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PROFILES OF TETRACYCLINE RESISTANT BACTERIA IN THE HUMAN INFANT DIGESTIVE SYSTEM THESIS Presented in partial Fulfillment of the Requirements for the Degree Master’s of Science in the Graduate School of The Ohio State University By Daniel Kinkelaar, B.A., B.S. ***** The Ohio State University 2008 Master’ Examination Committee: Approved by Professor Hua Wang, Advisor Professor Jeff Culbertson Professor Brian McSpadden-Gardener ________________________ Advisor Food Science and Nutrition Graduate Program iii ABSTRACT The rapid emergence of antibiotic resistant (ART) pathogens poses a serious threat to public health. A large antibiotic resistance (AR) gene pool has recently been found in commensal bacteria associated with many retail foods. Subsequently, the question is whether these foodborne ART bacteria through daily food consumption are responsible for the prevalence of ART bacteria in human digestive ecosystems. To address this issue, the ART bacteria profiles in fecal samples of infant subjects before solid food consumption were analyzed by total plate counting of ART bacteria and real-time PCR assessment of the tetM gene pool. Tetracycline-resistant (Tetr) bacteria were found in fecal samples from both breast and formula-fed babies shortly after birth without being exposed to the corresponding antibiotic. The numbers of ART bacteria within these subjects increased rapidly within four weeks before reaching a relatively stable level. The numbers of ART bacteria ranged from 5.5x105 to 1.9x108 CFU/g of sample and was maintained throughout the 12 month examination period. Similar shift of the tetM gene pool in the infant fecal samples was observed by real-time PCR. The tetM gene pool in all infant fecal samples, with the exception of the meconium samples, ranged at least from 9.0x105 to 9.0x108 copies per gram of sample. The data suggest that routes other than conventional food intake may have played a role in the initial colonization of ART ii iv bacteria in infants. The impact of the ART bacteria from conventional food intake on the dynamic shift of the ART bacteria in human digestive tract at later stages through occasional colonization and maybe horizontal gene transmission is yet to be revealed. iii ACKNOWLEDGMENTS I wish to thank my advisor, Hua Wang, for intellectual support and assistance which made this thesis possible. This research was supported by Dr. Wang’s Ohio State University start up fund. iv VITA September 11, 1972………………………………………..……Born - Parma, Ohio, USA 1995………………………………….....B.A Communications, The Ohio State University 2006………………………………………..B.S. Microbiology, The Ohio State University PUBLICATIONS Kinkelaar D, Wang HH. 2007. Antibiotic resistance development in human oral and gut ecosystems. The Center for Microbial Interface Biology Retreat. Abstract #45. Kinkelaar D, Wang HH. 2008. Profiles of tetracycline resistance bacteria in human microflora associated with infant digestive system. IAFP annual meeting. (P5-69). FIELDS OF STUDY Major Field: Food Science and Nutrition v TABLE OF CONTENTS Page Abstract………………………………………………………..……………………..…..ii Acknowledgements…………………………………………………...………………….iv Vita…………………………………………………………………………..…………....v List of Figures………………………………………………………………...………....viii Chapters 1. Introduction……………………………………………………….……………....1 1.1 Bibliography…………………………………………………………………4 2. Literature Review………………….……………………………………………...7 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 2.10 3. Introduction to antibiotics…………………………………..………………7 Application and uses for antibiotics…………………………..…………….9 Antibiotic resistant bacteria………………………………….…………….14 Exchange of genetic material……………………………...……..………..15 Vertical gene transfer...……………………………………………….….. 16 Horizontal gene transfer……………………………………………………….….17 Reservoirs for resistance genes………………...……………………….…20 Human microflora...…………………………………………………….…22 Means to assess bacterial populations...……………………………….…..29 Bibliography…………………………………………………………….…33 Profiles of tetracycline resistant bacteria in the human infant digestive system……………………….…...........................................................................38 3.1 Objectives………………………………………………………………….38 3.2 Introduction....……………………………………………………………...38 3.3 Material and methods………………………………………………………41 vi 3.3.1 3.3.2 3.4 3.5 3.6 4. Subjects and sampling……………………………………………41 Culturable microbial population assessment using conventional agar plating..…………………………………………………...…42 3.3.3 DNA template preparation……………………………………….42 3.3.4 Screening for representative tetr gene and identification of AR gene carriers……………………………………………………...44 3.3.5 Quantitative assessment of the tetM gene pool by Taqman real-time PCR…………………………………………………….45 Results……………………………………………………………………..47 3.4.1 Assessing culturable gut microflora by conventional plate counting………………………………………………....…47 3.4.2 Prevalence of Tetr bacteria in infant fecal samples………….…..49 3.4.3 Assessing the tetM gene pool by real-time qPCR………....…….50 3.4.4 The shift of ART bacterial population during infant development……………………………………………….53 Discussion…………………………………………………………………56 Bibliography……………………………………………………………....60 Conclusion and future developments……………………………………………63 4.1 Summary………………………………………………………………….63 4.2 Future studies …………………………………………………………….64 Bibliography……………………………………………………………………………..65 vii LIST OF FIGURES Figure Page 3.1 Variability in microbial recovery on selected bacterial media …………………48 3.2 Total and tetracycline resistant plate counts of 13 infant subjects………………49 3.3 Standard curve…………………………………………………………………...50 3.4 Extraction efficiency of tetM-specific qPCR……………………………………51 3.5 Validation of extraction by artificially spiking meconium with pure culture…...52 3.6 The distribution of tetM gene pool in 11 infant subjects…………………….......53 3.7 Total and resistant plate counts of twin over the course of a year…………….....54 3.8 The distribution of the tetM gene pool in twin subjects…………………………55 viii CHAPTER 1 INTRODUCTION Antibiotics are essential therapeutic tools for a wide variety of illnesses caused by bacterial infections. The rapid emergence of antibiotic resistant (ART) pathogens negates effective treatments and therefore is becoming a major threat to public health (2, 6, 19). In humans, the primary application of antibiotics is in therapeutic treatments caused by infections. Non-human uses include both curative and prophylactic treatments of companion and food animals, as well as applications in horticulture and aquaculture. The usage of antimicrobial agents in any of these applications selects for resistant populations (18). In addition to the direct propagation of the resistant bacteria, the exchange of mobile elements between commensal and pathogenic organisms facilitates the dissemination of the resistance genes across different bacteria, thus playing an important role in the rapid emergence of drug resistance (12, 14). The over prescribing of antibiotics to human and animal patients in clinical therapy is one potential rationale for the rapid increase in ART bacteria. The addition of antibiotics in sub-therapeutic doses to animal feed used in food animal production as a growth promoter has also been indicated as an important cause for this problem (5). 