RESISTANCE IN ADULT HUMAN GASTROINTESTINAL MICROFLORA - CAN IT TELL THE STORY OF RESISTANCE IN HUMANS?

THESIS

Presented in Partial Fulfillment of the Requirements for

the Degree Master of Science in the Graduate

School of The Ohio State University

By

Hanna H. Cortado, B. S.

The Ohio State University 2008

Committee members:

Professor Hua Wang, Adviser Approved by Professor Macdonald Wick

Professor Jeff Culbertson

Adviser Food Science and Nutrition Graduate Program

ABSTRACT

The acquisition of antibiotic resistance (AR) by clinically important is a worldwide human health concern. The rapid spread of AR has been attributed mainly to horizontal gene transfer events by resistance-encoding mobile genetic elements. Recently a large number of antibiotic resistant (ART) commensal bacteria were found in many retail foods. Characterization of the AR status of human gut microflora, involving infant subjects who have not been exposed to conventional solid foods as well as healthy adults with regular diet intake, can help reveal the potential impact of the food chain on disseminating AR to humans. In this study, adult human microflora associated with the gastrointestinal (GI) tract of 14 healthy individuals is examined for the presence of tetracycline-resistant bacteria and tetracycline-resistance conferring genes. Culture- dependent methods utilized in this study revealed a resistant count ranging from 106-109

CFU-mL-1, with total bacterial counts ranging from 106-1010 CFU-mL-1. A culture- independent approach was also optimized to assess the level of tetM gene pool in the sample microbiome. Among all subjects, the tetM gene pool range from 107 -109 tetM gene copy number per gram, as determined by TaqMan real-time quantitative polymerase chain reaction. Sixty-two out of 78 isolates tested (79%) were able to grow in media containing 140 μg-mL-1 tetracycline. Of these isolates, 39 carried one or more plasmids

ii and 8 distinct plasmid profiles were observed. Plasmid-carrying isolates were screened

for the efflux tet genes, tetK and tetL as well as the ribosomal protection genes tetM, tetQ

and tetW. The tetM gene was found to be most prevalent- observed in 51% (20/39) of the

isolates. Chromosomal and plasmid DNA of representative tetM-positive isolates were

probed for the 406 bp tetM gene in a DNA-DNA hybridization procedure. Preliminary results indicate the plasmid carriage of the tetM gene in some of the isolates examined.

Overall, results indicate a heavy ART bacterial load and considerable AR gene pool in

the GI tract of healthy human adults who have not taken in the past 3 months.

In some of the isolates, the tetM gene was found in plasmids, indicating that the human digestive tract could be a potential site for genetic transfer of AR determinants. The identified dominant AR gene carriers differ from those from infant subjects, and the AR gene pool is about 1-3 log higher than that found in infant subjects. The regular inoculation of the human digestive ecosystem of ART bacteria through the food chain is hypothesized as a significant contributor towards the dynamic shifting of ART isolates and enrichment of AR genes in the gut, as observed in this study.

iii ACKNOWLEDGEMENT

I wish to thank:

My adviser, Dr. Hua Wang, for her guidance and mentorship throughout this research

My committee members, Dr. Mick Wick and Dr. Jeff Culbertson for their time and support

Members of our lab,

Yingli Li, Dan Kinkelaar, Xinhui Li, Lu Zhang, Monchaya Rattanaprasert, Linlin Xiao,

Xiaojing Li and Andrew Wassinger for their technical assistance and encouragement

This research was supported by Dr. Wang’s OSU start-up fund.

iv VITA

August 6, 1982 ...... Born – Iloilo, Philippines

2003 ...... B. S. Molecular Biology and Biotechnology, University of the Philippines

2003 – 2005 ...... Research Assistant, Southeast Asian Fisheries Development Center, Tigbauan Iloilo, Philippines

2006 – present ...... Graduate Teaching and Research Associate, The Ohio State University

PUBLICATIONS

Research Publication

1. Hanna Cortado, Boris San Luis, Leobert dela Peña, Rosario Monsalud and Cynthia Hedreyda. 2005. Local Vibrio isolates exhibit molecular characteristics distinct from reference V. harveyi and V. campbellii strains. Science Diliman. 17(1): 23-30.

2. Fiona L. Pedroso, Evelyn Grace T. de Jesus-Ayson, Hanna H. Cortado, Susumu Hyodo and Felix G. Ayson. 2005. Changes in mRNA expression of grouper (Epinephelus coioides) growth hormone and insulin-like growth factor I in response to nutritional status. General and Comparative Endocrinology. 145(3): 237-246.

FIELDS OF STUDY

Major Field: Food Science and Nutrition

v TABLE OF CONTENTS Page Abstract ...... ii Acknowledgments ...... iv Vita ...... v List of Tables ...... vii List of Figures ...... viii

Chapters:

1. Introduction ...... 1

2. Literature Review ...... 6

3. Objectives ...... 33

4. Prevalence of Tetracycline - Resistant Bacteria and tet genes in the Human Gut ...... 34

4.1 Materials and Methods ...... 34 4.2 Results ...... 43 4.3 Discussion ...... 54

5. Conclusion and Future Development ...... 61

Appendix ...... 63 Bibliography ...... 65

vi LIST OF TABLES

Table Page

2.1 Tetracycline resistance genes and corresponding mechanisms...... 18

2.2 Major components of the human gastrointestinal (GI) microflora along the length of the GI tract ...... 23

4.1 Primer sequences used for tet screening of isolates ...... 41

4.2 tet gene screening of isolates from digestive microflora of healthy adult human subjects ...... 51

vii LIST OF FIGURES

Figure Page

2.1 Different routes of antibiotic resistance dissemination from animals to humans ...... 20

4.1 Total plate count and tetracycline-resistant count of bacteria isolated from fecal samples of 9 healthy human subjects ...... 44

4.2 Standard curve for determination of tetM gene copy numbers using TaqMan real-time PCR ...... 45

4.3 Validation of extraction efficiency ...... 47

4.4 Quantification of tetM gene copy numbers in fecal samples of 14 healthy human adults using real-time qPCR ...... 48

4.5 Assessment of antibiotic resistance in human digestive microflora by conventional plate counting and real-time PCR ...... 49

4.6 Representative plasmid profiles from isolates ...... 50

4.7 DNA-DNA hybridization for tetM detection ...... 52

viii CHAPTER 1

INTRODUCTION

During its era, antibiotics were deemed as miracle drugs, one that would put an end to the problem of morbidity and mortality from diseases, until the unwelcome emergence of antibiotic resistance (AR). In the United States alone, as much as $5 billion is spent annually towards treatment of infections caused by antibiotic resistant (ART) bacteria (5). As antibiotic resistance was primarily a human health concern, initial studies concluded that the misuse and overuse of antibiotics in the clinical setting were the main causes of AR emergence. However, an accumulating body of research has expanded this perspective to include antibiotic use in the absence of disease in agriculture and other environmental settings as potential contributors to the problem (4, 11, 14). It is not surprising that extensive measures are undertaken by various agencies throughout the world to address this serious concern. In 1996 the National Antimicrobial Resistance

Monitoring System (NARMS) was established as a collaboration between the Food and

Drug Administration (FDA), the U.S. Department of Agriculture (USDA) and the Center for Disease Control (CDC) to monitor the appearance and spread of antimicrobial resistance among enteric organisms in animals and humans as well as in retail meats (2).

In the European Union (EU), risk management tools include assessment of antimicrobial

1 agents and their potential influence on AR, and examination and surveillance of existing antibiotic resistance in animal pathogens, zoonotic bacteria and commensal bacteria (4).

Since its establishment, NARMS has reported an increasing trend in the carriage of AR in enteric species such as Salmonella, Campylobacter, Enterobactericea and

Enterococci. The most current report (2004) for human isolates specifies a considerable rise in resistance to clinically important antimicrobials. The report also highlights recent findings in newfound multi-drug resistance among isolates (1). The rapid emergence of

AR creates a need and priority for the scientific community to look more closely at the ecology of antibiotic resistance and the potential means of AR transfer. It is now common knowledge that antibiotics not only reach humans through the clinical route (direct application) but also by indirect means through various environmental compartments.

Antibiotics used in agriculture enter the ground water systems and could have an effect on food animals and plants that enter the food chain. The presence and impact of antibiotics in the environment is so widespread and all-encompassing that an accurate model for AR development is still yet to be determined. While one can still argue that antibiotic resistant (ART) pathogens isolated from retail foods represent a very small number of bacteria and therefore are not a significant source for HGT events, the recent discovery of a broad spectrum of ART commensals in many retail food items with mobile

AR genes transmissible to human residential bacteria and pathogens suggests that our previous understanding on the pathways and mechanisms in AR emergence, transmission and persistence likely is incomplete (15). The growing number of reports on ART bacteria found in foods that are directly consumed such as raw lettuce (12), ready-to-eat

2 turkey meat (6), seafood (16), and dairy products (3, 10) support the theory that the food chain may play a much more important role in disseminating the ART bacteria and the corresponding AR genes to the general public than the clinical exposure. It has been suggested that resistant commensal enteric bacteria may serve as AR reservoirs and have the potential to pass on resistant genes to other colonic resident bacteria or even pathogens that pass through the gut during the 24-48 hour transient incubation in the human digestive tract (8, 9). Indeed studies using animal models have demonstrated that the transmission of horizontal transmission of and resistance between Enterococcus faecium isolates likely happened (13). However, there are still major knowledge gaps in understanding the development of AR in human GI track and its correlation to AR in foods. Our laboratory had examined the prevalence of foodborne tetracycline-resistant commensal bacteria to illustrate the AR problem in the food chain

(7, 10, 15, Li XH and Wang, 2008 ASM General Meeting, Li XJ and Wang, 2008 IAFP

Annual Meeting). We will further investigate the prevalence of Tetr bacteria in adult and child subjects to reveal the potential correlation between AR in foods and AR in humans.

Analyses of the genetic background of the ART isolates from human subjects can further reveal the complexity of the ART bacteria, which may lead to an advanced understanding on the emergence, persistence and circulation of AR within the environment and the hosts.

3 BIBLIOGRAPHY

1. Center for Disease Control and Prevention. National Antimicrobial Resistance Monitoring System for Enteric Bacteria (NARMS): Human Isolates Final Report, 2004. Atlanta, Georgia: U.S. Department of Health and Human Services, CDC.

2. Centers for Disease Control and Prevention. 2007. http://www.cdc.gov/NARMS/. (viewed May 2008).

3. Flórez, A. B., M. S. Ammor, and B. Mayo. 2008. Identification of tet(M) in two Lactococcus lactis strains isolated from a Spanish traditional starter-free cheese made of raw milk and conjugative transfer of tetracycline resistance to lactococci and enterococci. Int J Food Microbiol. 121: 189-194.

4. Grugel, C. and J. Wallmann. 2004. Antimicrobial resistance in bacteria from food- producing animals: risk management tools and strategies. J of Vet Med B. 51: 419- 421.

5. Institute of Medicine. 1998. Antimicrobial drug resistance: issues and options. Workshop report. National Academy Press, Washington.

6. Khaitsa, M. L., R. B. Kegode, and D. K. Doetkott. 2007. Occurrence of antimicrobial-resistant salmonella species in raw and ready to eat turkey meat products from retail outlets in the midwestern United States. Foodborne Pathog Dis. 4: 517-525.

7. Lehman, M., and H. H. Wang. Abstr. 2006. Antibiotic resistance in ready-to-eat salad, abstr. 020B-05. Abstr. 2006 IFT Annu. Meet. Food Expo.

8. Lester, C. H., N. Frimodt-Moller, and A. M. Hammerum. 2004. Conjugal transfer of aminoglycoside and macrolide resistance between Enterococcus faecium isolates in the intestine of -treated mice. FEMS Microbiol. Lett. 235: 385-391.

9. Lester, C. H., N. Frimodt-Moller, T. L. Sorensen, D. L. Monnet, and A. M. Hammerum. 2006. In vivo transfer of a vanA resistance gene from an Enterococcus faecium isolate of animal origin to an E. faecium isolate of human origin in the intestines of human volunteers. Antimicrob Agents Chemother. 50: 596-599.

10. Manuzon.M.Y., Hanna, S.E, Luo, H. Yu. Z., Harper W.J. and H.H. Wang. 2007. Quantitative assessment of the tetracycline resistance gene pool in cheese samples by real-time TaqMan PCR. Appl Environ Microbiol. 73:1676-1677.

4 11. Molbak, K. 2004. Spread of resistant bacteria and resistance genes from animal to human- The public consequences. J of Vet Med. 51: 364-369.

12. Rodriguez, C., L. Lang, A. Wang, K. Altendorf, F. Garcia, and A. Lipski. 2006. Lettuce for human consumption collected in Costa Rica contains complex communities of culturable - and gentamycin-resistant bacteria. Appl Environ Microbiol. 72: 5870-5876.

13. Salyers, A. A., A. Gupta, and Y. Wang. 2004. Human intestinal bacteria as reservoirs for antibiotic resistance genes. Trends Microbiol. 12: 412-416.

14. Singer, R. S. and C. L. Holfacre. 2006. Potential impacts of antibiotic use in poultry production. Avian Dis. 50: 161-172.

