table 2.3). Phenazines are organic molecules that

exhibit broad spectrum activity against bacteria, fungi, and parasites that are produced by

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100

beneficial root colonizing Pseudomonas spp (Blankenfeldt et al., 2004). Further
upstream of bp 350 in the pRASZ sequence is the presence of a transposase gene (tnpA).
This implies that tet(31) may be resident on a transposon. Given that little is known
regarding the dissemination of tet(31) it is of further interest to investigate if the sequence
isolated on fosmid TC3 is resident on a transposon.

Other Genes of Interest Isolated on Fosmid Clones TC1-TC7. A number of
other genes of interest were sequenced from the ends of the original or sub-cloned
fosmids. In particular, transposase sequences indicative of the presence of a transposon
were identified near the end of fosmids TC1 and TC7 (Table 2.3). Furthermore, two
putative class 1 integrases were detected on TC4 and TC6. Integrases belong to the
tyrosine-recombinase family and facilitate the insertion of gene cassettes (containing a
gene and an attC site) into integrons at the attI site allowing expression driven by the Pc
promoter (Mazel, 2006). Although no tetracycline resistance genes have been found
residing inside integrons the finding of these sequences are consistent with previous
studies noting the linkage of integrons and transposons with tetracycline resistance
(Agerso & Sandvang, 2005; Bahl, Hansen, Goesmann, & Sorensen, 2007; Chopra &
Roberts, 2001).

Two strB genes encoding streptomycin phosphotransferases that confer resistance
to streptomycin were partially sequenced. Often strB is linked with strA and this gene is
required for high level resistance (Chiou & Jones, 1995; Sundin, 2002). Multiple
investigators have found the strA -strB genes linked to tetracycline resistance. The finding
of tet(31) in Aeromonas salmonicida plasmid pRAS2 was originally discovered due to an

investigation into strA -strB streptomycin resistance (L'Abee-Lund & Sorum, 2000).

101

Also, the study that discovered the first tetR(Y) repressor variant found strA-strB linked
in plasmid pABSS9 (Gordon et al., 2008b). Resistance linkage has also been observed in
isolates of E. coli with tet(B) on transposon Tn] 0 (Khachatryan, Besser, & Call, 2008),
with tet(M) in C itrobacter braakii, E. coli, and Providencia rettgeri, with tet(A) and
tet(W) in E. coli, and simultaneously with tet(O), tet(S), and tet(W) in Citrobacter
freundii (Srinivasan et al., 2008).

Fosmid TC6 has sequences with 100% amino acid identity to MerA and MerP
near one end (Table 2.3). These genes act in an operon, with a general structure of
merRT PAD, that confers mercury resistance by uptaking Hg(II) and reducing it to
gaseous Hg(O) allowing it to diffuse out of the bacterial cell (Barkay, Miller, & Summers,
2003). Specifically, merA encodes a cytosolic flavin disulfide oxidoreductase that uses
NAD(P)H as a reductant to reduce a Hg(II) dithiol derivative to Hg(O) (Barkay et al.,
2003). MerP is a small periplasmic mercury binding protein that is thought to act in
Hg(II) transfer by exchanging Hg(II) to two cysteine residues in a transmembrane helice
of MerT, an inner (cytosolic) membrane protein (Barkay et al., 2003).

Finding sequences of all of the above genes is consistent with the current
understanding of tetracycline resistance being: (a) commonly transferred by transposons
(Chopra & Roberts, 2001), (b) often linked with integrons and/or streptomycin resistance
genes (Srinivasan et al., 2008), and (c) the more recent understanding of co-selecting for
tetracycline resistance through metal resistance (Stepanauskas et al., 2006).

TC Resistant Bacterial Isolates. Bacterial isolates were cultured to complement
the metagenomic analysis of tetracycline resistance. As stated above, few investigators

have cultured resistant bacteria and identified the tetracycline resistance genes they carry.

102

Of the four studies that have attempted this, only one cultured isolates on agar media with
low nutrient concentrations (Ghosh & LaPara, 2007). This study alone represents 56% of
the unique gene/isolate combinations identified to date. The other three studies used
solely nutrient rich media (including LB, MacConkey agar, and TSA), short incubation
times (24-48 h), and high temperatures (35 — 37°C) (Agerso & Sandvang, 2005;
Srinivasan et al., 2008; Stine et al., 2007). Based on the observation that low nutrient
media and extended incubation time can increase viable cell counts (Davis, Joseph, &
Janssen, 2005) and recovered bacterial diversity, a strategy using such medias was
employed to try and recover greater breadth of tetracycline resistant bacteria from the site
# 21 soil.

With this in mind I attempted to recover isolates carrying tet(A), tet(C), tet(Y),

tet(30), and tet(31). As can be seen from Figure 2.7, LB yielded in the highest total

accumulation of colonies, followed by 1/10th R2A, and lastly, the SM media. Individual

analysis of each different media type revealed that pre-incubation of soil with the highest
level of tetracycline (1 mL of 40 ug/mL solution) consistently resulted in the highest

accumulation of resistant colonies demonstrating an enrichment of tetracycline resistant

microbes. For both LB and 1/10th R2A media, 50% of the colonies appeared on day 3,

the first day of visible cell growth. For media SM colonies did not appear until day 6

which is consistent with low nutrient concentrations. The observation that no visible

colonies appeared for the first 48 h on LB or 1/10th R2A indicates that 5 ug/mL

tetracycline was too high a concentration for many bacteria. Subsequent studies with

lower tetracycline concentrations may reveal a greater diversity of isolates.

103

Table 2.4 displays the 32 isolates selected for further analysis and their
phylogenetic assignment based on the Ribosomal Database Project (RDP) Classifier
(Cole et al., 2007). The diversity of isolates cultured was rather limited with only 11
genera represented. These included Microbacterium, Brevundimonas, Devosia,
Streptomyces, Pedobacter, Sphingobacterium, Luteimonas, Pseudomonas,
Stenotrophomonas, T hermomonas, and one unknown. Isolates were subsequently
screened by PCR for the presence of tet(A), tet(B), tet(C), tet(D), tet(B), tet(G), tet(H),
tet(J), tet(X), tet(Y), tet(Z), tet(30), tet(31), and clone 397. Five genes were found in the
isolates including tet(A), tet(C), tet(Y), tet(X), and tet(31).

Given the limited scope of this investigation a surprising number of novel
observations were made. This study represents the first isolation of tet(A) in
Microbacterium spp. (or any member of the Actinobacteria), tet(Y) in Pseudomonas spp.,
tet(C) and tet(31) in Stenotrophomonas spp., and tet(A) and tet(C) in T hermomonas spp.,
the first report of TC resistance in this microbe. This is also the first report of tet(Y) and
tet(31) being isolated from any cultured soil bacterium (Table 1.2).

The two Sphingobacterium spp. isolates were screened for tet(X) based on
Ghosh & LaPara’s (2007) finding that a soil-borne Sphingobacterium spp. harbored
tet(X). My discovery of tet(X) in Sphingobacterium spp. marks the second such finding.
The tet(X) gene was originally isolated from a Bacteroidesfragilis R plasmid but was
inactive in this strict anaerobe due to the protein’s requirement for oxygen (Guiney,
Hasegawa, & Davis, 1984). Transfer of tet(X) into aerobically grown E. coli allows the
protein to detoxify tetracycline (Speer & Salyers, 198 8). This 44-kDa cytoplasmic

soluble protein is a flavin dependent monooxygenase requiring FAD, NADPH,

104

Magnesium, and Oxygen to detoxify tetracyclines by regiospecifically hydroxylating
carbon 11a which causes this product to break down intracellularily (Yang et al., 2004).