1 To protect the efficacy of therapeutic antibiotics used in humans, the major health organizations such as the World Health Organization (WHO), Centers for Disease and Prevention (CDC), and the European Union (EU) have stressed the need to control the spread of ART bacteria (1, 7, 20). Several government entities have taken the corresponding actions. In 1999, the European Union banned the application of certain antimicrobial agents (tylosin, spiramycin, bacitracin, and virginiamycin) as growth promoters in chicken, swine, and beef production (6). Denmark voluntarily stopped using all antimicrobial agents as growth promoters in cattle, broilers, and pigs in the following year. Since 1999, no antibiotics have been used as growth promoters in food animals in Denmark (1, 13, 21). However, the impact of prophylactic usage of antibiotics is still a topic of debate in the United States, and control strategies have just begun to be implemented. In 1996 the National Antimicrobial Resistance Monitoring System (NARMS) was established as a group effort among the CDC, United States Department of Agriculture (USDA), and Food and Drug Administration (FDA), whose primary mission is to monitor antibiotic resistance (AR) in foodborne enteric pathogens (2). The antibiotic enrofloxacin, a fluoroquinolone used for controlling bacterial infections in poultry, was banned in the United States in 2005. This is the first new antibiotic in the United States that has been removed from food animal production because of the potential threat to public health (3). In the past decade, various ART pathogens have been isolated from retail food samples, particularly meat and poultry products (10, 15, 16) suggesting the possible transmission of AR to humans through the food chain. Nevertheless, pathogens only count for a very 2 small percentage in microbial population and they are not a significant source for horizontal transmission of AR. However, data from recent studies illustrated that commensal bacteria in ready-to-eat foods may be a much more important avenue in transmitting AR to the general public through the food chain (4, 54). Many retail foods were found carrying a large number of bacteria containing AR genes (4, 18). The AR genes present in these foods are readily consumed by humans and can potentially be transferred to the human residential bacteria associated with the digestive system. These resistance genes from food may further serve as a reservoir transmitting the AR genes to pathogens. Both commensal and beneficial bacteria were identified as AR gene carriers. The AR genes from foodborne bacteria can be transferred to oral and gut residential bacterium via natural transformation (9, 18). In addition, several EU groups found that ART bacteria were prevalent in oral and gut ecosystems from healthy humans without recent exposure to antibiotics, suggesting that colonization, propagation or transmission can be independent from the selective pressure of the antibiotic (8, 11, 17). A comprehensive understanding of both the major pathways is essential for the development of targeted control strategies to combat this public health challenge. 3 BIBLIOGRAPHY 1. Anderson, A.D., J. McClellan, S. Rossiter, and F.J. Angulo. 2003. Public health consequences of use of antimicrobial agents in agriculture. In: Knobler, S.L., Lemon, S.M., Najafi, M., Burroughs, T. (Eds.), Forum on Emerging Infections: The Resistance Phenomenon in Microbes and Infectious Disease Vectors. Implications for Human Health and Strategies for Containment—Workshop Summary. Board on Global Health, Institute of Medicine, Appendix A, pp. 231–243. 2. Centers for Disease Control and Prevention.2007. Get smart: know when antibiotics work. http://www.cdc.gov/narms/faq.htm#3 (viewed December 2007). 3. Davidson DJ. In the matter of enrofloxacin for poultry: withdrawal of approval of Bayer Corporation's new animal drug application 1 (NADA) 140828 (Baytril). In: FDA Docket No. 00N-1571; 2004. 4. Durán, G. M., and D.L. Marshall. 2005. Ready-to-eat shrimp as an international vehicle of antibiotic-resistant bacteria. J Food Prot. 68:23952401. 5. Environmental Defense Fund. 2001. http://www.edf.org/documents/619_abr_general_factsheet_rev2.pdf (viewed March 2008). 6. Food and Drug Administration, Center for Veterinary Medicine, April 28, 2000 HHS Response to House Report 106-157- Agriculture, Rural Development, Food and Drug Administration, and Related Agencies, Appropriations Bill. Executive Summary. 7. Fries, R.. 2004. Conclusions and activities of previous expert groups: the Scientific Steering Committee of the EU. J Vet Med B Infect Dis Vet Public Health. 51:403-7. 8. 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. 4 9. Jacobsen, L., Wilcks, A., Hammer, K., Huys, G., Gevers, D., and S. Andersen. 2007. Horizontal transfer of tet (M) and erm(B) resistance plasmids from food strains of Lactobacillus plantarum to Enterococcus faecalis JH2-2 in the gastrointestinal tract of gnotobiotic rats. FEMS Microbiol Ecol 59:158–166 10. Kolar., M., Pantcek, R, Bardon. J, Vagnerova,I., Typovska, H., Doskar, J. and I. Valka. 2002. Occurrence of antibiotic-resistant bacterial strains isolated in poultry. Vet. Med. 47:52-59. 11. Lancaster, H., Ready, D., Mullany, P., Spratt, D., Bedi, R. and M. Wilson. 2003. Prevalence and identification of tetracycline-resistant oral bacteria in children not receiving antibiotic therapy. FEMS Microbiol Lett. 228: 99-104. 12. Molbak, K. 2004. Spread of resistant bacteria and resistance genes from animal to human- The public consequences. J of Vet Med. 51: 364-369. 13. Randerson, J. 2003. Ban on growth promoters has not increased bacteria. New Scientist. 183:13-15. 14. Salyers, A.A., Gupta, A., and Y. Wang. 2004. Human intestinal bacteria as reservoir for AR. Trends Microbiol. 12: 412-416. 15. Van,T.T.H., Moutafis,G., Tran, L.T.,and P. J. Coloe. 2007. AR in FoodBorne Bacterial Contaminants in Vietnam. Appl Environ Microbiol. 73: 7906-7911. 16. Van Looveren, M., Daube, G., De Zutter, L., Dumont, J., Lammens, C., Wijdooghe, M., Vandamme, P., Jouret, M., Cornelis, M. and H. Goossens. 2001. Antimicrobial susceptibilities of Campylobacter strains isolated from food animals in Belgium. J. Anti Chemother. 48, 235-240. 17. Villedieu, A., Diaz-Torres, M.L., Hunt, N., McNab, R., Spratt, D.A., Wilson, M. and P. Mullany. 