15. Wang, H., M. Manuzon, M. Lehman, K. 9. Wan, H. Luo, T. Wittum, A. Yousef, and L. Bakaletz. 2006. Food commensal microbes as a potentially important avenue in transmitting antibiotic resistance genes. FEMS Microbiol. Lett. 254:226-231.

16. Durán, G. M., and D.L. Marshall. 2005. Ready-to-eat shrimp as an international vehicle of antibiotic-resistant bacteria. J Food Prot. 68:2395-2401.

5 CHAPTER 2

LITERATURE REVIEW

2.1 History of antibiotics and breadth of applications

The theory of “antibiosis” which is the inhibition or inactivation of a living

organism by another through the production of toxic substances was first described in

animals and plants in late 1800s, however, with the wide acceptance of the pathogenic

theory of medicine in the same century, the search for similar substances that would

inhibit disease-causing microorganisms followed (31). Although pyocyanase from

Pseudomonas aeruginosa was among the first antibiotics studied, penicillin from the

Penicillium mold gained much attention due to a demonstration by Alexander Fleming of the substance’s strong antibacterial activity against the then deadly Staphylococcus

aureus and minimal toxicity to humans (59). Following penicillin’s discovery was the introduction of streptomycin, broad spectrum agents like and , and cephalosporins. To date, known antibiotics are constantly being improved to address issues with toxicity and bacterial resistance by carrying out modifications in chemical structure or by creating novel, synthetic antimicrobials altogether.

Currently antimicrobials are classified by the Clinical Laboratory Standards

6 Institute into major structural categories as , aminopenicillins, β-lactams, cephalosporins, cephamycins, folate pathway inhibitors, , , phenicols, quinolones, sulfonamides and tetracyclins. Beta -lactams such as penicillins and cephalosporins are broad spectrum antibiotics that inhibit bacterial cell wall synthesis. Glycopeptides also target the cell wall by disrupting peptidoglycan crosslinks.

The mode of action for aminoglycosides involves binding to 50S ribosomal proteins.

Quinolones target DNA gyrases by binding to the alpha subunit. Macrolides reversibly bind the 50S ribosome and prevents peptide elongation (50). For human clinical applications, antibiotics serve prophylactic, empiric or therapeutic purposes. A current estimate by the Union of Concerned Scientists (UCS) reveals that in the United States, 3 million pounds of antibiotics are used for human medicine every year (57).

In food animals, antibiotics have had widespread use both therapeutically to treat disease and sub-therapeutically, over longer periods of time, to improve their rate of growth and efficiency of feeding. Antibiotics are also used to treat illness in domestic pets, such as dogs and cats, adding another 100-150 million animals to the total number ingesting antibiotics. The defined dosage for growth enhancement of food animals in the

US is < 200 g/ton of feed (31). The mechanisms responsible for growth promotion have not been fully elucidated but appear to include enhancement of vitamin production by gastrointestinal microorganisms, elimination of subclinical populations of pathogenic organisms, and increased intestinal absorption of nutrients (13). UCS reports in 2001 gave an estimate of 24.6 million pounds of antibiotics used non-therapeutically in animals, with 10.3, 10.5 and 3.7 million pounds contributed by swine, poultry, and cattle,

7 respectively (57). Public health officials are particularly concerned about the use of antibiotics in animals to promote growth because antibiotics used for growth promotion are administered in low doses over long periods of time to large groups of animals in the absence of diseases. This practice can allow animals to become reservoirs of antibiotic- resistant bacteria (58). In aquaculture, antimicrobial agents are used to treat infections caused by a variety of bacterial pathogens of fish including Aeromonas hydrophila, A. salmonicida, Edwardsiella tarda, Pasteurella piscicida, Vibrio anguillarum, and Yersinia ruckeri. The Center for Disease Control and Prevention, (CDC) however, spoke against the irresponsible use as these agents directly dose the environment, which results in selective pressures in the exposed ecosystem (9).

2.2 Discovery of tetracyclines and applications

For many years, tetracycline has been used widely in human therapy and livestock due to its low toxicity and broad spectrum activity. As a result, the tetracycline group is the second most widely used antibiotic after penicillins throughout the world.

Tetracycline targets a broad range of microorganisms including cell-wall free mycoplasmas, chlamydiae, mycobacterium, rickettsia, Helicobacter, Listeria and protozoan parasites such as Entamoeba histolytica, Giardia lamblia and Plasmodium falciparum. Tetracycline is therapeutically applied for combating a variety of illnesses including bacterial respiratory and urogenital tract diseases, periodontal, Lyme and rickettsial diseases (24). Even more current applications include treatment for atypical pneumonia, prophylaxis for traveler’s diarrhea, and as a topical treatment for acne (11).

8 Tetracyclines are also widely used in animal production for treatment of diseases as well as for growth enhancement of poultry, cattle, sheep and swine. In some cases for therapeutic treatment of large numbers of poultry reared on commercial farms, the antibiotics are added directly to feed or water or can be administered in aerosols (40).

Tetracyclines are also used for treatment of infections in domestic pets. In aquaculture, it is used to control infections in salmon, catfish and lobsters (58).

First generation antibiotics in the tetracycline class include , oxytetracycline, tetracycline, demethylchlortetracycline, , limecycline and . From 1965 to 1972, second generation methacycline, and were sequentially used. The most current tetracycline used today is already considered a third generation drug. Clinical activity of tetracycline can be attributed to the presence of a four-ring structure (tetracycle) where a variety of functional groups are attached (24). As these functional groups impact activity, substitutions to these groups are among the main modifications done to enhance the effectiveness of the drug.

Rolitetracycline and are products of addition of substituents to the amide nitrogen to make the antibiotic more soluble in water (63). The most current tetracycline today, is a derivative of minocycline and is produced by substitution in critical parts of the ring structure.

Tetracycline’s mode of action against target organisms is by a well established mechanism. By interacting with the ribosome, tetracycline interferes with the association of the organelle with aminoacyl-tRNA thereby preventing bacterial protein synthesis. In terms of drug uptake, that of gram negative enteric bacteria has been observed to involve

9 porin channels in the outer membrane. Tetracycline is a strong metal chelator and enters the charged outer membrane in an ion-complexed form. The molecule becomes uncharged in the periplasm and then diffuses into the inner membrane. Once in the cytoplasm, the antibiotic binds the 30S subunit of the ribosome causing structural changes in the 16S rRNA. This binding, however is reversible, which explains the drug’s bacteriostatic effect (11).

Administration of tetracycline to treat infections is commonly through the oral route. Absorption is in the stomach and small intestine and is influenced by the presence of food, milk or divalent cations, particularly calcium, with which tetracyclines form nonabsorbable chelates. Tetracycline generally penetrates moderately well into body fluids and tissues and is excreted in the urine. For instance, levels in sputum about 20% of those in serum, can be achieved, which explains why the tetracylines have a role in the treatment of respiratory tract infections. Tetracyclines also penetrate into the sebum and are excreted in perspiration, properties which contribute to their usefulness in the management of acne (16).

The extensive use of tetracycline in a broad range of applications has been blamed for the emergence and almost unstoppable rise in tetracycline resistance. The fact that it has been around for more than half a century makes it a good candidate for relating the environmental presence of the antibiotic with the breadth of antibiotic resistance gene pool across bacterial populations. Studying tetracycline resistance could provide a prototypical view of the use of antibiotics and their effects on bacterial populations (7).

10 2.3 Emergence and mechanisms of antibiotic resistance

After their discovery and proven therapeutic utility, antibiotics were

considered miracle drugs and so did consumers demand for rapid and convenient access

to it. The increase in consumer demand propelled the discovery of more antibiotics and

derivatives and their extensive use. However, shortly after clinical applications of

antibiotics were initiated, bacteria resistant to the antibiotic started to appear, first

exclusively in clinical settings but later on in communities as well. A classic example

would be the case of penicillin. In 1946, just a few years after penicillin was made public,

14% of the strains isolated from a hospital in London, where much of the early use of

penicillin had taken place, were resistant. This frequency increased to 59% in the same

hospital by the end of that decade. Initially resistant strains are only found from ill

patients in hospitals. However, they began to spread into the community. In 1970s,

resistant strains of two different microorganisms that were known to be penicillin-

susceptible, were found. These are the meningitis-causing Hemophilus influenzae and

Neisseria gonorrhoeae, the agent for gonorrhea. Resistance was affected by enzymatic

action and interestingly, the enzyme in N. gonorrheae was the same enzyme found in H. ifluenzaea. It is likely that the identical gene had been picked up by these two different kinds of bacteria. Today penicillin resistant gonorrhea strains plague every country in the world (31).

One the first cases of multi-drug resistance was reported in Japan. Dysentery-

causing Shigella strains isolated from hospital patients were found to be resistant to

tetracycline, sulfonamide, streptomycin and chloramphenicol. Escherichia coli, which

11 was part of the same patients’ normal microflora was also found to carry resistance to the same four antibiotics (46). Incidences of multi-drug resistance led scientists to the discovery of “R” factors carried on plasmids which have the potential to be transferred within and across bacterial populations in close contact with each other. Since then, discovery of more strains carrying resistance has not stopped. To circumvent resistance, second and third generation drugs were created. However, resistant isolates always seem to emerge. The most recent report by the CDC, highlights some of the most current findings in antibiotic resistance (AR) surveillance among enteric bacteria of human health concern. The 2004 report indicated an increase in resistance of clinically important strains to third generation drugs. Campylobacter resistance to flouroquinolone increased from 12.9% in 1997 to 19% in 2004. For non-Typhi Salmonella, resistance to cephalosporin was up to 3.4% from 0.2% in 1996. Quinolone resistance of Salmonella

Typhi isolates jumped from 18.7% in 1999 to 41.7% (10).

Initially, antibiotic resistance was not considered by clinicians and pharmacologists as a likely threat to the rising benefits of antibiotics. This is because resistance to antibiotics was first believed to only occur via natural mutation; the frequency of which was too minimal to be of any significant concern. However, a body of findings kept accumulating as the various mechanisms by antibiotics are ripped by the very target of its antimicrobial impact. One of the most common mechanisms found to affect certain antibiotic chemical structures is the inactivation of the antibiotic by enzymatic hydrolysis. Enzymes produced by resistant microorganisms catalyze the structural modification of therapeutic agents rendering them inactive. Examples of these

12 enzymes are β-lactamases which specifically target the β-lactam ring structure of antibiotics such as penicillins and cephalosporins by hydrolytic inactivation (15). To counter this type of resistance, efforts such as the production of β-lactam antibiotics with modified structures have been launched by pharmacologists to prevent enzyme substrate recognition. However, microorganisms producing β-lactamases were later on discovered to undergo a series of point mutations which contribute to its activity against a wider spectrum of drug substrate. As more β-lactam drugs were synthesized to evade enzyme action, the target bacteria counteracted by acquiring thr necessary gene mutations that lead to the production of yet another effective enzyme (33).

A similar resistance mechanism, in terms of drug covalent modification, is known to occur against aminoglycoside antibiotics. Resistant bacteria inactivate the drug by adenylation, phosphorylation and acetylation of the target antibiotic class (51). Unlike,

β-lactamases, however, natural mutation to accommodate a wider spectrum of antibiotic substrates has not been known to occur. Other known mechanisms include restricted import of antibiotics (e.g. penicillin binding proteins), active export of antibiotics (e.g. by membrane inserted ATP-dependent efflux systems) or target modification (e.g. methylation of 23S rRNA, mutation of amino acid sequence of topoisomerase) (32).

According to Institute of Medicine estimates, the annual cost of treating antibiotic-resistant infections may be as high as $5 billion (27). Experts site the widespread use of antibiotics in human medicine as the principal cause of resistance but they identify the use of antibiotics in animals raised for human consumption as contributing to antibiotic resistance in humans. It is generally agreed that a large

13 proportion of the antibiotics used in the United States is administered to animals raised for human consumption (58).

2.4 Acquisition and dissemination of antibiotic resistance determinants

The likely causal relationship between the widespread utilization of antibiotics and the subsequent rapid emergence of antibiotic resistance was supported by data from

Hughes and Datta which showed a comparison of Murray’s isolates from the pre- antibiotic era with those that were isolated after the application of antibiotics to human and animal therapy had been established (26). The former isolates showed very minimal resistance. Osterblad and co-workers observed an almost complete absence of resistant enterobacteria in the gut flora of wildlife populations with very little or no contact with either humans or anthropogenic antibiotics (37). It was therefore concluded that the unrestrained rise in species harboring antibiotic resistance genes is largely due to the large-scale use of antibiotics as they provide selective pressure to support the growth of resistant clones.

With the discovery of transferable R plasmids, it was generally hypothesized that the source of antibiotic resistance genes would be the microorganisms that have the natural ability to produce antibiotics. These genes and mechanisms are possessed by antibiotic-producing organisms as a means of autoprotection (40, 6). Protein and nucleic acid sequence comparisons done by Davies showed sequence similarity between antibiotic-producing isolates and resistant clinical isolates. The first evidence of resistance gene transfer involving antibiotic-producing streptomyces in a clinical setting

14 was demonstrated by Pang and co-workers (38). Other researchers, however, pointed to other possible origins and routes. In a study by Aminov and Mackie, the authors attempt to explain the ecology and evolution of antibiotic resistance genes using phylogenetic analyses. Based on phylogenetic data, the authors are convinced that other factors besides selection by antibiotics play significant roles in the spread of resistance. AR genes encoding RPPs (ribosomal protection proteins), such as those that confer resistance to tetracyclines, according to the authors came about as a result of early branching and long independent diversification from elongation factors (proteins involved in peptide synthesis) long before the modern antibiotic era and could not be due to gene transfer events from antibiotic producing microorganisms (2).