Interestingly, carriage in E. coli has been shown to confer increases in tigecycline
resistance. This resistance is conferred by hydroxylation of carbon 11a, resulting in
reduced affinity for magnesium and hence, reduced affinity for the ribosome (Moore,
Hughes, & Wright, 2005). This is concerning since tigecycline, a tetracycline (more
specifically a glycylcycline), was recently approved for use by the FDA in (Shlaes,
2006). Although resistance increases were slight, it has been hypothesized that mutations
leading to increased enzymatic activity and transfer to clinically relevant organisms could
be detrimental to tigecycline’s effectiveness (Moore et al., 2005).

Originally tet(X) was hypothesized to have little clinical relevance since it is
inactive in Bacteroides spp. (Chopra & Roberts, 2001), however its discovery in
Sphingobacterium spp. isolates from geographically distinct locations, though both from
farm soil impacted with tetracycline contaminated manure, should spark renewed interest
in this gene. The G+C% content of B. fragilis, the organism within which tet(X) was
originally isolated is approximately 42%, considerably higher than the 37% G+C content
of it’s tet(X) variant. Because of this Speers and colleagues hypothesized that B. fragilis
was not the original host (Speer, Bedzyk, & Salyers, 1991). The %G+C of the genomes
of studied members of the genus Sphingobacterium ranges from 37.3 to 44.2% (Yoo et
al., 2007) and this may be the first clue in determining if indeed Sphingobacterium spp.
are the native host for tet(X). Currently it is unknown if this gene is resident on a mobile

genetic element in Sphingobacterium spp. and hence warrants future investigation.

105

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L’Abee-Lund & Sorum (2000) first reported tet(3 1) in an R plasmid (pRAS2) from the
fish pathogen Aeromonas salmonicida subspecies salmonicida. The finding of tet(3 1) in
Stenotrophomonas spp. is the second discovery of this gene in a bacterial isolate and the
first from soil. This demonstrates how little is known regarding the distribution of many
of the tet resistance elements. The tet(C) gene was also found in Stenotrophomonas spp.
which brings the total number of tet genes found in this genus to five (tet(C), tet(G),
tet(L), tet(31), and tet(3 5)), all efflux genes (Table 1.1 and 1.2). Tetracycline resistance
in Stenotrophomonas spp. is particularly concerning given that one member of the genus,
Stenotrophomonas maltophilia, is an opportunistic nosocomial pathogen of increasing
importance. Infection with this organism is associated with significant morbidity and
mortality, particularly in immunocompromised or immunosuppressed patients (Safdar &
Rolston, 2007). Isolation rates of S. maltophilia associated with infectious diseases have
been increasing since the early 19705 (Senol, 2004). The two most common clinical
manifestations are bacteraemia and pneumonia, however, infection is also associated with
a number of other diseases (Senol, 2004).
CONCLUSIONS AND FUTURE DIRECTIONS

Although this study failed in its goal of recovering full coding sequences of
clones 397 and 492, it did achieve the goal of expanding the current understanding of the
dissemination of tetracycline resistance elements in soil environments. A number of
novel observations were made. The discovery of tet(30) on fosmid TC1 represents the
first time this gene has been linked with another TC resistance gene (tet(A)) and possibly
the first time a functional repressor protein has been identified for this gene. Similarly,

the finding of tet(3 1) on fosmid TC3 is only the second time this gene has been isolated

110

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112

and the first time from soil.

A number of other new gene/isolate associations were made. This study
represents the first time tet(Y) has been discovered in a culturable soil isolate (Table 1.2)
and the first identification of this gene in Pseudomonas spp. The finding of tet(A) and
tet(C) in T hermomonas spp. is the first identification of any tetracycline resistance
elements in any member of this genus. Discovery of tet(C) and tet(31) marks the first
finding of these genes in Stenotrophomonas spp. This is also the first time tet(3 1) has
been discovered in a genus other than Aeromonas spp. Lastly, the identification of tet(A)
in Microbacterium spp. is novel.

The reason that such a small survey was able to identify so many novel findings is
most likely due to the lack of studies that investigate soil environments and use molecular
methods to identify tetracycline resistance elements. Furthermore, when such studies do
identify resistance elements the search is rarely exhaustive. Few studies come close to
attempting to identify the presence of the 38 known tetracycline resistance elements. In
fact, in the current literature there is only one study that actively screened tetracycline
resistant soil isolates for the presence of tet(3 l) (Agerso & Sandvang, 2005) and no
studies that screened for tet(30) or tet(Y). Exhaustive search is time consuming and
expensive. I believe that the culture independent metagenomic approach used in this
study was the key to identifying these genes in soil environments. By first attempting
recovery of functional genes by this method the bias and problem of work reduction to
identifying genes by PCR was eliminated. It must be noted that although metagenomic
library construction eliminates primer bias, its downside is the potential inefficiency of

heterologous expression in the host cell. The presence of any RPPS are conspicuously

113

absent from the metagenomic library. These genes are thought to have originated from
Gram-positive microbes (Connell et al., 2003) and the lack of presence in this library may
be due to inefficient gene expression in E. coli.

It was postulated that cultivating bacteria on lower nutrient media would allow
recovery of novel gene/isolate combinations. Again, this has only been attempted once in

a survey of the cultivatable tetracycline resistant fraction (Ghosh & LaPara, 2007). In my

. th .
study few isolates were recovered on 1/10 R2A medla that were not recovered on LB

media (Table 2.4). Selection of appropriate media is a difficult decision and each type
has its own inherent biases. Unfortunately no isolates grown on the SM medium grew
when transferred to liquid culture. Future studies should use a more diverse selection of
low nutrient media to attempt to recover a breadth of novel isolates.

This investigation has opened the door for a number of interesting follow-up
studies. Of particular interest are:

1) Given the length of sequence coverage between the tet(31) region on

fosmid TC3 and plasmid pRASZ, determining whether this gene resides

on a mobile element.

2) Investigate if the tet(30) gene resident on fosmid TC1 has a functional
repressor protein.
3) Determine if the genes identified in isolates are present on mobile

elements such as transposons and plasmids. The knowledge of whether
tet(X) and tet(31) reside on mobile elements in Sphingobacterium spp.
and Stenotrophomonas spp. would be useful. The linkage of tet(A) and

tet(C) in Pseudomonas spp. and T hermomonas spp. implies that these

114

genes are on a mobile element. Further investigation of this is
warranted.

4) Given that tet(X) has been shown to confer slight resistance to
tigecycline in E. coli (Moore et al., 2005), it would be interesting to
conduct evolution experiments to determine if mutations can confer
higher levels of resistance, and if so, by what mechanism.

In conclusion, this study further exemplifies the role of soil environments as a
reservoir of tetracycline resistance genes. The finding of a number of novel observations
with a study of such limited scope demonstrates the lack of information regarding the
environmental distribution of tetracycline resistance, particularly in soil systems. It
appears that microbiologists are still at the tip of the ice-berg regarding the role of
resistance in the environment. Further characterization is necessary before any comment
can be made on the role of tetracycline residues promoting tetracycline resistance in the

environment.