2003. Prevalence of tetracycline resistance gene in oral bacteria. Antimicrob Agents Chemother. 47: 878-882. 18. Wang, H., Manuzon, M., Lehman, M., Wan, K., Luo, H., Wittum, T., Yousef, A., and L. Bakaletz. 2006. Food commensal microbes as a potentially important avenue in transmitting AR genes. FEMS Microbiol Lett. 254:226-231. 19. Wassenaar, T. 2005. Use of antimicrobial agents in veterinary medicine and implications for human health. Crit Views Microbiol. 31: 1555-169. 5 20. WHO. 2000. WHO Global Principless for the Containment of Antimicrobial Resistance in Animals Treated for Food: Report of a WHO Consultation, Geneva, Switzerland, June 5-9, 2000. 21. WHO. 2003. World Health Organization, Impacts of antimicrobial growth promoter termination in Denmark, The WHO international review panel’s evaluation of the termination of the use of antimicrobial growth promoters in Denmark. http://www.who.int/salmsurv/en/Expertsreportgrowthpromoterdenmark.pdf 6 CHAPTER 2 LITERATURE REVIEW 2.1 Introduction to antibiotics In 1887, Ernest Duchensne noted that bacteria growth was inhibited by the presence of Penicillium, a soil mold (24). Further studies were carried out by the British scientist, Sir Alexander Fleming in 1928, confirming that the presence of the mold disrupted the growth of the bacteria. It was later determined that a β-lactam moiety was the inhibitory compound disrupting the cell membranes of Gram-positive bacteria. It was determined that β-lactam antibiotics work by binding to the enzyme D,D- transpeptidase, which the cell uses to create cross-links the peptidoglycan in the cell wall. By preventing the crosslinks the membrane is compromised and cytolysis occurs due to osmotic pressure (15). Initially the mass production of penicillin was challenging. In 1942 there was only enough penicillin to treat 10 people and the first patient to successfully be treated by penicillin for streptococcal septicemia used half of the total supply at that time. In 1943, after a worldwide search found that a cantaloupe in the Peoria, Illinois market contained the highest and best quality penicillin, and the results from fermentation research on corn steep liquid at the Northern Regional Research Laboratory at Peoria, Illinois, led to large 7 scale production of the compound. These discoveries enabled the large scale application of antibiotics for the first time in World War II. Approximately 2.3 million doses were produced for the invasion of Normandy in 1944 (28). The successful application of penicillin in reducing war related infections drew great attention from the scientific community and triggered a new wave of scientific studies on the functionality of penicillin and searches for new antibiotics. By 1946 it became a standard treatment for bacterial infections caused by streptococci and staphylococci. Penicillin was effective on all sorts of infections caused by these two Gram positive bacteria such as strep throat, pneumonia, scarlet fever, septicemia and wound infections, reducing a significant amount of mortality. The introduction of new antibiotics, including tetracycline, streptomycin and chloramphenicol in the early 1950’s began the age of antibiotic chemotherapy, which provided a means to prevent a large number of bacterial infections from both Gram positive and Gram negative bacteria as well as intercellular parasites. The success of these antibiotics inspired scientists to develop synthetic antimicrobial agents such as sulfonamides or “sulfa drugs” and aminosalicylic acid or PAS to treat tuberculosis. The usages of antibiotics became widely accepted in the early 1950’s and were regularly administered as therapeutic agents to the general population (39). Soon after the use of penicillin on a large scale, microbial resistance to penicillin was detected. It was recently estimated that as many as 80% of the strains of Staphylococcus aureus today are resistant to penicillin, limiting its effectiveness to prevent infection (24). 8 Similar results were found in regard to resistance to tetracycline, streptomycin and chloramphenicol, with mounting evidence showing that multiple resistant genes could be passed between strains and species (50). In the event that a bacterial pathogen acquires resistance to an antibiotic, treatment becomes less- or non-effective. Therefore there is a great need for the development of new antibiotics. Effective treatment of infections requires a proactive approach in understanding how the antimicrobial agents work; as well as, why treatments fail, in order to stay ahead of microbial pathogens. 2.2 Application and uses for antibiotics Antibiotics can be classified in a number of ways, the broadest being its mode of action, such as, bactericidal or bacteriostatic. Bactericidal antibiotics kill the organism where as bacteriostatic antibiotic prevent the growth and reproduction of the bacterium by interrupting protein synthesis, cellular metabolism or DNA replication. Antibiotics can also be classified by the type and range of its target organisms. Broad spectrum antibiotics such as amoxicillin and levofloxacin can target a wide variety of diseasecausing bacteria including both Gram-positive and Gram-negative bacteria. Narrow spectrum antibiotics target a particular type of bacteria such as either Gram-positive or Gram-negative. Methicillin is an example of a narrow spectrum beta-lactam antibiotic derived from the penicillin used to treat beta-lactamase producing Gram-positive bacteria such as Staphylococcus aureus. Another common classification of antibiotics is based on the chemical structure of the antibiotic, such as tetracycline, macrolide or 9 aminoglycosides. Antibiotics can also be classified by their mode of administration such as topically, orally, intravenously or parentally (3, 32). Tetracycline, β-Lactam sulfonamides, penicillins, macrolides, fluoroquinolones, cephalosporins, aminoglycosides, streptogramins and chloramphenicols are common antibiotics that are used as prophylactic measures in the propagation of food animals as well as therapeutics to treat human illness. Therefore the bacteria that become resistant in animals to these drugs can also become resistant in humans (4, 9). Antimicrobial agents are often used for three primary reasons: to treat an identified bacterial infection, to treat those at risk of a bacterial infection, or as a growth promoter used as a food additive for animals meant for consumption (52). Antibiotics are administered orally, parentally or topically and are used in both human and veterinary medicine to treat and prevent disease (13). Antibiotics are also used to treat aquaculture and farm fields against pathogens. When used on farm fields in this manner they are referred to as pesticides. The drug resistance issue is very serious today. The emergence of ART bacteria in hospitals seems to be correlated to the