Although gene mutation and antibiotic selective pressure could eventually give rise to resistant clones, gene transfer events in the environment are generally considered as the main vehicles for the rapid dissemination of AR determinants. Known and established mechanisms of genetic exchange include DNA-mediated transformation, transduction and conjugation. In DNA-mediated transformation, naked DNA traverses the bacterial cell wall and is taken up by naturally competent recipient cells. This occurs in a wide variety of Gram-positive and Gram-negative bacteria as well as the eukaryote,

Saccharomyces cerevisiae. Transduction involves a virus that targets bacteria and attaches to the bacterial host via cell wall receptors. Viral infection causes DNA from the virus to be incorporated into the host genome or simply be replicated inside the host using the latter’s machinery. Conjugation occurs when compatible microorganisms come in contact with each other resulting in horizontal gene transfer in which plasmids or

15 chromosomes are transferred (36). A direct visualization of this DNA transfer event is presented in a 2008 paper by Babic et al. (4). The authors showed that conjugation between E. coli single cells is mediated by the donor plasmid, in this case the F plasmid, and that the fate of the transferred genetic element is recombination into the recipient chromosome (96% of recipients). Conjugation has been demonstrated to occur not only in closely related species but has been discovered to cross species and genus barriers.

Antibiotic resistance can also be spread by mobile genetic elements such as transposons. Transposons are segments of DNA that can move from one site to another in the chromosome or plasmid of the same cell. Hall and Stokes (1989) pointed to these transposable elements as the key players in the dissemination process. They introduced the integrons which are a particular class of transposons as being responsible for AR spread (54). Furthermore, Francia and co-workers showed that Tn21 integrase can act on secondary target sites at significant frequencies and so permit the fusion of two R plasmids by the interaction between the recombination hotspot of one plasmid and a secondary integrase target site on a second plasmid (21). This phenomenon could potentially explain the co-carriage of multiple resistance genes. Dual function transposons such as conjugative transposons, were also considered as important contributors to the rapid spread of AR as these elements have the ability to excise themselves and form circular intermediates that could integrate into the host genome after conjugation (12).

There are also findings of AR gene transfer facilitated by sub-inhibitory concentrations of antibiotics. In work done by Barr et al the presence of β-lactam

16 antibiotics enhance the transfer of tetracycline resistance plasmids in S. aureus by up to

1000-fold (5). In Bacillus species, pre-growth of donor isolates in non-lethal amounts of tetracycline increased the rate of conjugative transposition of Tn916 (53). A similar enhancement of conjugal transfer is observed by Torres and co-workers for Tn925 in

Enterococcus faecalis (56). These studies suggest that antibiotics may have a stimulatory role in gene transfer. Hastings et al. explained that DNA damaging antibiotics serve as stimuli for the mobilization of integrating conjugative elements for AR to other bacterial populations (25).

2.5 Tetracycline resistance

After its introduction in 1948, the tetracycline family has become the drug of

choice to treat various infections in humans and to enhance the growth of livestock.

However by 1953, the first incidence of tetracycline resistance in a clinical strain was

reported in Shigella dysenteriae, the causative agent for dysentery. Two years later, a

multidrug resistant Shigella was found (42). Today, a solid body of research has

documented tetracycline resistance in both Gram-positive and Gram-negative genera.

These resistant bacteria encompass classifications belonging to pathogens, opportunistic

and normal flora species, and were isolated from man, animals, food and the

environment. Documentation for these findings has been tackled in numerous scientific

paper reviews (44, 14, 43).

With increasing numbers of studies documenting bacterial resistance to

tetracycline, the scientific community is forced to take a closer look at potential

17 mechanisms of tetracycline resistance and how determinants are acquired and spread within and among microbial populations. To date, there are three known mechanisms for bacterial resistance to tetracycline. The first is by the use of an efflux pump to force the drug out of the bacterial cell. Efflux proteins located in bacterial membranes facilitate the export and therefore decrease in intracellular concentration of ribosome-damaging tetracycline. The second mechanism is through ribosomal protection in which the gene product, by causing structural changes, affects the release of tetracycline molecules from the ribosomal A site liberating the ribosome from the inhibitory effects of drug. The last one is less common and is through enzymatic inactivation of tetracycline (42, 20).

Nomenclature for tetracycline resistance genes and the mechanisms they belong to has been established and is summarized in extensive reviews by Chopra and

Roberts (11) and Roberts (44), as follows:

Efflux tet(A), tet(B), tet(C), tet(D), tet(E) tet(G), tet(H), tet(J), tet(V), tet(Y) tet(Z), tet(30), tet(31), tet(K), tet(L) tetA(P), tcr3, tet(33), tet(35), tet(38) tet(39) Ribosomal protection tet(M), tet(O), tet(S), tet(W), tet(Q) tet(T), tetB(P), tet, tet(32), tet(36) Enzymatic tet(X), tet(34), tet(37) Unknown tet(U)

Table 2.1: Tetracycline resistance genes and corresponding mechanisms.

18 It is widely accepted among scientists that the rapid spread of tetracycline resistance among bacteria is attributable to the localization of tet genes on plasmids and transposable genetic elements which could also code for their transfer. Along with tetracycline resistance, it is also common to find genes for resistance to other antibiotics and heavy metals in conjugative transposons (48). This could make maintenance of AR genes possible in the absence of selective pressure. The most common transposon associated with tetracycline resistance genes belong to the Tn916-Tn1545 family.

Roberts has hypothesized that the host range of specific tet genes is influenced by the type of genetic element the resistance gene is carried upon. Compared to those found on plasmids, tet determinants located on conjugative transposons have the potential to be more rapidly disseminated in a variety of host genera due to the more liberal transferability of this particular genetic element (44).

2.6 Food and AR genes

The food industry is an important user of antibiotics and in many industrialized countries the total amount of antibiotics used for food production exceeds the amount used in human medicine and is thus a significant factor in exposing bacteria to antibiotics, giving rise to AR strains (62). Ingestion of food containing resistant bacteria may lead to transfer of resistant elements to human microflora (35). The role of commensals in the dissemination of AR determinants has recently been recognized as significant reservoirs for antibiotic resistance (49, 60). The human health risk associated with the use of antibiotics in food animals has been long debated. However, various

19 evidences including recent findings on the prevalence of AR in foodborne commensal bacteria indicate that the food chain may be a very important route in transmitting AR to humans. Below is a figure from a review by Phillips et al. that illustrates the possible routes of transmission of AR microorganisms between humans and animals with food playing a central role (39).

Figure 2.1: Different routes of antibiotic resistance dissemination from animals to humans.

20 Studies which aim to asses the prevalence of ART bacteria and AR genes in food have shown a considerable resistant bacterial load in the foods tested (18, 60).

Quantitative real time PCR data from Manuzon et al (34) showed a high number of tetS gene copies in commercial cheeses. Rodriguez and co-authors found an abundance of oxytetracycline- and gentamycin- resistant bacteria in the culturable microbiota of iceberg lettuce for human consumption in 10 conventional farms in Costa Rica (45). In another study, Lactococcus lactis strains isolated from raw milk starter-free cheese were found to posses a plasmid-associated tetM gene encoded by a conjugative transposon

Tn916. In the subsequent conjugation study conducted by the authors, they found that, although the plasmid that carried resistance was not detected in transconjugants, tetM and

Tn916-related sequences were present which suggests that resistance was transferred through transposon mobilization (19). Scientists have also found tet genes in

Lactobacillus sakei, starter culture in fermented meat products. Ammor et al. recently found, in this strain, two tet genes, efflux pump-coding tetL and ribosomal protection coding tetM. The former is carried on a plasmid while the latter is associated with a transposon (3). Gevers et al (23) compared the prevalence and diversity of tetracycline resistant lactic acid bacteria in raw meat versus the final product of fermented dry sausage. Results showed that the fermentation process reduced lactic acid bacteria diversity to lactobacilli and genetic diversity to tetM.

21 2.7 Human gut and microflora

The human gastrointestinal (GI) tract is home to a diverse microbial ecosystem, colonized by a variety of microorganisms ranging from the beneficial to the potentially pathogenic. Although there are individual variations, below are generally the microorganisms, starting from the numerically dominant, that are found along the length of the GI tract and their estimated numbers. This table was obtained from a recent review of the intestinal microbiota by Tappenden and Deutsch (55).

22 Table 2.2: Major components of the human gastrointestinal (GI) microflora along the length of the GI tract.

Due to the sheer number of microorganisms present and the ideal nutritive environment of the human and animal gut, scientists have postulated that it can be a favorable place for gene transfer events. Bacteria that harbor resistance genes could serve as reservoirs for these genes and could pass them along to other bacteria that they come

23 in contact with via conjugation (47). Indeed the concern is when these genes are acquired

by pathogens that pass through the colon. Commensals turning resistant can also be a

problem when an individual becomes immune-compromised as some members of

commensals are often considered opportunistic pathogens.

Studies have shown that some of these gut microflora harbor transferable

genetic elements which could facilitate horizontal gene transfer. Dual function

mobilization factors such as conjugative transposons were found by Clewell and co-

workers to be present in Bacteroides, one of the predominant microbial components of

human colonic microflora. These conjugative transposons carry tetracycline resistance

(12). The same scenario was observed by Shoemaker et al. whose assessment study

reflected a prevalence of tetQ, ermF, ermG and ermB in colonic Bacteroides species,

isolated from both the community and hospitals (52).

As further proof of gene transfer occurring in the gut, some researchers were

able to obtain transconjugants out of recipient strains while in transit in the GI tract of

germ-free rats (28). Lester et al. investigated the transfer of resistance genes, particularly

erm(B)-Tn5405-like element and aac(60)-Ie-aph(200)-Ia from multi-resistant E. faecium

to sensitive E. faecium in the intestine of mice with intact normal flora. Both in vivo and in vitro demonstrations were conducted and in both cases, gene transfer was observed

(29). In a follow up study, the Lester et al. showed that transfer of vanA (for vancomycin

resistance) can occur from E. faecium isolated from chicken to E. faecium isolate found

in humans. Transfer occurred in 3 out of 6 human volunteers, even a multi-resistance

transfer to one (quinupristin-dalfopristin). This suggests that it is possible, upon intake,

24 for some strains carrying mobile elements to transfer this characteristic to other strains

that are part of the human microflora (30). This could pose a risk for immune-

compromised patients that could be susceptible to commensals.

2.8 Quantitative polymerase chain reaction (qPCR)

Methods for characterizing the genotype of tetracycline resistant isolates have

evolved from hybridization with conventional probes to more currently, real-time PCR

assays. DNA-DNA hybridization is based upon the use of probes - polynucleotides of

known tetracycline resistance determinants or genes and utilizing them to detect similar

genes in the bacterial genome (8). Frech and Schwarz employed this method to detect

tetracycline resistance genes in Salmonella enterica (22). However, this method is time consuming, labor intensive and exhibits low sensitivity. These limitations can be circumvented by the use of PCR-based approaches, which some studies have utilized to detect tetracycline resistance in Staphylococcus aureus (61) and other gram-negative

bacteria (1). In the mentioned studies, primers specific for the target tet genes were

designed and used in a conventional PCR amplification reaction. For simultaneous

detection of multiple genes, multiplex PCR is used, in which the different primers are

added in a single tube reaction. The differentiation among the amplicons of each primer

pair would lie in the detection of specific florescence associated with each amplification

product. In one study, a large number of tetracycline resistant clinical isolates, numbering

up to 107 was screened for 8 tetracycline efflux genes using multiplex real-time SYBR

Green I PCR assay. The sensitivity of the multiplex test assay varied from 10-1000 CFU

25 per PCR reaction. Melting point analysis was employed to differentiate the various real- time products (18). Fluorescence-based real-time assays such as SYBR Green PCR allow for identification of multiple target genes from a single tube reaction by melting point analysis without the need for time consuming gel electrophoresis.

Quantitative real-time PCR detects and quantifies the fluorescence signal emitted by accumulating PCR products. The amount of fluorescence following each cycle is proportional to the concentration of target DNA in the sample. Two of the most common detection chemistries used along with real-time PCR include SYBR Green and hybridization probes such as TaqMan. The former is an intercalating dye so signal from any double stranded product present in the tube is detected. This calls for a melt curve analysis to verify the homogeneity of the product. Hybridization probes on the other hand, utilizes probes that are designed according to the target nucleotide sequence so any signal detected could only come from the specific product. Real-time PCR eliminates post-PCR analysis and in contrast to end-point PCR, amplicons are detected during the exponential phase when reaction components are in abundance and the target can be more accurately quantified (58). Two major technical challenges in applying this method in real food or human sample analyses are 1) to effectively extract the DNA from the matrices and 2) to minimize the interference to the PCR amplification reaction due to inhibitory compounds from the matrices. Our laboratory has optimized the real-time PCR procedures to measure the AR gene pool in high-fat and high-protein food matrices as cheese products (34). Its applicability in assessing AR gene pools in human fecal samples needs to be further investigated.