115

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CHAPTER III
AN ANALYSIS OF TETRACYCLINE - CATION CHELATION AND ITS
RELATION T O BIOAVAILABILITY AND TETRACYCLINE EFFLUX GENE
EXPRESSION
INTRODUCTION
Tetracycline antibiotics are used for a variety of purposes including human

therapy (Smilack, 1999), animal therapy and growth promotion (Gaskins, Collier, &
Anderson, 2002), aquaculture (Cabello, 2006), and plant agriculture (McManus,
Stockwell, Sundin, & Jones, 2002). Ultimately un-altered tetracyclines make their way to
the environment through a variety of pathways including surface runoff (Pruden, Pei,
Storteboom, & Carlson, 2006), WWTP discharges (Karthikeyan & Meyer, 2006), and
manure application as fertilizer (Hamscher, Sczesny, Hoper, & Nau, 2002). Not
surprisingly, multiple investigators have measured tetracycline (TC) accumulation and
the presence of resistant bacteria and their genes in terrestrial and aquatic environments
including; soil (Ghosh & LaPara, 2007), surface water (Goni-Urriza etal., 2000),
groundwater (Chee-Sanford, Aminov, Krapac, Garrigues-Jeanj ean, & Mackie, 2001),
sediment (N eela, Nonaka, & Suzuki, 2007), and even dust particles inside concentrated
animal feeding operations (CAFOs) (Chapin, Rule, Gibson, Buckley, & Schwab, 2005;
Hamscher, Pawelzick, Sczesny, Nau, & Hartung, 2003) (Tables 1.1 — 1.5). These
observations have created concern that the environmental accumulation of TC residues
may be enhancing the development, maintenance, and transfer of TC resistance to human
and animal pathogens (Kummerer, 2004). This scenario would be especially problematic
because of (a) reduced effectiveness of TC to eliminate bacterial infections and (b) the

economic consequences of pathogen resistance.

121

Tetracyclines cross the outer membrane of gram-negative enteric bacteria through
OmpF and OmpC porin channels as positively charged metal-tetracycline coordination
complexes (Chopra & Roberts, 2001). In the periplasm the metal-tetracycline complexes
dissociate and the slightly lipophilic tetracycline molecules diffuse through the
cytoplasmic membrane (Chopra & Roberts, 2001). Transfer across Gram-positive
cytoplasmic membranes is assumed to occur in the same manner with the electroneutral,
lipophilic form being the species transferred (Chopra & Roberts, 2001). Uptake across
the cytoplasmic membrane is energy dependent and driven by the ApH component of the
proton motive force (Chopra & Roberts, 2001). Once in the cytoplasm, tetracyclines
reversibly bind to the 16S ribosomal RNA (rRNA) of bacterial ribosomes near the
acceptor A site, preventing aminoacyl-tRNA binding (White, Alekshun, McDermott, &
Levy, 2005). Peptide elongation is halted, and growth is subsequently inhibited.

To evade this grth inhibition, bacteria have developed, maintained, and
transferred tetracycline resistance genes (TcR) over the course of evolution. Currently
four genetic mechanisms are known; the two most widely disseminated being ribosomal
protection (RPPS), and tetracycline efflux (Chopra & Roberts, 2001). Tetracycline efflux
genes encode membrane bound pumps, regulated by tetracycline inducible repressor-
operator systems (TetR-tetO), that confer resistance to tetracycline by an energy-
dependent efflux of a magnesium chelated tetracycline molecule in exchange for a proton
(Chopra & Roberts, 2001). The RPPs quench TC’s effect on the bacterial cell by
dislodging TC from the ribosome in a GTP dependent manner (Connell, Tracz, Nierhaus,
& Taylor, 2003). The presence of these genes on mobile genetic elements such as

conjugative plasmids and transposons (Chopra & Roberts, 2001) has allowed the

122

horizontal transfer of resistance to diverse genera (Tables 1.1 — 1.5). Selective pressure,
provided by TC, can enhance horizontal transfer of TcR genes (Whittle, Shoemaker, &
Salyers, 2002). Furthermore, it has been demonstrated that once established tetracycline
resistance can persist even after the antibiotic is withdrawn (Langlois, Cromwell, Stahly,
Dawson, & Hays, 1983). Phenomenon such as compensatory mutations (Nguyen, Phan,
Duong, Bertrand, & Lenski, 1989), co-selection (Tuckfield & McArthur, 2007), cross-
resistance (Levy, 2002), and beneficial physiological functions of genes (Krulwich, Jin,
Guffanti, & Bechhofer, 2001) have all aided in the maintenance of resistance in the
absence of selective pressure.

In order for TC residues to exert their selective pressure they must be bioavailable
to the indigenous bacteria. Here, bioavailability is defined as the ability of tetracycline to
leave the environmental matrix, penetrate the microbial cell ’s membrane, and act on its
target. Cm'rently it is unknown what factors control the bioavailability of TC residues
found in environmental systems.

Tetracyclines are amphoteric, adaptable molecules that can form a variety of
different tautomers in solution (Sassman & Lee, 2005). They consist of a linear fused
tetracyclic nucleus (A, B, C, D) to which a variety of functional groups are attached
(Mitscher, 1978). 6-deoxy-6-demethyltetracycline is the minimum pharmacore that
retains antibacterial activity (Chopra & Roberts, 2001). The presence of three ionizable
functional groups allow tetracyclines to exist as four different species in solution
(cationic, zwitterionic, and two anionic species) with the relative distribution dependent
upon pH. For a review of pH dependent TC speciation see Sassman & Lee, 2005. A

significant amount of research has been conducted on tetracyclines’ ability to chelate

123

cations in solution. Current theory suggests that chelation occurs in the lower peripheral
region between 012 and 01 oxygen groups on the BA rings and the 010 and 011
oxygen of the DC rings for calcium; whereas the 011 carbonyl group and 012 enol group
is thought to bind magnesium (Nelson, 1998). The presence of divalent and trivalent
cations in solution with tetracycline tends to raise the minimum inhibitory concentration
(MIC) of diverse bacteria (Avery, Goddard, Sumner, & Avery, 2004; Chopra & Howe,
1978; Nanavaty, Mortensen, & Shryock, 1998). Furthermore, it has been demonstrated
that magnesium inhibits bacterial uptake of TC and that this effect is pH dependent
(Yamaguchi, Ohmori, Kaneko-Ohdera, Nomura, & Sawai, 1991). Therefore, it seems
likely that TC’s ability to chelate cation’s would prevent the antibiotic from entering the
cell and activating expression of the TC-efflux genes by de—repression the TetR protein.
All of the above evidence is qualitative and no studies have attempted to quantify the TC
— cation chelation effect and how it relates to gene expression in a live microbial cell.
Given that calcium, magnesium, sodium, and potassium are the predominant cations
present in soil systems it seems that the geochemical characteristics of a given
environment could modulate TC bioavailability and hence the effect on the native
microbial flora. Characterizing this dynamic is essential to understanding the risk of the
environmental accumulation of tetracyclines.

At present date only two studies have implicitly addressed the concept of TC
bioavailability in natural systems (Chander, Kumar, Goyal, & Gupta, 2005; Verma,
Robarts, & Headley, 2007). Neither of these studies provides any mechanistic
understanding of the factors that control tetracycline bioavailability. The development of

bioreporter technology over the past 15 years allows the question of what factors control

124

bioavailability to be addressed. A bioreporter (a.k.a. biosensor), in simple terms, is a cell
that has a reporter gene (typically gfp, lacZ, or bacterial luciferase) that responds to an
environmental stimulus through a fused promoter (Fig. 3.2). Bioreporters have been
employed to measure metals, antibiotics, temperature, water potential, and quorem
sensing compounds (Leveau & Lindow, 2002).

This study was designed to provide insight into how solution phase TC-cation
chemistry affects microbial cell TcR gene expression. This was accomplished by the use
of a whole-cell tetracycline biosensor developed by Dr. Soren Sorensen’s group at the
University of Copenhagen (Bahl, Hansen, & Sorensen, 2005), and a quantitative model
for promoter activity developed by Dr. Steven Lindow’s group at UC-Berkeley (Leveau
& Lindow, 2001). Briefly, the biosensor used in this work is a live Escherichia coli cell
containing a plasmid, pTGM, with a transcriptional fusion between a tetracycline
inducible promoter (Ptet(A)) and a gfi) gene (Bahl et al., 2005). Two important aspects of
this construct include:

o The architecture of the Ptet(A) promoter is completely maintained as it
was originally isolated from Tn10. Therefore, gfp expression is
proportional to the Ptet(A) activity that drives antibiotic resistance gene
expression in a natural setting.