overuse of antibiotics in the clinical setting. In the 1940’s, penicillin was very effective in treating Staphylococcus aureus infections. Currently, most strains of S. aureus are resistant to penicillin, limiting their use in therapeutic applications. Today, vancomycin, a powerful antibiotic, is readily used in treating S. aureus as well as Enterococcus infections. Resistant strains have already 10 emerged making treatment difficult (34). The Center for Disease Control and Protection (CDC) reports that in the United States nearly 2 million patients a year contract infections while staying in the hospital, of which around 90,000 patients die from these infections. More than 70% of these hospital acquired infections are resistant to at least one commonly used antibiotic causing longer hospital stays and higher health care costs (51). Hospitals provide a good environment for the emergence of ART bacteria. The close proximity of sick patients to one another, along with extensive use of antibiotics likely contributed to an environment rich in ART bacteria. The use of antibiotics in veterinary medicine has also been implicated in the rapid increase of ART organisms, which includes the use on pets, farm animals, and animals rose in aquaculture. Tetracycline resistant genes have been detected in pathogens, opportunistic pathogens as well as the normal microflora or commensal bacteria. The non-pathogenic (commensal) bacteria are believed to play an important role in creating reservoirs in each ecosystem (23, 37, 38, 47). The main diseases treated by veterinarians using antibiotics include enteric and pulmonary infections, skin and organ abscesses, and mastitis. Over the last 50 years an estimated one million tons of antibiotics have been released into the environment, of which approximately half were from veterinary and agricultural channels. Surveillance studies have indicated that there have been increased occurrences of resistance development in both pathogenic and commensal bacteria from farm animals (48). A recent study used tetracycline resistance probes to monitor swine 11 effluents into the environment from two swine production facilities. They looked at the impact of the swine fecal material on nearby lagoons and ground water. This study linked the tetracycline resistance genes as far as 250 meters from the source, suggesting the farm as the direct source of resistance genes into the environment. This study identified that ground water can serve as a potential vehicle transferring resistant genes from the farm to the food chain (11). Antibiotics are not limited to therapeutic or preventative applications. In agriculture, antibiotics are often supplemented in animal feed or water as a performance enhancer or as antibiotic growth promoters. Antibiotic growth promoters increase the growth rate of the animals as well as improve the feed efficiency in healthy well fed animals. Antibiotic growth promoters are commonly given to pigs, poultry and non-ruminating veal calves. Under optimal growth conditions the growth rate of the animal is increased by 2-4% (52). The mechanism of increased growth is not clear, but studies have shown that the use of antibiotics as growth promoters is most effective in young animals under good hygienic conditions. The mode of action for growth promoting antibiotics is unknown (35). It is assumed that the antibiotics cause multiple beneficial effects to the host, including: lethal or sub-lethal damage to pathogens; a reduction of toxins produced by these pathogens; a reduction in bacterial utilization of available essential nutrients; improved absorption of nutrients by reducing the thickness of the intestinal epithelium; reducing the intestinal mucosa epithelial cell turnover; and reducing intestinal motility (36). The addition of 12 antibiotics to animal feed alters the intestinal characteristics to resemble those seen in germ free animals (13). Tetracyclines are broad spectrum antibiotics comprised of four hydrophobically fused 6 member rings which create the general structure. The various derivatives are different at one or more of four sites on the ring structure which provide activity against a wide range of bacteria. They have been used extensively in therapy in humans as well as companion animals. Tetracycline and its analogues have also been extensively used as feed additives in sub-therapeutic levels for growth promotion in food animals in addition to treating diseases that effect field crops and fruit trees (42). A number of recent reports documented the detection of ART bacteria and AR gene dissemination beyond the range of selective pressure in the human ecosystems. One recent study examined the prevalence of tetracycline resistant bacteria in children that were never administered tetracycline and found that 100% of the subjects carried ART bacteria of which 11% on average of the cultivatable microflora was resistant to tetracycline (53). While another recent study examined the presence of the tetracycline resistance genes tetW, tetM, tetO, tetS, tetT, and tetB, in breastfed infants that were never administered antibiotics and found that every subject tested had ART bacteria in their fecal samples (20). Several main resistant strategies have been found in bacteria to interfere with the tetracycline action. There are currently 38 different tet and Otr genes identified, of which 23 genes encoded for energy dependant efflux pumps, 11 genes for ribosomal protection 13 proteins, 3 gene for an inactivation enzyme, and there is 1 gene with an unknown mechanism. Efflux pumps lower the concentration of the antibiotic concentration in cytoplasm of the cell, were as, ribosomal protection genes create peptides that protect the ribosome from the tetracycline binding while the degradation enzyme of tetracycline into non-toxic compounds (38). Of the genes coding for the ribosomal protection protein, tetM is the most prevalent. One of the reasons for the large distribution of this gene is that it is commonly located within a conjugative transposon which has a broad range of hosts (12). 