26 BIBLIOGRAPHY

1. Aminov, R. I., J. C. Chee-Sanford, N. Garrigues, B. Teferedegne, I. J. Krapac, B. A. White, and R. I. Mackie. 2002. Development, validation, and application of PCR primers for detection of tetracycline efflux genes of gram-negative bacteria. Appl Environ Microbiol. 68: 1786-1793.

2. Aminov, R.I., and R.I. Mackie. 2007. Evolution and ecology of antibiotic resistance genes. FEMS Microbiol Lett. 271: 147-161.

3. Ammor, M. S., M. Gueimonde, M. Danielsen, M. Zagorec, A. H. van Hoek, C. G. de Los Reyes-Gavilán, B. Mayo, and A. Margolles. 2008. Two different tetracycline resistance mechanisms, plasmid-carried tet(L) and chromosomally located transposon-associated tet(M), coexist in Lactobacillus sakei Rits 9. Appl Environ Microbiol. 74: 1394-1401.

4. Babic, A., A. Lindner, M. Vulic, E. Stewart, and M. Radman. 2008. Direct visualization of horizontal gene transfer. Science 319: 1533-1536.

5. Barr, V., K. Barr, M. R. Millar, and R. W. Lacey. 1986. β- lactam antibiotics increase the frequency of plasmid transfer in Staphylococcus aureus. J Antimicrob Chemother. 17: 409-413.

6. Benveniste, R., and J. Davies. 1973. Aminoglycoside antibiotic-inactivating enzymes in actinomycetes similar to those present in clinical isolates of antibiotic- resistant bacteria. Proc Natl Acad Sci. 70: 2276-2280.

7. Bryan, A., N. Shapir, and M.J. Sadowsky. 2004. Frequency and distribution of tetracycline resistance genes in genetically diverse, nonselected, and nonclinical Escherichia coli strains isolated from diverse human and animal sources. Appl Environ Microbiol. 70: 2503-2507.

8. Carey, C.M., J.L. Kirk, S. Ohja, and M. Kostrzynska. 2007. Current and future uses of real-time polymerase chain reaction and microarrays in the study of intestinal microbiota, and probiotic use and effectiveness. Can J Microbiol. 53:537-550.

9. Center for Disease Control and Prevention. 2007. http://www.cdc.gov/narms/faq_pages/11.htm (viewed Mar 2008).

10. Center for Disease Control and Prevention. National Antimicrobial Resistance Monitoring System for Enteric Bacteria (NARMS): Human Isolates Final Report, 2004. Atlanta, Georgia: U.S. Department of Health and Human Services, CDC.

27 11. Chopra, I., and M. Roberts. 2001. : mode of action, applications, molecular biology, and epidemiology of bacterial resistance. Microbiol Mol Biol Rev. 65: 232-260.

12. Clewell, D. B. and C. Gawron-Burke. 1986. Conjugative transposons and the dissemination of antibiotic resistance in streptococci. Annu Rev Microbiol. 40: 635- 659.

13. Committee on Drug Use in Food Animals. 1999. The use of drugs in food animals, benefits and risks. National Academy Press, Washington, D.C.

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

15. Davies, J. 1994. Inactivation of antibiotics and dissemination of resistance genes. Science. 264: 375-382.

16. Doan, T. L., H. B. Fung, D. Mehta and P. F. Riska. 2006. : a glycylcycline antimicrobial agent. Clin Ther. 28: 1079-1106.

17. Durán, G. M., and D.L. Marshall. 2005. Ready-to-eat shrimp as an international vehicle of antibiotic-resistant bacteria. J Food Prot. 68:2395-2401.

18. Fan W., T. Hamilton, S. Webster-Sesay, M.P. Nikolich, and L.E. Linder. 2007. Multiplex real-time SYBR Green I PCR assay for detection of tetracycline efflux genes of gram-negative bacteria. Mol Cell Probes. 21: 245-256.

19. Flórez, A. B., M. S. Ammor, and B. Mayo. 2008. Identification of tet(M) in two Lactococcus lactis strains isolated from a Spanish traditional starter-free cheese made of raw milk and conjugative transfer of tetracycline resistance to lactococci and enterococci. Int J Food Microbiol. 121: 189-194.

20. Fluit A.C., A. Florijn, J. Verhoef and D. Melatovic. 2005. Presence of tetracycline resistance determinants and susceptibility to tigecycline and minocycline. Antimicrob Agents Chemother. 49: 1636-1638.

21. Francia, M. V., P. Avila, F. de la Cruz, and J. M. Lobo. 1997. A hot spot in plasmid F for site-specific recombination mediated by Tn21 integron integrase. J Bacteriol. 179: 4419-4425.

22. Frech, G. and S. Schwarz. 2000. Molecular analysis of tetracycline resistance in Salmonella enterica subsp. enterica serovars Typhimurium, enteritidis, Dublin,

28 Choleraesuis, Hadar and Saintpaul: construction and application of specific gene probes. J Appl Microbiol. 89: 633-641.

23. Gevers, D., L. Masco, L. Baert, G. Huys, J. Debevere, and J. Swings. 2003. Prevalence and diversity of tetracycline resistant lactic acid bacteria and their tet genes along the process line of fermented dry sausages. Syst Appl Microbiol. 26: 277-283.

24. Handbook of experimental pharmacology, Vol 78. 1985. The tetracyclines. J. J. Hlavka and J. H. Boothe (eds). Springer Verlag, New York.

25. Hastings, P., S. Rosenberg, and A. Slack. 2004. Antibiotic-induced lateral transfer of antibiotic resistance. Trends Microbiol 12: 401-404.

26. Hughes, V.M., and N. Datta. 1983. Conjugative plasmids in bacteria of the ‘pre- antibiotic era. Nature 302: 725-726.

27. Institute of Medicine. 1998. Antimicrobial drug resistance: issues and options. Workshop report. National Academy Press, Washington.

28. Jacobsen, B.L., M. Skou, A.M. Hammerum, and L.B. Jensen. (1999) Horizontal transfer of the satA gene encoding A resistance between isogenic Enterococcus faecium strains in the gastrointestinal tract of gnotobiotic rats. Microb Ecol Health Dis. 11: 241–247.

29. Lester, C. H., N. Frimodt-Moller, and A. M. Hammerum. 2004. Conjugal transfer of aminoglycoside and macrolide resistance between Enterococcus faecium isolates in the intestine of streptomycin-treated mice. FEMS Microbiol. Lett. 235: 385-391.

30. Lester, C. H., N. Frimodt-Moller, T. L. Sorensen, D. L. Monnet, and A. M. Hammerum. 2006. In vivo transfer of a vanA resistance gene from an Enterococcus faecium isolate of animal origin to an E. faecium isolate of human origin in the intestines of human volunteers. Antimicrob Agents Chemother. 50: 596-599.

31. Levy, S.B. 1992. The antibiotic paradox: how miracle drugs are destroying the miracle. Plenum Press, New York.

32. Levy, S.B. 2002. Active efflux, a common mechanism for biocide and antibiotic resistance. Sym Ser Soc Appl Microbiol. 31:65-71.

33. Majiduddin, F. K., I. C. Materon, T. G. Palzkill. 2002. Molecular analysis of beta- lactamase structure and function. Int J Med Microbiol. 292: 127-137.

34. Manuzon, M. Y., S. E. Hanna, H. Luo, Z. Yu, W. J. Harper, and H. Wang. 2007. Quantitative assessment of the tetracycline resistance gene pool in cheese samples by

29 real-time TaqMan PCR. Appl Environ Microbiol. 73: 1676-1677.

35. Mathur, S and R. Singh. 2005. Antibiotic resistance in food lactic acid bacteria--a review. Int J Food Microbiol. 105: 281-295.

36. Nester, E. W., D. G. Anderson, C. E. Roberts, Jr., N. N. Pearsall and M. T. Nester. 2004. Microbiology: a human perspective, 4th ed. McGraw Hill, New York.

37. Osterblad, M., K. Norrdahl, E. Korpimaki, and P. Huovinen. 2001. Antibiotic resistance: how wild are wild mammals? Nature 409: 37-38.

38. Pang, Y., B.A. Brown, V.A. Steingrube, R.J. Wallace Jr., and M.C. Roberts. 1994. Tetracycline resistance determinants in Mycobacterium and Streptomyces species. Antimicrob. Agents Chemother. 38:1408-12.

39. Phillips, I., M. Casewell, T. Cox , B. De Groot, C. Friis, R. Jones, C. Nightingale, R. Preston, and J. Waddell. 2004. Does the use of antibiotics in food animals pose a risk to human health? A critical review of published data. J Antimicrob Chemother. 53: 28-52.

40. Piendl, W. and A. Bock. 1982. Ribosomal resistance in the producer organism Micromonospora purpurea. Antimicrob Agents Chemother. 22: 231-236.

41. Plumb, D. C. 1995. Veterinary drug handbook. Ames, Iowa.

42. Roberts, M. C. 1996. Tetracycline resistance determinants: mechanisms of action, regulation of expression, genetic mobility, and distribution. FEMS Microbiol. Rev. 19: 1-24.

43. Roberts, M. C. 2002. Resistance to tetracycline, macrolide-lincosamide- streptogramin, trimethoprim, and sulfonamide drug classes. Mol Biotechnol. 20: 261- 83.

44. Roberts, M. C. 2005. Update on acquired tetracycline resistance genes. FEMS Microbiol Lett. 15: 195-203.

45. Rodriguez, C., L. Lang, A. Wang, K. Altendorf, F. Garcia, and A. Lipski. 2006. Lettuce for human consumption collected in Costa Rica contains complex communities of culturable oxytetracycline- and gentamycin-resistant bacteria. Appl Environ Microbiol. 72: 5870-5876.

46. Rosen, B. P. 1999. Resolving the antibiotic paradox: progress in understanding drug resistance and development of new antibiotics. Kluwer Academic, New York.

47. Salyers, A. A., A. Gupta, and Y. Wang. 2004. Human intestinal bacteria as

30 reservoirs for antibiotic resistance genes. Trends Microbiol. 12: 412-416.

48. Salyers, A. A., N. B. Shoemaker, A. M. Stevens, and L. Y. Li. 1995. Conjugative transposons: an unusual and diverse set of integrated gene transfer elements. Microbiol Rev. 59: 579-590.

49. Salyers, A. and N. B. Shoemaker. 2006. Reservoirs of antibiotic resistance genes. Anim Biotechnol. 17:137-146.

50. Scott, G.M., and M.S. Kyi. 2001. Handbook of essential antibiotics. Harwood Academic, Amsterdam.

51. Shaw, K.J., P.N. Rather, R.S. Hare, and G.H. Miller. 1993. Molecular genetics of aminoglycoside resistance genes and familial relationships of the aminoglycoside- modifying enzymes. Microbiol Rev. 57:138-163.

52. Shoemaker, N. B., H. Vlamakis, K. Hayes, and A. A. Salyers. 2001. Evidence for extensive resistance gene transfer among Bacteroides spp. and among Bacteroides and other genera in the human colon. Appl Environ Microbiol. 67: 561-568.

53. Showsh, S. A. and R. E. Andrews, Jr. 1992. Tetracycline enhances Tn916-mediated conjugal transfer. Plasmid 28: 213-224.

54. Stokes, H.W., and R.M. Hall. 1989. A novel family of potentially mobile DNA elements encoding site-specific gene-integration functions: integrons. Mol Microbiol. 3: 1669-1683.

55. Tappenden, K. A. and A. S. Deutsch. 2007. The physiological relevance of the intestinal microbiota--contributions to human health. Am Coll Nutr. 26: 679S-683S.

56. Torres, O. R., R. Z. Korman, S. A. Zahler, and G. M. Dunny. 1991. The conjugative transposon Tn925: enhancement of conjugal transfer by tetracycline in Enterococcus faecalis and mobilization of chromosomal genes in Bacillus subtilis and E. faecalis. Mol Gen Genet. 225: 395-400.

57. Union of Concerned Scientists. 2006. http://www.ucsusa.org/food_and_environment/antibiotics_and_food/hogging-it- estimates-of-antimicrobial-abuse-in-livestock.html. (viewed Mar 2008).

58. United States General Accounting Office. Report to congressional requesters. April 2004. Antibiotic resistance. federal agencies need to better focus efforts to address risk to humans from antibiotic use in animals. Washington D. C.

59. Walsh, C. 2003. Antibiotics: actions, origins, resistance. ASM Press, Washington, D. C.

31 60. Wang, H., M. Manuzon, M. Lehman, K. 9. Wan, H. Luo, T. Wittum, A. Yousef, and L. Bakaletz. 2006. Food commensal microbes as a potentially important avenue in transmitting antibiotic resistance genes. FEMS Microbiol. Lett. 254:226-231.

61. Warsa, U. C., M. Nonoyama, T. Ida, R. Okamoto, T. Okubo, C. Shimauchi, A. Kuga, and M. Inoue. 1996. Detection of tet(K) and tet(M) in Staphylococcus aureus of Asian countries by the polymerase chain reaction. J Antibiot (Tokyo). 49: 1127- 1132.