0 The pTGM construct contains a tetracycline resistance gene, tet(M), to
inhibit tetracyclines from killing the cell (Bahl et al., 2005). This gene is a
ribosomal protection protein that dissociates TC from the ribosome upon
GTP hydrolysis (Connell et al., 2003). Therefore the intracellular

concentration of TC remains unchanged.

125

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Bioavailable tetracyclines, once in the cytoplasm, will bind the transcriptional
regulator, TetR, inducing a conformational change releasing it from the two operator
regions of the Ptet(A) promoter (Ramos etal., 2005). As a result the gfi) gene is
expressed (Figs. 3.1 and 3.2). In the present study this biosensor strain has been used to
quantitatively analyze the activity of the Ptet(A) promoter (which mimicks tetracycline
efflux gene expression) in the presence of tetracycline complexed with environmentally
relevant cations (magnesium, calcium, sodium, and potassium) at environmentally
relevant pH’s (5, 7 and 9) in the solution phase.

MATERIALS AND METHODS

Bacterial Strain & Culture Conditions. Escherichia coli strain MC4100/pTGM
(Bahl et al., 2005) was used as a whole-cell bacterial biosensor for this study. Plasmid
pTGM contains the Ptet(A) promoter originating from Tn10 fused to gfiymut3 encoding a
flow cytometry optimized gfp gene.

Culture media was as follows: 10 g tryptone (Accumedia, Lansing, MI), 5 g yeast
extract (Accumedia, Lansing, MI), and 0.5 g NaCl (J .T. Baker, Phillipsburg, NJ) per liter.
To this pH 5.0 included 100 mM 2-(N-morpholino)ethanesulfonic acid (MES, free acid;
Calbiochem, Darmstadt, Germany) buffer, pH 7.0 included 50 mM 3-(N-
morpholino)propanesulfonic acid (MOPS; Sigma-Aldrich Inc., St. Louis, MO) buffer,
and pH 9.0 included 100 mM 3-[(1,1-dimethyl-2-hydroxyethly)amino]-2-
hydroxypropanesulfonic acid (AMPSO, free acid; Research Organics, Cleveland, OH)
buffer. Good’s buffers were chosen because of their low interaction with metal ions

(Good et al., 1966).

128

For promoter activity experiments cells were inoculated and incubated at 30°C
overnight in the same type of media to be used for the assay the following day with 100
ug/mL ampicillin (Sigma-Aldrich Inc., St. Louis, MO) to maintain the pTGM plasmid.
The following morning an appropriate amount of media was transferred to a sterilized
1000 mL Erlenmeyer flask. To this 100 ug/mL ampicllin was added. The flask was
stirred well and three 25 mL aliquots were transferred to sterile 125 mL Erlenmeyer
flasks to measure leaky expression of the Ptet(A) promoter in the absence of tetracycline .
To the remaining media an appropriate amount of tetracycline (Sigma-Aldrich Inc., St.
Louis, MO) was diluted from a 5 mg/mL stock solution and added to achieve a final
concentration of 0.1 ug/mL (100 ppb). The flask was again stirred and 25 mL aliquots
were transferred to three sterile 125 mL flasks representing 100% Ptet(A) expression at
0.1 ug/mL tetracycline. Media was then subdivided to five 80 mL fractions in 250 mL

Erlenmeyer flasks. To these appropriate amounts of 1M stock solutions of cations

(MgC12, CaC12, NaCl, KCl; J .T. Baker, Phillipsburg, NJ) were added to achieve final

cation concentrations of 0.1 mM, 0.125 mM, 0.25 mM, 0.375 mM, 0.5 mM, 1 mM, 5
mM, and 10 mM. Media from these 80 mL flasks was then subdivided into three 125 mL
Erlenmeyer flasks each receiving 25 mL of media. This extensive procedure was done to
reduce variation introduced into the assay due to pipeting error. After all flasks were
aliquoted for the days’ experiment they were incubated in a horizontal shaker (150 rpm)
at 30°C to equilibrate for 30 minutes.

After equilibration a 1/ 100 dilution of the overnight culture of the Escherichia
coli strain MC4100/pTGM bioreporter was made into each 125 mL Erlenmeyer flask

with the appropriate assay media. Cultures were then grown in triplicate exponentially in

129

125 mL Erlenmeyer flasks in at 30°C while shaking at 150 rpm. 1 mL aliquots were
periodically taken for analysis by spectroflourimetry (gjpmut3 excitation = 488; emission
= 511) and spectrometry (OD600) performed with a SpectraMax M2 spectroflourimeter

(Molecular Devices, Sunnyvale, CA).

Experiments in which MgC12 and EDTA ((ethylenedinitrilo)tetraacetic acid,

disodium salt, dehydrate; J .T. Baker, Phillipsburg, NJ) were added mid-culture were set
up as described above. At appropriate times (as indicated in Figure 3.14) magnesium
chloride was added from a 1M stock to achieve a final concentration of 10 mM.
Similarly, EDTA was added from a 250 mM stock to achieve a final concentration of 10
mM.

Promoter Activity Calculations. Promoter activity is arrived at by using
equation 13 (P = fss x u x (l + (p/m))) from Leveau & Lindow (2001). Fluorescent
steady state (fss) is calculated by the slope of an F’OD plot (OD600 plotted against
relative fluorescence units (RFU)). Growth rate (a) is determined by plotting the log of
OD600 by time (hours) and taking the slope of that line. A value of 1.54 h‘1 was used for
the GFP maturation constant (m) (Leveau & Lindow, 2001). A value for promoter
activity is arrived at with units RNU/OD/h (relative non-fluorescent units of GFP per OD
unit X unit time). Scatter plots were made and linear regression was performed with
Microsoft Excel. All R-squared values ranged from 0.95 - 0.999 (data not shown). All
graphs for the present study were created with Microsoft Excel.

1 Analysis of intracellular TC. Reagents and Chemicals. All chemicals were at least
of analytical reagent grade, and deionized water was used throughout, unless stated

otherwise. Tetracycline hydrochloride was purchased from Sigma-Aldrich Inc., St. Louis,

130

MO. HPLC grade Methanol was purchased from J .T.Baker, Phillipsburg, NJ. McIlvain
buffer (pH 3.8) consisted of 10.9 g of disodium hydrogen phosphate, 37.2 g
ethylenediarninetetraacetic acid (EDTA) sodium salt, and 12.9 g citric acid monohydrate
per 1L of water. All tetracycline hydrochloride solutions were protected from light and
prepared fresh before each experiment.

Tetracycline was extracted from cell pellets with an Oasis HLB solid phase
extraction (SPE) column (Waters Inc., Milford MA) and analyzed with an LC-20 solvent
delivery unit (Shimadzu, Kyoto, Japan) coupled with an API 3200TM LC/MS/MS
(Applied Biosystems, Foster City, CA).

Sample Preparation. After cell cultures’ promoter activity was analyzed (OD600 of
approximately 0.8) cell cultures were centrifuged at 10,000 rpm to pellet cells and were
subsequently washed twice with PBS buffer. Supernatant was removed and cell pellets
were freeze dried overnight. Freeze dried cells were mixed with lOmL of McIlvain
buffer, vortexed for 1 min, and then sonicated for 10 min. This solution was centrifuged
at 10,500 rpm for 20 min. Supematants were filtered through membranes (0.45 um pore
size) to eliminate cell material. Centrifugation and filter steps were repeated and filtrate
was used for analysis.