2.3 Antibiotic resistant bacteria Four years after the mass production and distribution of antibiotics the first report of penicillin resistant Staphylococcus. aureus was documented (24). Fleming warned that overuse of antibiotics would reduce the antimicrobial potency, but it wasn’t until the 1970’s when multi-drug resistant strains were published that public attention became focused on the spread on AR (52). There are three common ways that organisms can be protected from antibiotics: the organism can be intrinsically resistant where the mode of action of the antibiotic does not have an effect on the microbe; the microbe can have genetic material that encodes for protection; or a random mutation in the DNA of the microbe can occur providing 14 protection from the action of the antibiotics (38, 42, 50). Antibiotics target specific sites in bacterial cells, such as the cell wall or the ribosome. Cells may be inherently resistant by lacking the specific target site, or it may acquire resistance by genetic mutation or obtain genetic material from another bacterium, which provides protection to the target site. 2.4 Exchange of genetic material Exchanging genetic materials among bacteria enabled them to evolve quicker than by independent mutation alone. Examples of its medical impact include the rapid emergence and dissemination of AR-encoding plasmids, switching between two alternative antigenic forms of flagellin filament protein (flagellar phase variation) in Salmonella, and the antigenic variation of surface antigens in Neisseria and Borrelia, etc. (21). Different mechanisms are implemented to introduce donor DNA into recipient bacteria through processes such as transformation, transduction or conjugation. DNA is usually unstable in the recipient. The transferred data can be retained in the recipient cells either as part of an independent replicon (plasmid) maintained under certain selective pressure or stabilization mechanisms, or by integrated into the genome of the recipient through recombination (21). Transmission between organisms generally occurs at a rate of 10-6 to 10-8 transconjugants/donor CFU. The frequency is correlated to the compatibility 15 between the organisms and the availability of the AR genes. Transfer events further enable these organisms to disseminate genetic attributes effectively within the community to enhance the ecosystem development and are more often between same or similar species (45). Overall the transfer of the genetic material is often beneficial to the recipient organism. Genetic transfer between organisms plays a crucial role in microbial evolution in shaping the structure of microbial communities. 2.5 Vertical gene transfer AR evolves naturally by random mutations including point mutations, deletions, insertions or rearrangements in the DNA that confers protection to the organism by blocking the targets of the antibiotic. These mutations are passed down to daughter cells and are known as vertical gene transfer. The increase in the prevalence of AR is a product of natural selection. The antibiotic does not technically cause the resistance, but it creates a situation in which a resistant variant can withstand the action of the antibiotic, while killing the defenseless bacteria. This process selects for resistant bacteria allowing them to multiply and become the predominant microorganism (24, 50). Most antibiotics are derived from natural origin, which is usually produced by either bacteria or fungi. Mutations occur naturally at a frequency of around 10-8 to 10-10. Even though the frequency of mutation is low, the presence of antibiotics can accelerate the 16 domination of resistant bacteria by acting as a selective agent. Genetic mutants are often stable and can vertically disseminate to daughter cells. 2.6 Horizontal gene transfer On the other hand, certain genetic materials that encode for resistance of antibiotics are carried on a plasmids or transposons and can easily be acquired and spread to other microbes, transmitting the gene horizontally through the population. The resistance genes encode for cellular machinery that offers protection to the organism by either creating a peptide to block the action of the antibiotic, by generating an efflux pump to remove the antibiotic from the cytoplasm, or by encoding an enzyme that degrades the antibiotic, rendering it harmless (12, 38, 42). Horizontal gene transfer is a means for spreading AR genes from organism to organism, and pathogens are more likely to obtain ART genes from non-pathogenic commensal organisms rather than from another pathogen (40, 54). Horizontal gene transfer is the transmission of genetic material from one organism to another. Naturally, genetic material can be transferred by one of three methods: conjugation, transformation, or transduction. Conjugation is the exchange of genetic material in the form of a plasmid by cells touching. Transformation is the absorption of naked DNA from the environment 17 and integrating it into a plasmid or the genomic DNA. Transduction is the mis-packing of the virus head with the genetic material of the host bacteria and then is released during the lytic cycle. Conjugation and transformation are the most common means in which ART genes are transferred (19, 38). Horizontal gene transfer has been indirectly observed among Bacteroides spp. and other genera in the human colon. The colon provides a suitable environment for the potential of antibiotic resistanceAR genes to disseminate between commensal organisms and potentially to pathogens (43). Horizontal gene transfer of tetQ occurred between bacterial genera that normally colonize different hosts and used a comparative approach to examine the extent of gene dissemination between species of Bacteroides spp. This study displayed that it is possible for different genera of Bacteroides spp. to transfer genes even though they are known to colonize different hosts (33). Horizontal gene transfer has also been observed in vitro between commensal and pathogenic members of the Enterobacteriaceae. Genetic material conferring resistance was readily transmitted suggesting that transmission is commonplace among members of Enterobacteriaceae, a dominant member of the human microflora maintaining a genetic reservoir for antimicrobial resistance (7). Lactococcus lactis, a commensal bacterium that is widely distributed in nature, as well as, foodprocessing environments, is also susceptible to various gene transmissions. (17). Luo et al (2005) found that a clumping lactococcal strain, previously known for the high frequency transmission of important fermentation traits such as lactose utilization and casein-hydrolyzing proteinase, is also able to acquire the AR genes effectively. Furthermore, this inherited high frequency conjugation mechanism can further 18 disseminate the broad-host-range drug resistance-encoding plasmid pAMβ1 at a frequency 10,000 times more than strains without the mechanism. This suggests commensal bacteria as such may not only serve as the AR gene pool, but potentially as facilitator significantly enhanced the spread of the AR genes in microbial ecosystems (25). Recently a number of reports documented the detection of ART bacteria and AR gene dissemination beyond the range of selective pressure in both animal and human ecosystems. Villedieu et al. (2003) examined the prevalence of tetracycline resistant bacteria in children that were never administered tetracycline and found that 100% of the subjects carried ART bacteria of which 11% on average of the cultivatable microflora