62. Wegener, H. C., F. M. Aarestrup, L. B. Jensen, A. M. Hammerum and F. Bager. 1999. Use of antimicrobial growth promoters in food animals and Enterococcus faecium resistance to therapeutic antimicrobial drugs in Europe Emerg Infect Dis. 5: 329-335.

63. Zhanel, G. G., K. Homenuik, K. Nichol, A. Noreddin, L. Vercaigne, J. Embil, A. Gin, J. A. Karlowsky, and D. J. Hoban. 2004. The : a comparative review with the tetracyclines. Drugs 64: 63-88.

32 CHAPTER 3

OBJECTIVE

Commensals in the environment are gradually being recognized as playing a significant role in the maintenance and spread of antibiotic resistance among microbial populations by serving as reservoirs for AR determinants and potential facilitators for AR dissemination (7). The objective of this study is to determine the prevalence of antibiotic resistance, using tetracyline resistance as an example, in the commensal microflora of the healthy adult human digestive system. This will be accomplished by using conventional culture methods as well as culture-independent means to asses and quantify the resistance gene pool, and by analyzing the genetic background of selected ART isolates from human subjects. These data, in combination with results from another AR study our laboratory involving human infant subjects, will contribute to an advanced understanding on the emergence, persistence and circulation of AR in both the environment and the hosts.

33 CHAPTER 4

Prevalence of Tetracycline – Resistant Bacteria and tet Genes in the Healthy Adult Human Gut

4.1 Materials and Methods

Sample collection and enumeration of tetracycline-resistant populations.

After agreeing to participate in the study, subjects were asked to provide the following

information to establish individual background: gender, age, type of diet, ethnicity and

whether or not they live with pets, following the approved OSU IRB protocol

#2006H0083. Each subject was given a commode collection container (Fisher Scientific,

Fairlawn, NJ) for handling-free collection of fecal samples. Samples were analyzed

within the next 4 hours to reduce the loss of viable counts of microorganisms. One gram

of sample was weighed out in a sterile tube and resuspended uniformly in 9 mL sterile

distilled water to obtain a 10-1 initial dilution. Ten-fold serial dilutions, with distilled

water as diluent, were prepared to reach plating dilutions of 10-5, 10-6, 10-7 for agar plates

containing 16 μg-mL-1 tetracycline (Fisher Biotech, Fair Lawn, NJ) and 10-6, 10-7 and 10-

8 for agar plates without tetracycline. One-hundred μL of each diluted sample was spread-

plated on two sets of plating media, one set without antibiotic for total plate count and the

other set with tetracycline to recover resistant isolates. Based on results from a parallel

34 study involving child subjects, Columbia Blood Agar (CBA, BD & Company, Sparks,

MD) supplemented with 5 % defibrinated sheep’s blood (Colorado Serum Company,

Denver, CO) was used to enumerate viable bacterial isolates from the fecal samples.

Plates were incubated anaerobically using an O2-consuming and CO2-generating

GasPak™ envelope in a BBL™ GasPak™ jar (Fisher) for up to 48 h at 37°C, after which

plate counts of resistant and non-resistant bacterial populations were obtained and

recorded. Raw counts were converted to CFU-g-1 to enumerate tetracycline-resistant and total bacteria count populations for each sample. For each subject, approximately 30 isolates from the antibiotic plate were picked and inoculated on fresh CBA-Tet plates and saved as seeds for further genetic analyses.

Amplification of tetM standard fragment using polymerase chain reaction

Conventional PCR was used to amplify the tetM gene fragment that was used as a

template for the development of the standard curve for quantitative real-time PCR of the

tetM gene in fecal samples. Primers in this reaction were designed according to the tetM

sequence for Enterococcus sp. found NCBI. The primer pair tetM FP 5’-

CGAACAAGAGGAAAGCATAAG-3’and realRP 5’-CAAACAGGTTCACCGG-3’ was

used to amplify a 1257 bp segment of the tetM gene of the tetM+ isolate Enterococcus sp.

ACF-R1-1. This isolate was obtained from an infant fecal sample. To prepare the

template, DNA was extracted from the isolate using a previously described bead beat and

boil method (12). Following manufacturer recommendation (Invitrogen) PCR recipe for a

50μL reaction is as follows: 5 μL of 10X PCR buffer, 2 μL of 50mM MgCl, 2 μL of

35 10mM dNTPs, 0.3 μL each of 10μM primers and 0.3 μL 500 U Taq polymerase. Thermal

amplification was carried out in an iCycler (Bio-Rad Laboratories, Hercules, CA) thermal

cycler under the following conditions: initial denaturation at 95°C for 3 minutes, 35

cycles of: denaturation at 95°C for 1 min., annealing of primers at 50°C for 30 s, and

product extension at 68°C for 1 min. A final extension step at 68°C for 5 min was

followed, after which the products were put on temperature hold of 10 °C. After PCR and

visualization of the product, the fragment was purified using a QIAquick PCR

Purification Kit (Qiagen, Valencia, CA). This purified 1257 bp PCR product served as

the template in a real-time PCR reaction used to generate a standard curve for tetM and to quantify tetM gene copies present in samples.

Total DNA extraction from fecal samples

Total microbiome DNA was extracted from the fecal sample using the method of Yu and

Morrison (12) with minor modifications. Specifically, to loosen sample matrix, 0.2 g of

sample was transferred to a sterile 15 mL culture tube containing 0.2 g of 0.5 mm

zirconia beads and 4 mL distilled water. The tube was subjected to vortexing at

maximum speed for 1 minute. One mL of the resuspended sample was transferred to a 2

mL screw cap tube containing 0.3 g of 0.1 mm zirconia beads. The tube was centrifuged

at 5400 x g for 5 minutes to collect sample pellet. The supernatant was discarded. To pool sample, the remaining sample resuspension was transferred to the same screw-cap tube in

1 mL portions until all the sample was transferred. Another deviation from the Yu-

Morrison method was the application of two rounds (instead of one) of homogenization

36 for 3 minutes at maximum speed on a Mini-Beadbeater (BioSpec Products, Bartlesville,

OK, USA). After the extraction procedure, DNA was stored in AE Buffer (10 mM Tris-

Cl; 0.5 mM EDTA; pH 9.0) (Qiagen) at -20°C until ready for use.

Quantitative Taqman real-time polymerase chain reaction for tetM detection in

fecal samples

Generation of the standard curve. A Taqman PCR protocol was optimized for detection of the tetM gene in the sample microbiome. After preparation of the tetM standard

fragment as described above, its DNA concentration was measured using a pico green

assay in a nanodrop spectrophotometer (ND 3300 flurospectrometer, Wilmington, DE).

The DNA concentration, given in ng-μL-1, was converted to copies per μL using the

formula:

copies per µL = [DNA concentration / MW] x 6.022 x 1023 copies/mol-1, where MW is

the calculated molecular weight of the 1257 bp fragment.

Ten-fold dilutions (10-1 to 10-7) of the PCR product were made. The calculated copy number (copies per μL) was entered for each of the dilutions. From each of the dilutions,

1.5 μL template was added to a reaction containing 2.5 μL 10X Reaction Buffer, 1.5 μL

50 mM MgCl, 1.0 μL 10 mM dNTPs, 1.0 μL of 10 μM tetM specific TaqMan probe, 1.0

μL of each 10 μM primers and 0.3 μL 500U Taq polymerase in a real-time PCR run with

the following thermal cycling conditions - initial denaturation: 95°C for 1 min; 40 cycles

of denaturation: 95°C for 1 min, annealing: 53°C for 30s, extension: 68°C for 1 min; final

37 extension: 68 °C for 1 min; hold: 10°C. The real time primer pair is tetM-specific

Taqman primers realFP 5'-GAACATCGTAGACACTCAATTG -3', and realRP 5'-

CAAACAGGTTCACCGG-3’. These primers amplify a 169 bp fragment. The tetM- specific probe (5’-FAM-CGGTGTATTCAAGAATATCGTAGTG-BHQ-3’) were designed following procedures described previously (3).

Validation of extraction procedure. Extraction efficiency was assessed using DNA obtained from Enterococcus sp. ACF-R1-1 (Kinkelaar and Wang, 2008 IAFP). The single-colony derived overnight culture was serially diluted from 10-1 to 10-5. DNA was extracted from each dilution using the modified Yu and Morrison DNA extraction procedure, described in the previous section. The resulting extracts were subjected to the real-time amplification reaction and gene copies were quantified against the machine- generated standard curve. Reaction components and run conditions were similar to those used for the standard.

The same reaction components and real-time PCR conditions were also applied for quantification of tetM gene copies from fecal samples. However, due to the presence of

PCR inhibitors such as proteins, acids, metabolic products, etc., the samples needed to be diluted down ten-fold in order to observe the target amplicon for real-time PCR.

Minimum inhibition concentration (MIC) profiles of ART isolates.

Ten to 20 randomly selected isolates, representative of the microbial population from each fecal sample, were chosen for observation of resistance to increasing amounts of

38 tetracycline. The isolates were grown in Columbia broth (BD & Company, Sparks, MD)

overnight at 37°C under anaerobic condition. The overnight culture was diluted down to

2x10-2 dilution. This was the source of the inoculum in the wells which comprised half of

the total plate volume. The final concentration of the inoculum in the each well was 1%.

The tetracycline concentrations tested were 140, 70, 45 and 20 μg-mL-1. The MIC

procedure was carried out in a 96-well microtiter plate in which each well contained 1%

inoculum from an overnight culture of the isolate and the antibiotic amount in the specific

concentration tested. Wells containing the growth medium only, without any inoculum or

antibiotic were used as negative controls. Wells containing the growth medium and 1%

inoculum, without tetracycline were used as positive controls. The cultures were allowed

to grow at 37°C in anaerobic conditions for 12-24 hours after which the wells were

visually inspected for turbidity which indicates growth of the isolate.

Total DNA extraction from isolates

Individual colonies were picked from the seed plate and grown overnight anaerobically in

Columbia broth (CB, BD & Company) for total DNA extraction, where the method of

Anderson and McKay (1) was used. This procedure is relatively rapid and enables the

purification of large-size plasmid DNA. In this method, pelleted cells from a 10-mL

overnight culture were resuspended in 380 μL 6.7% sucrose-50mM Tris-1mM EDTA, pH 8.0. Cell lysis was carried out using a combination of lysozyme, sodium dodecyl sulfate (SDS), and vortexing at maximum speed. DNA and proteins were precipitated by the addition of sodium hydroxide and subsequently, phenol saturated with 3% sodium

39 chloride. DNA was separated from proteins by applying chloroform-isoamyl alcohol

(24:1 ratio) reagent. DNA was isopropanol-precipitated and resuspended in sterile distilled water.

Detection of tet genes in resistant isolates using conventional PCR

To assess the genotype of the tetracycline-resistant isolates in the samples, PCR was employed to amplify regions in the genetic material of the isolates that would correspond to a known tetracycline resistance gene. The presence of these genes would reveal the possible type of tetracycline resistance mechanism in the microorganism. Primers were designed based on known sequences from the NCBI database. The primer sequences used to amplify specific tet genes and the corresponding amplicon size are summarized below:

40 Size Primer name Sequence (5’→3’) Tm °C (bp)

tetM-FP GTGGACAAAGGTACAACGAG 59.6 406 tetM-RP CGGTAAAGTTCGTCACACAC 60.6

tetKF65 AGGATAGCCATGGCTACAAG 60.6 981 tetKR1045 ACAAGGAGTAGGATCTGCTG 58.4

tetWF510 CGACATAGAAGCATGGGATG 63.0 983 tetWR1492 TACAGTCCGTTACGTTCCAG 60.2

tetLF431 TTGGATCGATAGTAGCC 60.1 908 tetLR1340 GTAACCAGCCAACTAATGAC 56.1

tetQF320 TGCTTGATGGAGCAGTCCTC 62.0 817 tetQR1136 ATCACCTTGCTTCTCTCTTC 58.0

Table 4.1: Primer sequences used for tet screening of isolates. Melting temperatures for primers as well as the amplicon size are indicated.

Similar PCR formulation and thermal reaction conditions as the amplification of the tetM fragment for real time standard were applied, except for the annealing temperatures which were adjusted according to melting temperatures of the specific tet primers. The annealing temperature used for each primer pair is within 5°C lower than the primer melting temperature. After PCR, the amplicons were subjected to agarose gel

41 electrophoresis (AGE) and post-run ethidium bromide staining. For AGE, 10 μL of

products were loaded onto wells of 1% agarose gel and run at 100V in a 1X TAE (40 mM

Tris-acetate, 1 mM EDTA) buffer for 20 minutes. A 1 kb DNA ladder (Invitrogen) was

included in the run for estimation of product sizes. The gel was stained in 0.5 μg ethidium bromide solution per mL for 10 minutes and then destained in distilled water. Products were viewed under UV light using a transilluminator.

Amplification of 16S rRNA gene of isolates

A 1.5 kb fragment of the 16S rRNA gene of representative isolates were amplified using

conventional PCR previously described (3). The primer pair 16S-up 5’-

AGAGTTTGATCCTGGCTCCG-3’ and 16S-down 5’-TACCTTGTTACGACTT-3’ was

used. The 16S rRNA gene sequences of the isolates were compared to those deposited in

the NCBI database for determination of identities.