Solid Phase Extraction. Before tetracycline extractions, SPE cartridges were
preconditioned with 3 mL of methanol, followed by 3 mL of 0.1N of hydrochloride acid
and 6 mL of pure water. Sample filtrate (10 mL) was passed through cartridges at the
flow rate of 2 mL per min. After sample loading, the cartridges were washed with 3 mL
of pure water and dried with nitrogen gas for 30 min. Tetracycline was eluted with 10 mL

of a methanol — water mixture (1:1) containing 150 ppm EDTA.

131

Liquid Chromatograpy-Mass Spectrometry. The tetracycline elutions (10 uL) were
separated by an LC-20 solvent delivery unit (Shimadzu, Kyoto, Japan) using a Gemini
C18 column (5 pm, 50 * 2.00 mm; Phenomenex Inc., Torrance, CA). LC mobile phase A
consisted of 95% water, 5% methanol, 20mM heptafluorobutyric acid (HF BA), and 2
mM ammonium acetate. LC mobile phase B consisted of 95% methanol, 20 mM
heptafluorobutyric acid (HFBA), and 2 mM ammonium acetate. The flow rate was 0.5
mL per min. The gradient is shown in Table 3.1. LC was directly interfaced to the
electrospray ionization source (ESI) coupled with Applied Biosystems API 3200TM
LC/MS/MS system. Two ion pairs were used to confirm the analyte (tetracycline), and
one ion pair (445.000/410.000) was used for the purpose of quantitation.

Epiflourescent Microscopy. Overnight cultures of E. coli strain MC4100/pTGM
were grown at 30°C and 150 rpm in standard LB (Accumedia, Lansing, MI)
supplemented with an appropriate amount of tetracycline (Sigma-Aldrich Inc., St. Louis,
MO) and magnesium chloride (J .T. Baker, Phillipsburg, NJ) (Figs. 3.1 and 3.3). Cultures
were then diluted appropriately and epifluorescent images of 10 uL wet mounts were
taken with a Zeiss Axioskop 2 Microscope (Carl Zeiss Inc., Thomwood, NY) fitted with
a Spot 2 cooled CCD camera (Diagnostic Instruments, Sterling Heights, MI), a Zeiss
Universal Arc-lamp Power Supply with mercury burner (Carl Zeiss Inc., Thomwood,

NY), and using Zeiss Filter set 09 (Carl Zeiss Inc., Thomwood, NY) for GFP detection.

132

Table 3.1. Liquid Chromatography Separation Gradient for Analysis of
Intracellular Tetracycline.

 

 

 

 

 

 

 

Time %B
0 5

1 5

3 95
5.5 100
6 5

8 7

 

 

 

 

133

RESULTS AND DISCUSSION

Verification of Functionality of Bioreporter. Upon receiving the E. coli
MC4100/pTGM biosensor (Courtesy of Dr. Lars Hansen, University of Copenhagen) it
was necessary to demonstrate that the GFP response was dependent on the concentration
of tetracycline present, and simply not an on-off switch. To do this the biosensor was
grown overnight in the presence of increasing concentrations of tetracycline. The
following morning cultures were diluted and imaged by epifluorescent microscopy.
Figure 3.1 shows a typical result of this experiment. As expected the brightness of GFP
increases with increasing tetracycline concentration.

Multiple observations have been made that suggest that TC — cation complexes
increase the minimum inhibitory concentrations (MICs) because tetracycline is unable to
enter the cell under such conditions (Avery et al., 2004; Nanavaty et al., 1998). If this is
true, then gfpmut3 gene expression from the pTGM plasmid should be reduced when the
bioreporter is grown in media with tetracycline and cations. Furthermore, cation
concentration dependent titration of this response should be possible. To confirm this
assumption, overnight cultures of the E. coli strain MC4100/pTGM whole cell biosensor
were grown in the presence of 0.1 ng/mL (100 ppb) tetracycline and various
concentrations of magnesium chloride (0, 0.1, 0.5, 1, 5, and 10 mM). Again, the
following morning cultures were diluted and imaged via epifluorescent microscopy (Fig.
3.3). As expected, the brightness of GF P decreases with increasing magnesium chloride
concentration.

Determining the Range of Linear Response in Promoter Activity at pH 5.0,

7.0 and 9.0. Given that the substrate being measured (TC) has the ability at sufficient

134

 

Figure 3.3. Fluorescent Images of pTGM Bioreporter in the Presence of Increasing
Magnesium Chloride Concentrations.

concentration to inhibit the response (GFP protein), it was first necessary to determine the
range of TC concentrations in which the response remained linear. Based on previous
investigations into the induction of TetR from the tet operator (tetO) it is expected that
the plot of TC concentration versus promoter activity (P) (calculated as described in
Materials and Methods) should have a sigmoid shape (Lederer, Takahashi, & Hillen,
1995). If experiments were conducted at tetracycline concentrations outside of the linear
range it would under or overestimate the percentage change in promoter activity due to
decreasing or increasing cation concentrations, respectively. As expected the plots at pH

5.0, 7.0 and 9.0 all are sigmoid (Figs. 3.4 to 3.6) and demonstrate a clear linear range.

135

Based on these plots 0.1 ug/mL (100 ppb) tetracycline was selected as the concentration
at which further experiments were conducted.
Cation Chelation Effect on Promoter Activity. Tetracycline in solution can

complex with cations, anions, and biopolymers (Durckheimer, 1975). The following

. . . . 3+ 3+ 2+ 2+ 2+
cations complex in order of decreasrng affinity: Fe > A1 = Cu > C0 = Fe >

Zn2+ > Mn2+ > Mg2+ > Ca2+ (Nelson, 1998). Previous observations of multivalent

cation’s ability to raise minimum inhibitor concentrations (MICs) of tetracyclines suggest
that cation complexation prevents tetracycline from entering the cell and acting on the
bacterial ribosome. From these observations it can be postulated that at a constant TC
concentration increasing the cation concentration should decrease promoter activity.

Solution chemistry studies have shown that at pH 5.0 the predominant tetracycline

species is HZTC. As pH increases towards 7.0 a greater percentage of the TC is in the

form of HTC-, and further pH increases towards 9.0 results in a mix of HTC- and TCZ-

species. Furthermore, studies by Werner et al. (2006) demonstrate that a raise in pH
corresponds with a raise in cation chelation by TC. Based on these observations it can be
predicted that at pH 5.0 cations would have minimal effect on promoter activity because
they would be unable to complex with tetracycline, and hence, decrease uptake across the
membrane. However, as pH shifts higher reduction of promoter activity due to cation
chelation would become stronger and measurable. Given the observation of cation
affinity for TCs (Nelson, 1998) and that sodium was unable to raise TC MICs

(N anavaty, Mortensen, & Shryock, 1998), the following experiment was conducted with

the expectation that the effect in decrease in promoter activity would be as follows for pH

136

7.0 and 9.0 (greatest effect to least effect): Mg2+ > Ca2+ > Na+ = K+. It was also

expected that at pH 5.0 Mg2+ and Ca2+ would be unable to repress promoter activity.

To determine the effect that these cations have on tetracycline bioavailability in
solution, 12 separate experiments were conducted (Fig. 3.7 — 3.10). Promoter activity is
plotted as a percentage of full induction on the y-axis (full induction, or 100%, meaning
promoter activity at 0.1 ug/mL TC with no cation) and cation concentration is plotted on
the x-axis. Cation concentrations tested included 0, 0.1, 0.5, 1, 5, and 10 mM.