was resistant to tetracycline. Gueimonde et al (2006) examined the presence of a number of tetracycline resistance genes including tetW, tetM, tetO, tetS, tetT, and tetB, in breastfed infants that were never administered antibiotics and found that every subject tested had ART bacteria in their fecal samples. A recent study used germ free rats and spiked them with a strain of tetracycline resistant Lactobacillus planterium and a nonresistant Enterococcus faecalis to examine genetic transfer in vivo. Initially no selective pressure was administered to the rats, until several weeks into the study. Prior to administering the tetracycline some of the rats digestive tracts were examined to find that genetic transfer occurred prior to selective pressure. They also found that once antibiotics were administered that there was only a slight increase in transmission (22). These studies suggest that selective pressure may not be required for the colonization of ART bacteria and transmission of AR genes if the growth environment is ideal. 19 2.7 Reservoirs for resistance genes The emergence of AR in pathogens in the hospital environment has generally been the focus of study. For a long time it was believed that the wide application of antibiotics in such environment is responsible for the spread of AR genes. This view is currently believed to be too narrow. First, in terms of dissemination pathways, globally, an estimated 50% of all antimicrobials used serve veterinary and agricultural purposes. The use of antibiotics in veterinary medicine has contributed to the spread of AR and includes the use on pets, farm animals and animals raised in aquaculture (48). In addition to human therapy, the prophylactic use of antibiotics in agriculture affects the food animals, the environment, and the water ways. Food animals consume antibiotics through applications to their feed and water sources and shed antibiotic residues, as well as viable bacteria with functioning resistance genes throughout the fields and waterways. The human body has also been implicated as a potential reservoir for AR genes with food as a significant link between the antibiotic genes found in the farm as well as in the human digestive tract. It has been well documented that ART pathogens found in chicken, beef and pigs can be detected in the meats sold in supermarkets (41). Campylobacter and Salmonella are examples of food-borne pathogens becoming resistant in both human and veterinary medicine (55). First reported in the Netherlands the use of fluoroquinolones, in poultry farming (enrofloxacin) and in human medicine (ciprofloxacin) caused the increase in 20 fluoroquinolone resistant Campylobacter illness (1). A temporal increase in resistance was observed between the administration of fluoroquinolones and the increase in resistant populations (44). Horizontal transfer of the AR genes in the human body between commensal bacteria and transient bacteria may be responsible for the phenomenon. Second, pathogens only account for a very small percentage in the human microbial community, majorities are commensal bacteria. The chance of a pathogen acquiring AR genes directly from another pathogen is very small. Therefore improved understanding of the size of the AR gene pool in commensal bacteria and the roles of commensal bacteria in the spread of antimicrobial resistance are pivotal in assessing the emergence of resistance in pathogens. The bacteria in the human gut are mostly benign and were believed to only transfer genes between themselves. Bacteria that are transient through the gut can also acquire resistance genes through conjugation. In 2004, 7 sprouts samples were screened for various ART enterobacteria (8). The sprouts were determined to carry between 107-109 CFU/g of coliform bacteria, of which remarkably 107 CFU/g were resistant to various antibiotics. Many of the isolates maintained multiple drug resistance. Upon examination of the seed no resistant bacteria were obtained suggesting that the growing environment provided these bacteria (8). Another recent study examined ART bacteria in many ready to eat foods, which illustrates the prevalence of resistant commensal bacteria and resistance genes in many retail foods. The extent at which ART food-borne commensal bacteria has been recently examined and the presence of 102-107 CFU of ART bacteria per gram of food has been detected. 21 Many of these foods are consumed without further cooking or processing, and through daily intake, humans are regularly inoculated with ART bacteria, including opportunistic pathogens as well as commensal bacteria. Some of these bacteria include Pseudomonas spp. Streptococcus spp., Enterococcus spp., and Staphylococcus spp.. The high concentration of resistant bacteria suggests that food can be a potential avenue for transmitting the resistance genes (30, 54). Many of these organisms that have been isolated from ready-to-eat foods can survive in low pH environments, such as that of the human stomach and gastrointestinal tract. Many of these AR genes are maintained on conjugative plasmids or transposon which can readily be transmitted to potential pathogens creating a potential reservoir for pathogens to acquire AR genes through gene transfer and dissemination (40). 2.8 Human microflora The gastrointestinal (GI) tract is a specialized tube divided into various well-defined anatomical regions which extends from the lips to the anus. The indigenous bacteria of the human GI tract are not randomly distributed throughout the gastrointestinal tract but instead are found at population levels and in species distributions that are characteristic of specific regions of the tract (oral, stomach, small intestine and large intestine). The oral cavity provides a favorable habitat for many bacteria both beneficial and disease causing. The bacteria benefit from the high availability of nutrients and epithelial debris as well as the continual moisture provided by the salivary glands (50). The GI tract has an increasing pH from stomach to large intestine, which creates an increasing gradient of 22 indigenous microbes. There are characteristic spatial distributions of organisms within each gut compartment and at least 4 microhabitats: the intestinal lumen, the unstirred mucus or gel layer that covers the epithelium of the entire tract, the deep mucus layer found in intestinal crypts, and the surface of the epithelial cells (5). The human body hosts a complex microflora that contains more than 400 species of bacteria as well as eukaryotic fungi, protists, virus and methanogenic archaea, of which most are uncultivatable. The cultivatable bacteria are mostly anaerobes and are represented by 3040 species. The human gut has been estimated to contain 1011 bacteria cells/gram feces (26). The distinction between indigenous and exogenous microbes is critical to an ecological understanding of colonization, succession, and mechanisms of interaction between intestinal microbes and their host. In general the term indigenous implies these microbes colonize part of GI tract and occupy all niches and habitats available. Where as the exogenous species are not established and are detected in passage through the GI tract via food, water or environment. Cleary there is some gray area between the indigenous and exogenous, since there are some pathogens that are indigenous and can coexist in their host without illness unless the ecosystem is disrupted. Also, some microbial species may be indigenous to one habitat but non-indigenous to another, through which it normally passes after it is shed from its native habitat (26). Therefore the make up of the normal flora is continually changing and is affected by various factors including genetics, age, stress, nutrition and diet of the individual. 