DNA-DNA hybridization (Southern blot) analysis of ART isolates

Total DNA extracted from resistant isolates using the Anderson and McKay method was

used for hybridization with digoxygenin-labeled tetM gene probe. The main steps for

Southern blot include DNA probe labeling, membrane transfer, fixation, washing and detection. First, DNA was run in a 0.8 % agarose gel, visualized and transferred to a nylon membrane following the Southern blotting protocol indicated in Cold Spring

Harbor Protocols (2). The type of membrane used is a positively charged nylon membrane and the type of transfer was an alkaline transfer procedure. The probe was

42 labeled using DIG DNA Labeling Detection Kit from Roche (Roche Diagnostics,

Penzberg, Germany). The PCR-generated DNA fragment used as probe was run along

with the samples in the gel to serve as positive control. DNA from a tetM-free

Enterococcus sp. OG1RF strain was used as negative control.

4.2 Results

Enumeration of total and tetracycline-resistant populations in fecal samples

Nine fecal samples were used to enumerate total and tetracycline-resistant bacteria.

Across all samples, results (Fig. 4.1) indicate a total plate count that ranged from 6-10 log

CFU-g-1 fecal mass. The total plate count ranged from 3.0 x 106 CFU-g-1 (sample E) to

2.1 x 1010 CFU-g-1 (sample I). Resistant plate counts were in the range of 6 to 9 log CFU-

g-1. Although in all plate counts, total counts were always higher than the resistant counts,

a less than 1 log difference between the two counts was observed in 7 out of 9 samples

(Samples E, F, H, I, K, L, and N). Sample G has within 1 log difference in resistant and

total counts while sample J has a total plate count 3 logs higher than the resistant count.

The results suggest approximately 0.1-10% of the bacterial population in adult fecal

samples are Tetr.

43 12

10

8 L m

r e p 6 U F C

g o l 4

2

0 E F G H I J K L N Subjects

Figure 4.1: Total plate count (blue bars) and tetracycline-resistant count (orange bars) of bacteria isolated from fecal samples of 9 healthy human subjects using Columbia

Blood Agar (CBA) and CBA added with 16 μg/mL tetracycline. Both media were supplemented with 5% defibrinated sheep’s blood

Quantitative real-time polymerase chain reaction for tetM detection in fecal samples

Fig. 4.2 shows the standard curve developed for quantitative real-time PCR determination of tetM gene copy numbers present in each sample. The original (undiluted) DNA concentration of the 1257 bp standard template was determined to be 1.8 ng-mL-1

44 equivalent to 1.90 x 109 copies per mL using the formula indicated in the Methods part of this chapter. Threshold cycle values (Ct) of each of the seven standard dilutions were determined from real-time run. Each point represents the mean of three measurements.

The curve shows decreasing Ct values with increasing copy numbers. The curve shows an R squared value of 0.995 which indicates an acceptable fit of the data points in the curve generated.

40

y = -4.292x + 44.703 35 R2 = 0.995

30 )

t 25 C (

e l c y c

20 d l o h s e r

h 15 t

10

5

0 0 1 2 3 4 5 6 7 8 9 log tet M copy number

Figure 4.2: Standard curve for determination of tetM gene copy numbers using

TaqMan real-time PCR. Each data point represents a mean of three measurements.

Standard deviations are all within 10%.

45 The tetM containing Enterococcus isolate, ACFR1-1 from infant fecal sample was used

to determine the correlation between the resistant count of the isolate and tetM gene copy

numbers. DNA was extracted from ten-fold serial dilutions of an overnight culture of the

isolate which had a tetracycline-resistant count of 1.90 x 109 resistant CFU-mL-1

determined by plate count. Gene copies from each of the dilutions were determined

against the standard in a real-time PCR run. Fig. 4.3 shows linear curve with a positive

slope that indicates a directly proportional relationship between the tetracycline-resistant

CFU-mL-1 and tetM gene copies per mL. For each log increase in resistant CFU/ml, there is a close to 1 (0.9543) log increase in copy number. Each data point is a mean- representation of three measurements. However, the tetM gene copy numbers by real- time PCR were approximately one log lower than the plate count numbers, suggesting the

DNA extraction efficiency is no more than 10%. The graph was generated using EXCEL

Windows Microsoft program.

46 6.5 y = 0.9543x - 0.9119 6 R2 = 0.9978 l m

r

e 5.5 p

r e b

m 5 u n

y

p 4.5 o c

e

n 4 e g

M t

e 3.5 t

g o l 3

2.5 4 4.5 5 5.5 6 6.5 7 7.5 log tetracycline-resistant CFU per ml

Figure 4.3: Validation of extraction efficiency. The curve shows the directly proportional

correlation between tetracycline-resistant plate count and tetM gene copy numbers in the

isolate Enterococcus sp. ACFR1-1.

Using the developed real-time PCR method, the tetM gene pool in fecal samples from 14 adult subjects was assessed. The gene pool was quite large in all the subjects examined, with the values differed only within one log difference (Fig. 4.4). Fig. 4.5 shows a

47 comparison of tetracycline-resistant CFU-ml-1 obtained from plate count and tetM gene copy numbers per gram determined from real-time PCR in each sample. Five samples (F,

H, J, L, N) have only within 1 log difference between the two values. The rest of the four samples (E, G, I, K) have a difference of more than 1 but less than 2 logs, suggesting the consistency of the data by the two methods.

12 g

r e p 10 r e b

m 8 u n

y

p 6 o c

e

n 4 e g

M

t 2 e t

g o

l 0 A B C D E F G H I J K L M N Subjects

Figure 4.4: Quantification of tetM gene copy numbers in fecal samples of 14

healthy human adults using real-time qPCR. Values represent means of two

measurements.

48 12 g

r

e 10 p

r e b m u n 8 y p o c

e n e g

6 M t e t

g o l

r 4 o

U F C

R t e

T 2

g o l

0 E F G H I J K L N Subjects

Figure 4.5: Assessment of antibiotic resistance in human digestive microflora by conventional

plate counting and real-time PCR. Orange bars, log tetracycline-resistant CFU per g; aqua

bars, log tetM gene copy number per g.

Minimum inhibition concentration (MIC) profiles of ART isolates.

Although 10-20 isolates per sample were picked from the seed plates (90-270 total), only a total of 78 isolates (from 5 subjects) were able to be grown on CB broth medium I expect that is discussed in the discussion section. Of these isolates, 62 (79%) showed tetracycline resistance of more than 140 μg-mL-1.

49 Plasmid profiles of AR isolates

Total DNA was extracted from 86 isolates from 6 subjects. Of these, 39 were found to

contain one or more plasmids. Eight different plasmid profiles were identified from the

extracts. In Fig. 4.6, plasmid of a representative isolate from each of the different profile

groups is shown. The first profile (labeled as F-3) in the figure is the most common

among the isolates (22/39), followed by profile H-3 (6/39), profile H-5 (3/39) and profile

L-6 (3/39), and profile H-9 (2/39). The remaining 3 isolates have unique plasmid profiles.

F-3 H-3 H-5 H-9 L-6 H-20 F-5 H-1

16,210 bp

5,012 bp 16,210 bp

2,067 bp

5,012 bp

2,972 bp

Figure 4.6: Representative plasmid profiles from isolates.

50 16S rRNA gene sequences of representative isolates

The 16S rRNA gene sequence of 11 representative isolates from 5 subjects showed high

homology with the genera: Escherichia (9/11), Shigella (1/11) and Enterococcus (1/11).

Detection of tet genes in resistant isolates

The 39 plasmid-carrying isolates were screened for the presence of 5 tet genes, tetK, tetL,

tetM, tetQ and tetW. DNA templates from the isolates were first group-screened using five isolates per tube. When a group comes up positive for a particular tet gene, each of

the 5 isolates in the group was individually screened for the same gene. Table 4.2 shows

the breakdown of the number of tet positive isolates from the subjects. Among the 39

isolates, 20 or 51% tested positive for tetM. This gene appears to be the most prevalent in

the isolates examined. The gene tetW on the other hand was found in only 2 of the

isolates. Seventy-four percent (29/39) of the isolates was found to carry at least one of the

6 tet genes tested. Of these, 58% (17/29) carry two or more of the tet genes, the most

common of which is tetL/M.

51 Subject tetK tetL tetM tetQ tetW A B C D E F 1 3 2 G H 4 6 9 I J K 5 5 3 L 1 2 2 M N 1 Total 4 13 20 5 2 % carrying 10 33 51 12 5 gene

Table 4.1: tet gene screening of isolates from digestive microflora of healthy adult human subjects. Isolates from subjects highlighted in gray were not able to grow in the chosen broth medium, Columbia broth, and so were not examined.

Southern blot analysis of ART isolates

Only preliminary result for DNA-DNA hybridization is presented in this study. Nine tetM positive isolates with different plasmid profiles were used for this initial run. Fig.

4.7 shows a number of very low-signal positives after the denatured DNA from the isolates were hybridized with a 406 bp tetM gene probe.

52 L 1 2 3 4 5 6 7 8 9 10 11

Figure 4.7: DNA-DNA hybridization for tetM detection. Plasmids from tetM gene positive isolates were probed for digoxygenin-labeled 406 bp tetM. L, 1 kb ladder; lanes 1-9 are isolates K-4, K-7, L-3, H-4, H-24, H-19, H-18, H-22 and H-20, respectively; lane 10, tetM- Enterococcus sp. OG1RF (negative control); lane 11, tetM amplicon used as probe (positive control).

53 4.3 Discussion

Proper measurement of microbial population from complex matrices such as fecal samples is a challenging issue. Not only many of the bacteria are anaerobic and are hard to culture in vitro, gut flora usually contain 300-500 species of bacteria so it is very difficult to find the proper media and incubation conditions to recover all of them.

Therefore, culture-dependent methods can hardly reveal the full picture of AR in fecal samples and thus culture-independent methods such as real-time PCR is sought. On the other hand, fecal samples contain various compounds that can inhibit the enzymes involved in DNA extraction and PCR amplification. Furthermore, many adult fecal samples have very tight structure and the contents vary among subjects. Therefore optimizing procedures for DNA extraction and method validation are also essential for the proposed study. In this study, we have developed a Taqman-real-time PCR-based method and successfully used it to assess the tetM gene pool in adult fecal samples. The size of the gene pool ranged from 10-7 to 10-10 gene copy number per gram sample as indicated in Fig. 4.4. It is worth noting that the efficiency of the measurement, largely dependent upon the DNA extraction efficiency, is still low. The pure culture validation study showed that the efficiency is no more than 10%, if the strain used in the study only has one copy of the plasmid in each cell. Because all adult samples carry very high load of tetM gene already, we were not able to directly spike the fecal samples and validation the combined efficiency with extraction and PCR amplification, which is expected to be even lower than 10%. Based on results from another study using infant subjects, the efficiency was found to be within the range of 0.1-10%. We anticipate the efficiency in

54 adult sample measurement will be lower than those from infant subjects, due to the

structure and complexity of the adult fecal samples.

In this study, prevalence of ART bacteria and AR genes in human digestive

microflora were observed. This is not surprising given the long and prophylactic history

of tetracycline. The antibiotic has been in widespread use for decades and has therefore

long provided selective pressure resulting in the emergence and maintenance of resistant

bacteria in various environmental compartments: soil, water, food animals and plants. In

the light of the recognition of food as a potential avenue for carrying resistant

microorganisms and resistance genes into the human oral and digestive ecosystems (10),

finding ART bacteria and AR genes in the human colon contents is almost inevitable.

Although information as to the identity of representative resistant isolates and the kind of

resistance genes they carry are provided in this study, it is still an underestimation of the

entire resistance scenario. Growth and isolation of resistant microbial populations

analyzed relied on culture dependent methods which leaves some if not the majority of

the colon microflora unexamined. However, findings from other investigators revealed

that in the numerically dominant colon bacteria, Bacteriodes spp., tetracycline-resistance was also present and found to be increasing in prevalence through time (11). Also in our study, quantitative real-time PCR was utilized to asses the gene pool of the ribosomal protection protein tetM, as this resistance-conferring gene was found to be present in

many of the isolates. Culture-independent methods such as real time qPCR provide the

advantage of total quantification of specific genes from the entire sample microbiome,

not just from culturable ones. The tetM gene copy number per gram of sample from

55 subjects in this study range from 7 to 9 logs and no significant variation across the different subjects was found. Could this be the same situation for the rest of the population? Subjects in this study are within the age range of 20-35 years old and all are healthy, have not used antibiotics in the past 3 months and are on regular diet. Indeed, our sample is limited in both size and diversity for a definitive conclusion to be made. It would also be worth investigating how ART bacteria and AR gene profiles in the human gastrointestinal tract vary with differences in diet, age, exposure to antibiotics, among other environmental factors. Although only one gene was examined in this part of the study, the suitability of real-time qPCR was assessed and the protocol for quantifying resistance gene pool in a sample matrix was optimized, which sets the stage for examining other genes in the future.