As expected, at pH 7.0 and 9.0 the presence of divalent cations calcium and magnesium
strongly decreases promoter activity. Comparison of the magnesium and calcium plots at
pH 7.0 reveals that the effect of magnesium is stronger than that of calcium (discussed
below). At a glance, it appears that at both pH 7.0 and 9.0 a 50% reduction in promoter
activity is reached earlier when magnesium is present in the medium than when calcium
is present. Given that at pH 9.0 the TC solution species become predominantly more
negatively charged, and hence should have a greater affinity for cations, one might expect
that at pH 9.0 the effect of magnesium should be greater than the effect of magnesium at
pH 7.0, and similarly for calcium. It appears that there may be some slight evidence of
this happening with magnesium; however, large measurement variability at pH 9.0
prevents this comparison from being made. This increase in relative variability at pH 9.0
and 5.0 for normalized values is due to the decrease in raw promoter activity values. It is
possible that cellular stress responses due to grth at pH extremes is causing the
significant decrease in promoter activity at pH 5.0 and 9.0 as compared to pH 7.0 at the
same TC concentration (Maurer, Yohannes, Bondurant, Radmacher, & Slonczewski,

2005) (Fig. 3.11). Despite this, it is still clear that at pH 5.0 the effect of magnesium and

137

calcium is strongly minimized (Fig. 3.7 — 3.10). This is expected given that affinity for

cations towards TC reduces with reducing pH (J in et al., 2007). The predominant species

of TC at pH 5 would be H2TC, a neutral species in which pKal is negatively charged,

pKa2 is neutral, and pKa3 is protonated. As pKa2 and pKa3’s functional groups become
deprotonated at higher pH there is a corresponding increase in cation affinity. It is also
observed that sodium and potassium fail to reduce promoter activity at all pH’s, as would
be expected if they were unable to complex with TC.

Effect of Magnesium Chloride an Tetracycline Uptake. To conclusively
determine that the reason added magnesium chloride reduced promoter activity was due
to decreased cellular uptake of TC, an experiment was conducted in which LC-MS/MS
was used to measure the intracellular TC concentration after promoter activity
measurement in the presence of magnesium chloride. Cells were grown in the presence
of TC or TC + 10 mM magnesium chloride in LB at pH 7.0 and promoter activity was
measured. As can be seen from Figure 3.12 and 3.13, the presence of 10 mM magnesium
chloride in the media greatly decreased the promoter activity and the intracellular TC
concentration, as would be expected if reduced promoter activity was due to reduced TC
accumulation inside cells. The intracellular concentrations for cells grown in the
presence of 10 mM magnesium chloride were 6.72 pg TC/ g dry cells (standard deviation
of 0.23) compared to 105.37 pg TC/g dry cells (standard deviation of 8.69). Based on the
percentage decrease in promoter activity, an intracellular concentration of 0.70 ug TC/ g
dry cells (in other words 0.66% of fully induced) would have been expected for the cells
grown in the presence of 10 mM magnesium chloride. It is known that biopolymers bind

TC and it is possible that the presence of magnesium increases this effect making

138

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washing steps unable to remove extracellular bound TC leading to a greater
measured intracellular TC concentration (Durckheimer, 1975).

Modulation of Promoter Activity by addition of Magnesium Chloride or
EDTA Mid-Growth. Another experiment was designed to corroborate the results of
experiments displayed in Figures 3.12 and 3.13 with the expectation that if magnesium
can chelate tetracycline and reduce cellular uptake, one should be able to modulate GF P
expression by adding magnesium chloride during mid-culture growth. Magnesium
chloride (final concentration of 10 mM) was spiked into medium containing 0.1 ug/mL
TC with the expectation that upon entry, the magnesium ions would complex with TC
and gjp expression would decrease to a level similar to a culture that was grown
continuously in the presence of 0.1 pg/mL TC and 10 mM magnesium chloride.
Similarly, if EDTA (known for its ability to chelate divalent cations) was spiked into a
culture grown in the presence of 0.1 ug/mL TC plus 10 mM magnesium chloride, it
would be expected that after EDTA addition gjp expression would increase. This would
be due to EDTA chelating magnesium stronger than TC thereby freeing TC to enter the
cell and activate gene expression.

Promoter activity was analyzed in 4 different conditions: no TC (negative control;
black diamonds, solid line), 0.1 ug/mL TC (black squares, solid line), 0.1 ug/mL TC plus
10 mM magnesium chloride (black triangles, solid line), and 0.1 ug/mL TC plus 10 mM
magnesium chloride plus 10 mM EDTA (black circles, solid line). Two separate cultures
were started for the 0.1 ug/mL TC and 0.1 ug/mL TC plus 10 mM magnesium chloride
conditions. At an appropriate time (indicated by the arrows, Fig. 3.14) 10 mM

magnesium chloride was added to one of the 0.1 ng/mL TC cultures (open squares,

142

pH5

pH7

pH9

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Figure 3.7. Promoter Activity Data for Magnesium at pH 5, 7, and 9. % promoter
activity on the Y axis, mM concentration of corresponding X axis. Performed in
triplicate, error bars represent 1 standard deviation.

143

 

 

 

 

 

 

 

 

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Figure 3.8. Promoter Activity Data for Calcium at pH 5, 7, and 9. % promoter
activity on the Y axis, mM concentration of corresponding X axis. Performed in
triplicate, error bars represent 1 standard deviation.

144

 

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activity on the Y axis, mM concentration of corresponding X axis. Performed in
triplicate, error bars represent 1 standard deviation.

145

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triplicate, error bars represent 1 standard deviation.

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dashed line) and 10 mM EDTA was added to one of the 0.1 pg/mL plus 10 mM
magnesium chloride cultures (open circles, dashed line).

As expected, after addition of either magnesium chloride or EDTA the slope of the F’OD
plot (representing fluorescent steady state) immediately changes to a level similar to
cultures grown continuously under the same conditions (Fig. 3.14). The presence of 10
mM EDTA greatly increases the slope of the POD plot (black circles and open circles)
compared to induction at 0.1 ug/mL TC (black squares). This is likely due to EDTA’s
ability to chelate cations that are not added, but are present in the assay media that
complex with some amount of the TC reducing gene expression (black squares).

A Closer Comparison of the effect of Magnesium and Caclium at pH 7.0. It
is known that TC — cation complexation is reversible (Durckheimer, 1975). Based on this
observation it is hypothesized that at a constant TC concentration each incremental
increase in the concentration of divalent cation should have incrementally less effect on
the percentage reduction in promoter activity (i.e. exponential decay). This would occur
because each increase in cation concentration corresponds with a decrease in the amount
of “free” TC molecules surrounding each individual cation. Therefore, each incremental
cation has less opportunity to interact with free, un-complexed TC than the previous
cation.

Recently, J in et al. (2007) used isothermal titration calorimetry to study the
binding of calcium and magnesium to TC. They found that each cation binds with
distinct stoichiometry; one calcium per TC and one magnesium per two TC molecules.
Furthemore, they conclude that this stoichiometry is invariant with pH (J in et al., 2007).

This means that at a given TC concentration the cation concentration required to give any

148

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percentage decrease in promoter activity should be twice as much for calcium as
compared to magnesium. If this holds true under the present experimental conditions,
and measurements are made in the linear response range for promoter activity, then if one
fits exponential decay functions to scatter plots of cation concentration (x-axis) vs.
percentage promoter activity(y-axis) for both magnesium and calcium it can be predicted
that the ratio of calcium to magnesium concentration should equal 2 at any given y-value.

To examine this relationship an experiment was conducted at pH 7.0 with cation
concentrations of 0.125, 0.25, 0.375, and 0.5 mM. Higher concentrations (i.e. 5 and 10
mM) were not used because at high cation concentrations the response (promoter
activity) is depressed enough that it is outside of the linear range (Fig. 3.5). As expected

plots of the data yielded curves that fit an exponential decay model well (Fig. 3.15). The

equation for magnesium: y = 100e-2'M4x. The equation for calcium: y = 100e-1'1693x.