23 The gastrointestinal tract of normal human fetus is sterile at birth. During the birthing process and rapidly after, the microbes from the mother and environment quickly colonize the gut of the newborn. After the first week of life the establishment of a stable bacterial flora is developed (16). Infants born by cesarean delivery can also be exposed to the microbiota of their mother, but initial exposure is most likely due to environmental isolates from equipment, air, and other infants, with the nursing staff serving as vectors for transfer (26). The establishment of the human microflora is not a simple process of succession, rather it is a complex process influenced by microbial interactions with the host, as well as the internal and external factors. Regardless of the mode of birthing or environment of the infant, the development of the microbial flora is connected to the anatomical development of the intestinal tract, peristalsis, bile acids, intestinal pH, immune response, mucosal receptors, and microbial interactions (16). The diet of the infant directly affects the bacterial composition of the gut. These commensal bacteria originate from the nipple and surrounding skin as well as the milk ducts in the breast. The breast fed infants are initially colonized with more Bifidobacteria spp. than formula fed infants, but also contain more staphylococci, streptococci, corynebacteria, lactobacilli, micrococci, and propionibacteria. Formula fed infants also contain these bacteria; however, studies have found these infants to host higher amounts of other anaerobes such as Clostridium spp., Bacteroides spp., Enterobacter Spp. and Enterococcus spp. (26). Cooperstock and Zedd used the 4 phase model to describe the development of the infant intestinal microflora. Phase 1 is the initial acquisition phase which occurs over the first 24 few weeks of life. Phase 2 is the period of solely consuming milk. Phase 3 is the time between the beginning of supplementation and the cessation of breast feeding and phase 4 is the conversion to adult biota patterns upon the completion of weaning. Interestingly, during phases 3 and 4, after introduction of solid food and weaning, the differences between breast-fed and formula-fed infants are lost and the fecal microbiota resembles that of adults by about the second year of life (14). The succession of bacteria varies from the individual, the mode of birth, the mode of feeding and environment, however after the initial acquisition, developmental succession occurs during weaning, when feeding modes shift from a liquid to a solid diet. The human body is a complex ecosystem and is composed of many organisms that can thrive in multiple regions and are not limited to only one. The human gastrointestinal tract is a complex bioreactor that hosts hundreds of species of organisms. The human intestinal microflora is essentially an “organ” that regulates epithelial development, provides nutrition and instructs innate immunity yet it is poorly understood. Characterization of this immense ecosystem is only the beginning step in understanding its role in the health and disease of the host (15). This optimal environment makes the entire system susceptible to genetic transfer event of resistance genes from region to region or organism to organism. The major regions of the body hosting bacteria include the skin, respiratory tract, oral cavity, stomach, small and large intestine and the urogenital tract. 25 The skin, respiratory tract and urogenital tract are not regions that are of primary concern in the genetic transfer of AR genes from farm to human through the food chain. Nevertheless, the human body works as a system and each region influences the other regions. The skin is the largest organ of the human body that comes in direct contact with the environment. However the skin is a dry region of the body and most of the commensal bacteria are located near the warm moist areas such as armpits, perineum, palms of the hands and toe webs. Drier areas such as the face, arms, trunk and legs are less colonized. These regions are inhabited by bacteria such as Staphylococcus epidermidis, Micrococcus species, and Corynebacterium species (2). The respiratory tract is colonized in the upper region, inhaled organisms attach to the mucous membranes of the nose throat and pharynx. Examples of some of the bacteria found in the upper respiratory tract include Streptococcus salivarius, Staphylococcus aureus, and Neisseria species (10). Unlike the upper respiratory tract, the lower tract including the trachea and lungs does not contain bacteria in healthy individuals. Microbes are trapped and pushed to the upper respiratory tract by tiny hairs called cilia. The urogenital tract is free of bacteria with the exception of the urethra and the vagina. The urethra can harbor such as S. epidermidis, E. coli, and Proteus mirabilis and adult women tend to have Lactobacillus acidophilius. Prepubescent girls and women that have experienced menopause tend to have more species of bacteria such as Staphylococcus, Streptococcus, and Corynebacterium present than Lactobacillus acidophilius (27). 