Although the observation of resistance prevalence in colonic content is not a direct proof of the involvement of food in the spread of AR, it is an indication of the heavy ART bacterial load and high AR gene pool in the human digestive ecosystem resulting from the oral route. The human gut microbiota is composed of the individual’s resident microflora, surviving bacteria from swallowed material including food and sometimes pathogens from infections. By comparing results from the infant study

(Kinkelaar and Wang, 2008 AIFP), we found the Tetr population in adult fecal samples are approximately 1-3 log higher than that from infant subjects. While Enterococcus sp. and Streptococcus sp. were found to be dominant in the culturable Tetr population from infant fecal samples, Escherichia, Shigella and Enterococcus were found to be the major ones from adult fecal samples. These data suggest that the ART bacteria in conventional

56 foods consumed by adults may contribute to the dynamic shift of the ART bacteria and

increased AR gene pool as seen in adult subjects. Whether our results indicate a dynamic

gene transfer occurring in the human gut is still yet to be determined. The occurrence of

gene transfer in the gastrointestinal ecosystem is however demonstrated by studies

conducted in animal models such as gnotobiotic rats (4) and antibiotic treated mice (5). In

a recent study Mater and co-workers observed the acquisition of vancomycin resistance

from an enterococci by a probiotic Lactobacillus acidophilus commercial strain during

digestive transit in mice (8). Furthermore, one human study reports an in vivo transfer of

a vanA resistance gene from an Enterococcus faecium isolate of animal origin to an E.

faecium isolate of human origin in the intestines of human volunteers (6). A growing body of research also documents the cross-transfer of resistance genes even between very distantly related species even crossing genus boundaries. The ease of transfer of some of these specific genes has been attributed to mobile genetic elements that facilitate the accelerated acquisition and dissemination of these genes. Herein lays the importance of characterizing the genetic location of resistance genes. Roberts has hypothesized that the carriage and spread of AR determinants is dictated by where in the bacterial genome the gene is found (9). The latter part of this study is an attempt to characterize the resistance genotype of the tetR isolates that we were able to grow. Different plasmid profiles were observed after total DNA extraction of the representative isolates from the different subjects. In this study, multiple tet genes, including tetL, tetM, tetQ and tetW were found

in fecal isolates by PCR. This indicates the presence of a variety of tetracycline resistance

mechanisms in the isolates and that tetracycline resistance was not only confined to a

57 particular plasmid profile (or strain) but is actually present in different strains. Since gene transfer in the gut environment is carried out mostly by conjugation, it would be interesting to find out whether plasmid-borne resistance genes can be transferred to other strains as well.

58 BIBLIOGRAPHY

1. Anderson, D. G. and L. L McKay. 1983. Simple and rapid method for isolating large plasmid DNA from lactic Streptococci. Appl Environ Microbiol. 46: 549-552.

2. Cold Spring Harbor Protocols. 2006. doi:10.1101/pdb.prot4040.

3. Connor, C., Luo, H., McSpadden-Gardrener, B.B., and H.H. Wang. 2005. Development of a second rapid detection system for Alicyclobacillus spp. using real-time PCR. Int J. Food Microbiol. 99:229-235.

4. Jacobsen, B.L., M. Skou, A.M. Hammerum, and L.B. Jensen. (1999) Horizontal transfer of the satA gene encoding streptogramin A resistance between isogenic Enterococcus faecium strains in the gastrointestinal tract of gnotobiotic rats. Microb Ecol Health Dis. 11: 241–247.

5. Lester, C. H., N. Frimodt-Moller, and A. M. Hammerum. 2004. Conjugal transfer of aminoglycoside and macrolide resistance between Enterococcus faecium isolates in the intestine of streptomycin-treated mice. FEMS Microbiol. Lett. 235: 385-391.

6. Lester, C. H., N. Frimodt-Moller, T. L. Sorensen, D. L. Monnet, and A. M. Hammerum. 2006. In vivo transfer of a vanA resistance gene from an Enterococcus faecium isolate of animal origin to an E. faecium isolate of human origin in the intestines of human volunteers. Antimicrob Agents Chemother. 50: 596-599.

7. Luo, H., Wan, K., H.H. Wang. 2005. High-frequency conjugation system facilitates biofilm formation and pAMβ1 transmission by Lactococcus lactis. Appl Environ Microbiol. 71: 2970–2978.

8. Mater, D. D., P. Langella, G. Corthier, and M. J. Flores. 2008. A probiotic Lactobacillus strain can acquire vancomycin resistance during digestive transit in mice. J Mol Microbiol Biotechnol. 14: 123-127.

9. Roberts, M. C. 1996. Tetracycline resistance determinants: mechanisms of action, regulation of expression, genetic mobility, and distribution. FEMS Microbiol. Rev. 19: 1-24

10. Salyers, A. and N. B. Shoemaker. 2006. Reservoirs of antibiotic resistance genes. Anim Biotechnol. 17:137-146.

59 11. Shoemaker, N. B., H. Vlamakis, K. Hayes, and A. A. Salyers. 2001. Evidence for extensive resistance gene transfer among Bacteroides spp. and among Bacteroides and other genera in the human colon. Appl Environ Microbiol. 67: 561-568.

12. Yu, Z. and M. Morrison. 2004. Improved extraction of PCR-quality community DNA from digesta and fecal samples. Biotechniques. 36:808-12.

60 CHAPTER 5

CONCLUSION AND FUTURE DEVELOPMENT

In summary, this work revealed the large pool of Tetr bacteria and tetM gene pool in adult human subjects. This result is in agreement with observations from previous studies of a high level of resistance genes among colonic bacteria (30, 36, 37). A qPCR method was optimized to quantify the AR gene pool from adult fecal samples. A significant portion of the Tetr isolates carried various plasmids and had the tetracycline

MIC level of no less than 140 µg per mL. The tetM gene in a number of isolates was plasmid-encoded. The mobility of the plasmids needs to be further investigated.

The finding of a large ART bacterial population from the fecal samples is not surprising because aside from the fact that most healthy human adults have been exposed to antibiotics at some point in their life, the human gut is an ideal place for bacterial conjugation, a major driving force for gene transfer events. A prerequisite for conjugation is cell to cell interaction and this is readily accomplished in an environment of heavy microbial load such as the human gut. Also, the colon is moist and rich in nutrients, which is another favorable situation for HGT. There is however a need to assess the extent of gene transmission that occurred in the colon. How is one to determine whether the resistant genes were acquired by isolates while in transit in the colon or are these

61 genes already possessed by their hosts even prior to their entry into the human digestive system, through food perhaps? Is it also possible that these genes are carried by the resident microflora of the human host? Which isolates are the donors and which ones are the recipients? The task of answering these questions is rather difficult considering the limitations of the study. Given unlimited resources, ample monitoring time, a thorough knowledge of an individual’s normal microflora, an ideal situation would have been to feed subjects with a controlled diet, one that has been characterized for non-resistant as well as ART bacteria and AR gene profiles and then recover these isolates in the form of fecal samples and observe changes in the profiles. However, this study provides some insight as to the usefulness of quantitative PCR in assessing the gene pool level of a particular gene. This is a targeted approach and can be applied to other AR genes as well.

This would be useful in determining gene copy threshold levels for HGT to take place.

62 APPENDIX

Gene Cloning and Expression of Lactococcus lactis clumping protein CluA

Prior to working on the study tetracycline resistance in humans, I have initially worked on cloning and expression of CluA, a cell wall associated protein in Lactococcus

lactis involved in cell to cell interaction and aggregation and high frequency transfer of

the sex factor. In this study, the cluA gene was cloned in a manner that the cell wall

anchor protein is removed and replaced with histidine sequences to facilitate purification

of protein upon expression. The method used was that of Stenz, et al (69). Two fragments

of the cluA gene were amplified from the chromosome of Lactococcus lactis MG1363

using polymerase chain reaction. The 3642 bp fragment was amplified using the primers

Mclubfp 5’-CGCGGATCCATGAAAAAAACATTGAGAGAC-3’ and MDLC2

5’CTAATGATGATGATGATGATGTGAACCTCTTGGGACAAGTGAACCTGTGA

TTTTTTCAATCACG-3’. Mclubfp has a BamHI restriction site while MDLC2 has sequences that code for six histidine residues. The 271 bp fragment was amplified using the primers 5’-CATCATCATCATCATTAGTGCTGAGGAATATCTTCAG-3’ and

DEXT3 5’-GACTCGAGGATATCAATAAGGTAATGAG-3’. The forward primer for

this fragment contains sequences for histidine and the reverse primer contains an XhoI

site. The PCR products were purified using a PCR purification kit (Qiaquick, QIAGEN).

63 Both products were used as template to amplify a 3743 bp crossover product using

Mclubfp and DEXT3 primers. The crossover product was subjected to restriction enzyme digestion with BamHI and XhoI and ligated to a similarly digested commercial cloning vector, pCR 2.1 using a TOPO cloning kit (Invitrogen, USA) and E. coli DH5α competent cells. Transformants were screened using a commercial plasmid extraction kit

(QIAGEN).

For expression of CluA in L. lactis, a nisin-inducible vector pMSP3535 (9) was used. Ideally, upon induction of 1 μg/mL nisin, the CluA protein will be expressed and secreted out of the cell into the growth medium due to the lack of the cell wall anchor signal. The protein can then be purified from the supernatant using a Ni-His affinity column. However, this part of the study was unsuccessful. Further investigation is needed.

64 BIBLIOGRAPHY

1. Aminov, R. I., J. C. Chee-Sanford, N. Garrigues, B. Teferedegne, I. J. Krapac, B. A. White, and R. I. Mackie. 2002. Development, validation, and application of PCR primers for detection of tetracycline efflux genes of gram-negative bacteria. Appl Environ Microbiol. 68: 1786-1793.

2. Aminov, R.I., and R.I. Mackie. 2007. Evolution and ecology of antibiotic resistance genes. FEMS Microbiol Lett. 271: 147-161.

3. Ammor, M. S., M. Gueimonde, M. Danielsen, M. Zagorec, A. H. van Hoek, C. G. de Los Reyes-Gavilán, B. Mayo, and A. Margolles. 2008. Two different tetracycline resistance mechanisms, plasmid-carried tet(L) and chromosomally located transposon-associated tet(M), coexist in Lactobacillus sakei Rits 9. Appl Environ Microbiol. 74: 1394-1401.

4. Anderson, D. G. and L. L McKay. 1983. Simple and rapid method for isolating large plasmid DNA from lactic Streptococci. Appl Environ Microbiol. 46: 549-552.

5. Babic, A., A. Lindner, M. Vulic, E. Stewart, and M. Radman. 2008. Direct visualization of horizontal gene transfer. Science 319: 1533-1536.

6. Barr, V., K. Barr, M. R. Millar, and R. W. Lacey. 1986. β- lactam antibiotics increase the frequency of plasmid transfer in Staphylococcus aureus. J Antimicrob Chemother. 17: 409-413.

7. Benveniste, R., and J. Davies. 1973. Aminoglycoside antibiotic-inactivating enzymes in actinomycetes similar to those present in clinical isolates of antibiotic- resistant bacteria. Proc Natl Acad Sci. 70: 2276-2280.

8. Bryan, A., N. Shapir, and M.J. Sadowsky. 2004. Frequency and distribution of tetracycline resistance genes in genetically diverse, nonselected, and nonclinical Escherichia coli strains isolated from diverse human and animal sources. Appl Environ Microbiol. 70: 2503-2507.

9. Bryan, E. M., T. Bae, M. Kleerebezem, and G. M. Dunny. 2000. Improved vectors for nisin-controlled expression in gram-positive bacteria. Plasmid. 44: 183-190.

65 10. Carey, C.M., J.L. Kirk, S. Ohja, and M. Kostrzynska. 2007. Current and future uses of real-time polymerase chain reaction and microarrays in the study of intestinal microbiota, and probiotic use and effectiveness. Can J Microbiol. 53:537-550.

11. Center for Disease Control and Prevention. 2007. http://www.cdc.gov/narms/faq_pages/11.htm (viewed Mar 2008).

12. Center for Disease Control and Prevention. National Antimicrobial Resistance Monitoring System for Enteric Bacteria (NARMS): Human Isolates Final Report, 2004. Atlanta, Georgia: U.S. Department of Health and Human Services, CDC.

13. Centers for Disease Control and Prevention. 2007. http://www.cdc.gov/NARMS/. (viewed May 2008).

14. Chopra, I., and M. Roberts. 2001. Tetracycline antibiotics: mode of action, applications, molecular biology, and epidemiology of bacterial resistance. Microbiol Mol Biol Rev. 65: 232-260.

15. Clewell, D. B. and C. Gawron-Burke. 1986. Conjugative transposons and the dissemination of antibiotic resistance in streptococci. Annu Rev Microbiol. 40: 635- 659.

16. Cold Spring Harbor Protocols. 2006. doi:10.1101/pdb.prot4040.

17. Committee on Drug Use in Food Animals. 1999. The use of drugs in food animals, benefits and risks. National Academy Press, Washington, D.C.

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

19. Connor, C., Luo, H., McSpadden-Gardrener, B.B., and H.H. Wang. 2005. Development of a second rapid detection system for Alicyclobacillus spp. using real- time PCR. Int J. Food Microbiol. 99:229-235.

20. Davies, J. 1994. Inactivation of antibiotics and dissemination of resistance genes. Science. 264: 375-382.

21. Doan, T. L., H. B. Fung, D. Mehta and P. F. Riska. 2006. Tigecycline: a glycylcycline antimicrobial agent. Clin Ther. 28: 1079-1106.