The ratio of -2.144x to -1.1693x equals 1.83 with a standard deviation of 0.27 and a 95%
confidence interval of [1.65, 2.01] (a = 0.05). This is in good agreement with Jin et a1.
(2007) and as can be seen from the exponential decay plots for magnesium and calcium
at any y-value the x-value for calcium is always 1.83 times magnesium (Fig. 3.15).
There are multiple possible explanations for our inaccuracy and deviation from a
value of 2.0. First, J in et al. (2007) observed that the presence of sodium can have an
effect on calcium and magnesium affinity for TC depending on the pH. Although the
assay media is standard at any given pH, if sodium affects calcium and magnesium’s
affinity for TC differentially (as is demonstrated by J in et al. (2007)) this would skew the
shape of the exponential decay curves. Also, even though the culture media is buffered

the E. coli MC4100/pTGM it has been observed that throughout exponential growth the

151

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cells raise the medium pH to final values of approximately 7.1 (data not shown).
Differences between pH changes in separate batch cultures would introduce variance. It
is also possible that stoichastic fluctuations in gene expression in cell populations
between cultures could introduce variance. There is growing evidence that a growing
population of cells’ gene expression is not uniform and random fluctuations likely help
populations adapt to various environmental stresses (Losick & Desplan, 2008).
Observation of induced cells via flow cytometry at multiple time-points taken throughout
exponential growth confirms the presence of distinct populations; cells that express gfi)
and cells that do not (data not shown). Between culture variation in the proportion of cells
expressing gfp will introduce variance. Furthermore, TC is known to bind biopolymers
(Durckheimer, 1975). The assay was conducted in a rich media (see Materials and
Methods) and it is possible that media components binding free TC led to an uneven
distribution of TC upon subdividing the media to separate culture flasks.

If indeed the binding stoichiometry for calcium and magnesium is invariant with pH then
this relationship should also hold at pH 5.0 and 9.0. However, at pH 5.0 TC’s affinity for

cations would be extremely low given that the predominant species in solution is the

neutral form H2TC. It is likely impossible to detect this relationship at pH 5.0 with the E.

coli MC4100/pTGM bioreporter. In fact, J in et al. (2007) were unable to detect any
binding of calcium and magnesium at pH 5.0 with a 20-fold excess amount of cation

using isothermal titration calorimetry. At pH 9.0 TC would be more negatively charged

than at pH 7.0 with HTC- and TCZ“ being the solution species present. Given the

observation that calcium and magnesium affinity is higher for TC at higher pH due to

more negative charge it would be expected that the exponential decay curves would be

153

steeper than at pH 7.0. A 50% reduction in promoter activity should occur at a lower
cation concentration at pH 9.0, when compared to the same cation at pH 7.0.
Unfortunately, the cellular stress imposed on the bioreporter cells by growth at pH 9.0
precludes any accurate measurement of this effect. Visual comparison of the pH 7.0 and
9.0 graphs for magnesium in Figures 3.7 — 3.10 indicates that this may be occurring,
however, this does not appear to hold for calcium. This may be simply due to the
inability to obtain sufficiently accurate measurements at pH 9.0. If accurate measurement
were possible under present assay conditions it is expected that at pH 9.0 the ratio of
calcium to magnesium concentration would be less than 2.0. J in et a1. (2007) were able
to show that the presence of soditun alters the stoichiometry of magnesium binding
slightly at pH 8.5 and exhibits a larger increase at pH 9.5.
CONCLUSIONS AND FUTURE DIRECTIONS

It has been demonstrated that the E. coli MC4100/pTGM whole-cell biosensor is
an effective tool for investigating the bioavailability of tetracycline in the solution phase
under varying conditions (pH, cation, cation concentration). Most importantly, the
activity of the biosensor responds in a predictable manner based on tetracycline solution
chemistry. In summary, lowering pH increases the bioavailability of tetracycline in the
presence of calcium and magnesium. As pH increases, magnesium and calcium bind to
tetracycline and decrease its bioavailability. Futhermore, at pH 7.0 and 9.0 magnesium
exerts a stronger effect than calcium. In contrast, monovalent cations such as sodium and
potassium fail to bind TC at any pH and reduce bioavailability.

Often, bioreporters are looked at as a fast and cost-effective solution to

determining the presence and quantity of the substrate of interest in various matrices.

154

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Multiple groups have genetically modified bacterial cells to achieve lower detection
limits (Nivens et al., 2004). However, this overlooks the advantage inherent in utilizing a
live bacterial cell to assay bioavailability. That is, the conditions affecting gene
expression in a cell residing in a natural context can be examined quantitatively.
Tetracyclines have been measured in manure, soil, sediment, surface water, and
groundwater. Typically concentrations range from less than 1 ppb in groundwater and
surface waters to approximately 300 ppb in soil and up to 40 ppm in manure (Hamscher
etal., 2002; Kay, Blackwell, & Boxall, 2004; Kolpin et al., 2002). Minimum inhibitory
concentrations of tetracyclines for microbial cells are commonly in the ppm range. In
other words, with the exception of manure, three orders of magnitude more concentrated
than is found in environmental systems. Because of this difference, and the observation
that tetracyclines bind strongly to various clay minerals (Sassman & Lee, 2005), it is
often assumed that concentrations in environmental systems are too low to exert a
significant effect on microbial cells. However, this assumption may not always be valid.
Verma et a1. (2007) spiked river and wetland water with tetracycline and analyzed
inhibition of protein synthesis of the native microbial flora by 3H leucine incorporation.
They were able to demonstrate a highly significant reduction in protein production (p <
0.001) at a concentration as low as 10 ppb. Interestingly, it took a concentration of 4000
ppb to achieve a significant (p < 0.02) reduction in protein synthesis in wetland water
(V errna, Robarts, & Headley, 2007). Furthermore, they estimated that the actual “free”
concentration of tetracycline was approximately 5 0% of the spiked concentration. This
study demonstrates two points. First, differences in complex environmental matrices can

have vast consequences to tetracycline bioavailability. Second, the assumption that low

156

tetracycline concentrations fail to exert any effect on the native microbial flora may be
false depending on the characteristics of the natural system.

Not only is it possible that environmentally relevant concentrations of
tetracyclines can inhibit protein synthesis, but it has also been demonstrated that low
levels of tetracycline can stimulate the horizontal transfer of some tetracycline resistance
elements (Doucet-Populaire, Trieu-Cuot, Dosbaa, Andremont, & Courvalin, 1991; Rice,
Marshall, & Carias, 1992; Salyers, Shoemaker, & Li, 1995). Dr. Abigail Salyer’s group
has studied conjugative transposons in Bacteroides spp. and observed that low levels of
tetracycline can increase transfer of these transposons harboring the tet(Q) gene 100 to
1000-fold (Salyers, Shoemaker, & Li, 1995). The mechanism is not yet well understood,
and little research regarding similar transfer in environmental isolates has been
conducted, however, it seems plausible that this may occur in natural settings outside of
human and animal gastrointestinal tracts. This should raise concern that low level TC
contamination may facilitate the dissemination of resistance genes throughout the
bacterial domain.

It is also unknown if low TC levels in environmental systems can induce
tetracycline resistance gene expression. As described before, expression of the
tetracycline efflux pumps is controlled by the tetracycline inducible repressor protein
TetR. This type of control is conserved in all tetracycline-resistance encoding efflux
pumps in gram-negative bacteria (Roberts, 1996). Over-expression of these pumps will
destroy membrane potential and kill the cell (Eckert & Beck, 1989). Therefore, it is
imperative that expression is tightly regulated and only induced upon binding to

tetracyclines. This has selected for the evolution of a sensitive expression system in

157

which tetracycline binds to TetR with a 1000-fold higher affinity than to the bacterial
ribosome (Hinrichs et al., 1994), thereby allowing the cell to express the efflux pump and
reduce the intracellular tetracycline concentration before it can exert its negative effect on
the cell. Combined with the observation that 10 ppb tetracycline can significantly inhibit
protein synthesis of native microbial flora in a specific aquatic system (V erma et al.,
2007) it can be presumed that resistance gene expression would likely occur at a
bioavailable concentration much lower than 10 ppb.

It is also of interest to test the effect that cations have on various tetracycline
degradation products. Particularly relevant is anhydrotetracycline which has been
demonstrated to be present in soil (Aga et al., 2005). Anhydrotetracycline is an atypical
tetracycline that exhibits modest antibacterial activity but does not bind the ribosome.
Current research suggests that it disrupts the bacterial cytoplasmic membrane to exert is
bacteriocidal effect (Chopra & Roberts, 2001). The structural differences of
anhydrotetracycline reduce its binding capacity to ribosomes, however, it retains affinity
for TetR and induces gene expression roughly 1000X stronger than TC (Lanig, Othersen,
Beierlein, Seidel, & Clark, 2006). Although seemingly counterintuitive, E. coli strains
harboring multicopy plasmids with tet(B) are less resistant to 5a,6-anhydrotetracycline
than TC sensitive strains (Moyed, Nguyen, & Bertrand, 1983). This decreased resistance
is likely due to over-expression of efflux pumps that then destroy the membrane
potential. By using anhydrotetracycline (which does not affect protein synthesis) to
induce tet(B) expression Lenski et a1. (1989) demonstrated that there was a fitness
reduction associated even with low-level expression of the tet(B) gene. In the absence of

induction the tet(B) gene had no detectable effect on fitness (Nguyen et al., 1989). Oddly

158

enough, it appears that some TC degradation products in soil, if bioavailable, could
actually exert a negative selection pressure on carriage of the tetracycline efflux genes.

The following are proposed as further objectives to add light to the phenomenon
presented above:

1) Testing the effect of clay-mineral systems on cation complexed

tetracycline bioavailability.

Multiple investigators have documented the presence of TC residues in manured
agricultural soils (Brambilla et al., 2007; Hamscher, Pawelzick, Hoper, & Nau, 2005).
TC residues remain in soil for long periods and repeated manure application can cause
TC residues to accumulate (Hamscher et al., 2002). The observation that TCs can exist
in cationic complexes with divalent cations in solution at environmentally relevant pH,
and that soil sorption of TC is dependent on cation exchange capacity as well as pH
(Sassman & Lee, 2005) implies that geochemical characteristics of a given soil can have
vast consequences on the bioavailability of TC to the indigenous microbial flora.

2) Repeating the experiments presented above with other tetracyclines and
degradation products to determine their bioavailability (e. g. OTC, CTC,
and anhydrotetracycline).

It has been demonstrated that OTC, CTC, and anhydrotetracycline all bind cations
different than TC (Lambs, Decock-Le Reverend, Kozlowski, & Berthon, 1988; Wessels,
Ford, Szymczak, & Schneider, 1998). Therefore it is likely that these differences have
significant effects on bioavailability. Specifically, it is relevant to study the

characteristics of OTC and CTC since these are the two antibiotics most commonly used

159

in agriculture and aquaculture (McManus et al., 2002; Sarmah, Meyer, & Boxall, 2006)
and anhydrotetracycline for reasons stated above.

3) Transferring the Ptet(A)-flpmufi construct into a broad-host range
plasmid and mobilizing it into diverse Gram-negative genera to test if,
how, and what genetic differences affect bioavailability.

4) Developing a Gram-positive tetracycline biosensor.

One obvious bias of the E. coli MC4100/pTGM reporter strain is that the gin
construct is expressed in a Gram-negative bacterium. This is reasonable considering that
the majority of tetracycline efflux genes (which the reporter mimics the expression of)
are found in Gram-negatives, however, it is possible that membrane differences between
separate Gram-negative genera could affect tetracycline bioavailability. Most of the
work regarding tetracycline uptake by bacterial cells has been done with E. coli and this
bacterium will not be dominant in the majority of environmental settings. It is also likely
that differences exist between tetracycline uptake of Gram-negative and Gram-positive
genera.

There are two obvious ways to extend the observations presented in the present
study to other bacteria. First, transferring the pTGM bioreporter construct into a broad-
host range plasmid such as RSFlOlO (Labes, Puhler, & Simon, 1990) and expressing it in
diverse Gram-negative bacteria. The second way is to construct a new reporter to test
bioavailability of TCs to Gram-positive cells. The recent discovery that the Gram-
positive efflux genes tet(Z) and tet(33) are regulated by repressor proteins with homology

to TetR suggests that one should be able to construct a biosensor similar to E. coli

160

MC4100/pTGM for study of Gram-positives (Guillaume, Ledent, Moens, & Collard,
2004)

5) Testing how tetracycline resistance gene expression works in stressed,

dormant cells.

Lastly, it is important to realize that microbial cells present in natural settings are
not growing similar to exponential grth in the lab. As such, experiments should be
designed to delineate what stress factors control the expression or repression of
tetracycline resistance genes in the natural environment.

In conclusion, the utilization of whole-cell tetracycline biosensors can add to the
scientific community’s understanding of the phenomenon described above. Although
traditionally biosensors have been employed as an efficient way to detect contaminants
they also are effective tools for examining the intersection of geochemistry and biology
that is likely to determine gene expression in natural systems. Ultimately, further studies
such as those outlined above will allow a better determination of the risk associated with

the environmental contamination of tetracycline residues.

161

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Aga, D. S., O'Connor, 8., Ensley, S., Payero, J. 0., Snow, D., & Tarkalson, D. (2005).
Determination of the persistence of tetracycline antibiotics and their degradates in
manure-amended soil using enzyme-linked immunosorbent assay and liquid
chromatography-mass spectrometry. J Agric Food Chem, 53(18), 7165-7171.

Bahl, M. I., Hansen, L. H., & Sorensen, S. J. (2005). Construction of an extended range

whole-cell tetracycline biosensor by use of the tet(M) resistance gene. FEMS
Microbial Lett, 253(2), 201-205.

Brambilla, G., Patrizii, M., De Filippis, S. P., Bonazzi, G., Mantovi, P., Barchi, D., et a1.
(2007). Oxytetracycline as environmental contaminant in arable lands. Anal Chim
Acta, 586(1-2), 326-329.

Cabello, F. C. (2006). Heavy use of prophylactic antibiotics in aquaculture: a growing
problem for human and animal health and for the environment. Environ
Microbiol, 8(7), 1 137-1144.

Chapin, A., Rule, A., Gibson, K., Buckley, T., & Schwab, K. (2005). Airborne multidrug-
resistant bacteria isolated from a concentrated swine feeding operation. Environ
Health Perspect, 113(2), 137-142.

Chopra, 1., & Roberts, M. (2001). Tetracycline antibiotics: mode of action, applications,
molecular biology, and epidemiology of bacterial resistance. Microbiol Mol Biol
Rev, 65(2), 232-260 ; second page, table of contents.

Connell, S. R., Tracz, D. M., Nierhaus, K. H., & Taylor, D. E. (2003). Ribosomal
protection proteins and their mechanism of tetracycline resistance. Antimicrob
Agents Chemother, 47(12), 3675-3681.

Durckheimer, W. (1975). Tetracyclines: chemistry, biochemistry, and structure-activity
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