22. Durán, G. M., and D.L. Marshall. 2005. Ready-to-eat shrimp as an international vehicle of antibiotic-resistant bacteria. J Food Prot. 68:2395-2401.

66 23. Fan W., T. Hamilton, S. Webster-Sesay, M.P. Nikolich, and L.E. Linder. 2007. Multiplex real-time SYBR Green I PCR assay for detection of tetracycline efflux genes of gram-negative bacteria. Mol Cell Probes. 21: 245-256.

24. Flórez, A. B., M. S. Ammor, and B. Mayo. 2008. Identification of tet(M) in two Lactococcus lactis strains isolated from a Spanish traditional starter-free cheese made of raw milk and conjugative transfer of tetracycline resistance to lactococci and enterococci. Int J Food Microbiol. 121: 189-194.

25. Fluit A.C., A. Florijn, J. Verhoef and D. Melatovic. 2005. Presence of tetracycline resistance determinants and susceptibility to tigecycline and minocycline. Antimicrob Agents Chemother. 49: 1636-1638.

26. Francia, M. V., P. Avila, F. de la Cruz, and J. M. Lobo. 1997. A hot spot in plasmid F for site-specific recombination mediated by Tn21 integron integrase. J Bacteriol. 179: 4419-4425.

27. Frech, G. and S. Schwarz. 2000. Molecular analysis of tetracycline resistance in Salmonella enterica subsp. enterica serovars Typhimurium, enteritidis, Dublin, Choleraesuis, Hadar and Saintpaul: construction and application of specific gene probes. J Appl Microbiol. 89: 633-641.

28. Gevers, D., L. Masco, L. Baert, G. Huys, J. Debevere, and J. Swings. 2003. Prevalence and diversity of tetracycline resistant lactic acid bacteria and their tet genes along the process line of fermented dry sausages. Syst Appl Microbiol. 26: 277-283.

29. Grugel, C. and J. Wallmann. 2004. Antimicrobial resistance in bacteria from food- producing animals: risk management tools and strategies. J of Vet Med B. 51: 419- 421.

30. Gueimonde, M., S. Salminen, and E. Isolauri. 2006. Presence of specific antibiotic (tet) resistance genes in infant faecal microbiota. FEMS Immunol Med Microbiol. 48: 21-25.

31. Handbook of experimental pharmacology, Vol 78. 1985. The tetracyclines. J. J. Hlavka and J. H. Boothe (eds). Springer Verlag, New York.

32. Hastings, P., S. Rosenberg, and A. Slack. 2004. Antibiotic-induced lateral transfer of antibiotic resistance. Trends Microbiol 12: 401-404.

33. Hughes, V.M., and N. Datta. 1983. Conjugative plasmids in bacteria of the ‘pre- antibiotic era. Nature 302: 725-726.

34. Institute of Medicine. 1998. Antimicrobial drug resistance: issues and options.

67 Workshop report. National Academy Press, Washington.

35. Jacobsen, B.L., M. Skou, A.M. Hammerum, and L.B. Jensen. (1999) Horizontal transfer of the satA gene encoding streptogramin A resistance between isogenic Enterococcus faecium strains in the gastrointestinal tract of gnotobiotic rats. Microb Ecol Health Dis. 11: 241–247.

36. Kanami, N., F. Nowrouzian, I. Adlerberth, and A. E. Wold. 2006. Tetracycline resistance in Escherichia coli and persistance in the infantile colonic microbiota. Antimicrob Agents Chemother. 50: 156-161.

37. Kazimeirczak, K. A., H. J. Flint, and K. P. Scott. 2006. Comparative analysis of sequences flanking tet(W) resistance genes in multiple species of gut bacteria. Antimicrob Agents Chemother. 50: 2632-2639.

38. Khaitsa, M. L., R. B. Kegode, and D. K. Doetkott. 2007. Occurrence of antimicrobial-resistant salmonella species in raw and ready to eat turkey meat products from retail outlets in the midwestern United States. Foodborne Pathog Dis. 4: 517-525.

39. Lehman, M., and H. H. Wang. Abstr. 2006. Antibiotic resistance in ready-to-eat salad, abstr. 020B-05. Abstr. 2006 IFT Annu. Meet. Food Expo.

40. Lester, C. H., N. Frimodt-Moller, and A. M. Hammerum. 2004. Conjugal transfer of aminoglycoside and macrolide resistance between Enterococcus faecium isolates in the intestine of streptomycin-treated mice. FEMS Microbiol. Lett. 235: 385-391.

41. Lester, C. H., N. Frimodt-Moller, T. L. Sorensen, D. L. Monnet, and A. M. Hammerum. 2006. In vivo transfer of a vanA resistance gene from an Enterococcus faecium isolate of animal origin to an E. faecium isolate of human origin in the intestines of human volunteers. Antimicrob Agents Chemother. 50: 596-599.

42. Lester, C. H., N. Frimodt-Moller, T. L. Sorensen, D. L. Monnet, and A. M. Hammerum. 2006. In vivo transfer of a vanA resistance gene from an Enterococcus faecium isolate of animal origin to an E. faecium isolate of human origin in the intestines of human volunteers. Antimicrob Agents Chemother. 50: 596-599.

43. Levy, S.B. 1992. The antibiotic paradox: how miracle drugs are destroying the miracle. Plenum Press, New York.

44. Levy, S.B. 2002. Active efflux, a common mechanism for biocide and antibiotic resistance. Sym Ser Soc Appl Microbiol. 31:65-71.

45. Luo, H., Wan, K., H.H. Wang. 2005. High-Frequency Conjugation System Facilitates Biofilm Formation and pAM_1 Transmission by Lactococcus lactis. Appl

68 Environ Microbiol. 71: 2970–2978.

46. Majiduddin, F. K., I. C. Materon, T. G. Palzkill. 2002. Molecular analysis of beta- lactamase structure and function. Int J Med Microbiol. 292: 127-137.

47. Manuzon, M. Y., S. E. Hanna, H. Luo, Z. Yu, W. J. Harper, and H. Wang. 2007. Quantitative assessment of the tetracycline resistance gene pool in cheese samples by real-time TaqMan PCR. Appl Environ Microbiol. 73: 1676-1677.

48. Mater, D. D., P. Langella, G. Corthier, and M. J. Flores. 2008. A probiotic Lactobacillus strain can acquire vancomycin resistance during digestive transit in mice. J Mol Microbiol Biotechnol. 14: 123-127.

49. Mathur, S and R. Singh. 2005. Antibiotic resistance in food lactic acid bacteria--a review. Int J Food Microbiol. 105: 281-295.

50. Molbak, K. 2004. Spread of resistant bacteria and resistance genes from animal to human- The public consequences. J of Vet Med. 51: 364-369.

51. Nester, E. W., D. G. Anderson, C. E. Roberts, Jr., N. N. Pearsall and M. T. Nester. 2004. Microbiology: a human perspective, 4th ed. McGraw Hill, New York.

52. Osterblad, M., K. Norrdahl, E. Korpimaki, and P. Huovinen. 2001. Antibiotic resistance: how wild are wild mammals? Nature 409: 37-38.

53. Pang, Y., B.A. Brown, V.A. Steingrube, R.J. Wallace Jr., and M.C. Roberts. 1994. Tetracycline resistance determinants in Mycobacterium and Streptomyces species. Antimicrob. Agents Chemother. 38:1408-12.

54. Phillips, I., M. Casewell, T. Cox , B. De Groot, C. Friis, R. Jones, C. Nightingale, R. Preston, and J. Waddell. 2004. Does the use of antibiotics in food animals pose a risk to human health? A critical review of published data. J Antimicrob Chemother. 53: 28-52.

55. Piendl, W. and A. Bock. 1982. Ribosomal resistance in the gentamicin producer organism Micromonospora purpurea. Antimicrob Agents Chemother. 22: 231-236.

56. Plumb, D. C. 1995. Veterinary drug handbook. Ames, Iowa.

57. Roberts, M. C. 1996. Tetracycline resistance determinants: mechanisms of action, regulation of expression, genetic mobility, and distribution. FEMS Microbiol. Rev. 19: 1-24

58. Roberts, M. C. 2002. Resistance to tetracycline, macrolide-lincosamide- streptogramin, trimethoprim, and sulfonamide drug classes. Mol Biotechnol. 20: 261-

69 83.

59. Rodriguez, C., L. Lang, A. Wang, K. Altendorf, F. Garcia, and A. Lipski. 2006. Lettuce for human consumption collected in Costa Rica contains complex communities of culturable oxytetracycline- and gentamycin-resistant bacteria. Appl Environ Microbiol. 72: 5870-5876.

60. Rosen, B. P. 1999. Resolving the antibiotic paradox: progress in understanding drug resistance and development of new antibiotics. Kluwer Academic, New York.

61. Salyers, A. A., A. Gupta, and Y. Wang. 2004. Human intestinal bacteria as reservoirs for antibiotic resistance genes. Trends Microbiol. 12: 412-416.

62. Salyers, A. A., N. B. Shoemaker, A. M. Stevens, and L. Y. Li. 1995. Conjugative transposons: an unusual and diverse set of integrated gene transfer elements. Microbiol Rev. 59: 579-590.

63. Salyers, A. and N. B. Shoemaker. 2006. Reservoirs of antibiotic resistance genes. Anim Biotechnol. 17:137-146.

64. Scott, G.M., and M.S. Kyi. 2001. Handbook of essential antibiotics. Harwood Academic, Amsterdam.

65. Shaw, K.J., P.N. Rather, R.S. Hare, and G.H. Miller. 1993. Molecular genetics of aminoglycoside resistance genes and familial relationships of the aminoglycoside- modifying enzymes. Microbiol Rev. 57:138-163.

66. Shoemaker, N. B., H. Vlamakis, K. Hayes, and A. A. Salyers. 2001. Evidence for extensive resistance gene transfer among Bacteroides spp. and among Bacteroides and other genera in the human colon. Appl Environ Microbiol. 67: 561-568.

67. Showsh, S. A. and R. E. Andrews, Jr. 1992. Tetracycline enhances Tn916-mediated conjugal transfer. Plasmid 28: 213-224.

68. Singer, R. S. and C. L. Holfacre. 2006. Potential impacts of antibiotic use in poultry production. Avian Dis. 50: 161-172.

69. Stentz, R., K. Jury, T. Eaton, M. Parker, A. Narbad, M. Gasson, and C. Shearman. 2004. Controlled expression of CluA in Lactococcus lactis and its role in conjugation. Microbiology. 150: 2503-2512.

70. Stokes, H.W., and R.M. Hall. 1989. A novel family of potentially mobile DNA elements encoding site-specific gene-integration functions: integrons. Mol Microbiol. 3: 1669-1683.

70 71. Tappenden, K. A. and A. S. Deutsch. 2007. The physiological relevance of the intestinal microbiota--contributions to human health. Am Coll Nutr. 26: 679S-683S.

72. Torres, O. R., R. Z. Korman, S. A. Zahler, and G. M. Dunny. 1991. The conjugative transposon Tn925: enhancement of conjugal transfer by tetracycline in Enterococcus faecalis and mobilization of chromosomal genes in Bacillus subtilis and E. faecalis. Mol Gen Genet. 225: 395-400.

73. Union of Concerned Scientists. 2006. http://www.ucsusa.org/food_and_environment/antibiotics_and_food/hogging-it- estimates-of-antimicrobial-abuse-in-livestock.html. (viewed Mar 2008).

74. United States General Accounting Office. Report to congressional requesters. April 2004. Antibiotic resistance. federal agencies need to better focus efforts to address risk to humans from antibiotic use in animals. Washington D. C.

75. Walsh, C. 2003. Antibiotics: actions, origins, resistance. ASM Press, Washington, D. C.

76. Wang, H., M. Manuzon, M. Lehman, K. 9. Wan, H. Luo, T. Wittum, A. Yousef, and L. Bakaletz. 2006. Food commensal microbes as a potentially important avenue in transmitting antibiotic resistance genes. FEMS Microbiol. Lett. 254:226-231.

77. Warsa, U. C., M. Nonoyama, T. Ida, R. Okamoto, T. Okubo, C. Shimauchi, A. Kuga, and M. Inoue. 1996. Detection of tet(K) and tet(M) in Staphylococcus aureus of Asian countries by the polymerase chain reaction. J Antibiot (Tokyo). 49: 1127- 1132.

78. Wegener, H. C., F. M. Aarestrup, L. B. Jensen, A. M. Hammerum and F. Bager. 1999. Use of antimicrobial growth promoters in food animals and Enterococcus faecium resistance to therapeutic antimicrobial drugs in Europe Emerg Infect Dis. 5: 329-335.

79. Yu, Z. and M. Morrison. 2004. Improved extraction of PCR-quality community DNA from digesta and fecal samples. Biotechniques. 36:808-12.

80. Zhanel, G. G., K. Homenuik, K. Nichol, A. Noreddin, L. Vercaigne, J. Embil, A. Gin, J. A. Karlowsky, and D. J. Hoban. 2004. The glycylcyclines: a comparative review with the tetracyclines. Drugs 64: 63-88.

71