26 The indigenous bacteria of the mouth, stomach, small and large intestine are beneficial to body by synthesizing vitamins, preventing pathogens from colonizing, inhibiting the growth of exogenous bacteria, stimulating tissue growth and cross-reactive antibody production (50). Although beneficial to the body they can carry genes that encode for AR. When these commensal bacteria come in contact with bacteria from animals, environment, food, soil or pathogens they may transfer these genes. If pathogens obtain AR genes, it becomes more difficult to treat the infections they cause. The oral cavity is a favorable environment for bacteria to thrive on the constant supply of food and epithelial tissue. The mouth provides a low oxygen environment and harbors organisms such as Streptococcus mitis, Proteus species, and Veillonellae species (10). The stomach has the least amount of colonized bacteria due to the highly acidic environment and host organisms such as, acid-tolerant Lactobacillus and Streptococcus species (27). The intestines are the part of the GI tract that descends between the stomach and the rectum. The intestines are split into two major components the small and large intestines. The lining of the small intestine is coated by a simple columnar epithelial tissue, which is the portion of the GI tract where most of the nutrients from digested food are absorbed through folds in the intestinal wall called plicae circulares. Plicae circulares have fingerlike tissues called villi that protrude from the folds that are covered further with finer 27 hairs called micovilli which together increase the surface area of the small intestine. The increase in surface area makes the small intestine an efficient organ for the secretion of enzymes and the absorption of nutrients (49). The small intestine is comprised of 3 major parts: the duodenum, jejunum and ileum and the pH gradually increase from 4-5 at the duodenum to 7-8 in the ileum (18). The duodenum regulates the emptying of the stomach and is responsible for most of the chemical breakdown of food in the small intestine. Most of the nutrients are absorbed in the jejunum and the ileum, which include the passive transport of fructose and the active transport of amino acids, small peptides, vitamins and glucose. The ileum functions to absorb the remaining products that were not absorbed in the jejunum as well as vitamin B12 and bile salts (31). The constant movement through the small intestine makes it difficult for bacteria to colonize because they get washed out very quickly. As a result the concentration of bacteria in the small intestine remains relatively low with a concentration of approximately106 bacteria per ml. The predominant organisms colonizing the small intestine are comprised mostly of lactobacilli, streptococci, staphylococci and yeasts although there are the presence of some coliforms and anaerobes are present in low concentration (50). The large intestine extends from the ileocecal valve to the anus and has the largest concentration and diversity of commensal bacteria ranging in the billions. The pH of the large intestine is relatively neutral roughly around 7.0 and hosts over 700 species of bacteria existing which perform a variety of functions. These functions include the absorption of short-chain fatty acids metabolized by bacteria from undigested 28 polysaccharides. These bacteria also produce vitamins such as vitamin K and Biotin. There is also a production of cross reactive antibodies produced by the immune system against the normal flora which are effective against pathogens thereby preventing infection or invasion. The large intestine is coated by a mucus layer which also protects the intestine from attacks by the colonic commensal bacterial (46). Examples of the major commensal bacteria species living in the large intestine are Bacteroides spp., Lactobacillus spp., Clostridium spp., Bifidobacterium spp., Staphylococcus spp., Enterococcus spp., Salmonella spp., Klebsiella spp., Enterobacter spp. and Escherichia spp. (50). 2.9 Means to assess bacterial populations Bacterial detection and population measurements are traditionally achieved by cultivation the samples in liquid media or plating on bacterial agar plate with proper dilutions followed by incubation under optimal conditions. However, examining the bacteria from human digestive tract is problematic because most of the microflora is not cultivatable in vitro. Also, many of these microbes inhabit the gut in lower concentrations or are slow growers that have specific nutritional requirements and go undetected by standard plating methods. Selective enrichment is a means of cultivating organisms that have low population concentrations or bacterium that are slow growing by providing specific nutritional requirements and environmental conditions that are optimal for their growth. 29 Although selective enrichment is a powerful tool for detecting target organisms, it creates a detection bias due to its selectivity and is only valuable for detection not assaying populations (27). Culture-independent methods, particularly, nucleic acid-based analysis is becoming one of the most useful tools for detection and population analysis in recent years. It can provide quantitative and qualitative analysis on organisms that were previously not examined by extracting the DNA of the entire population regardless of the ability to cultivate. Many molecular tests have been developed to assay DNA and rRNA, revolutionizing the way the human gut is examined. NCBI databases have been applied to analyze results from cloning and sequencing of rDNA, fluorescence in situ hybridization and dot blot hybridization using oligonucleotide probes and species or group specific PCR. These applications have developed an accumulation of sequence data, which have been used to identify the un-cultivatable bacteria. The 16S rDNA is a conserved region and has been used to reconstruct phylogenetic trees. PCR allows experimenters the ability to amplify genes to a detectable concentrations and real time PCR technology provides a means to continually monitor quantitative changes especially for sub-dominant species. A recent study compared the sensitivity of quantification studies between two 5’nuclease assays and dot-blot hybridization using rDNA probes. They concluded that both real-time assays achieved similar results and both were faster, less expensive and had superior sensitivity in comparison to dot-blot hybridization (29). 30 This study suggests that real time PCR has a great potential in the analysis of human microbiota. Conventional PCR can be viewed as a quantitative amplification of DNA of limited precision that is analyzed by an endpoint analysis. There are three basic steps in the amplification process, the melting step which separates the double stranded DNA into single strands, the annealing step which binds Taq polymerase and primers to the single stranded DNA, and the elongation step which adds the complementary nucleotides to the single stranded DNA rendering a double stranded copy at a rate of 2n per reaction where n is the number of cycles. Essentially, real time PCR is the same as conventional PCR; with the addition of a fluorescent molecule to the PCR reaction and an optical module to the thermocycler to detect the increase in fluorescent signal. The main difference between the two methods is that conventional PCR analyzes the end product where as real-time detects amplification during the reaction. The fluorescent chemistries used in real-time PCR are either DNA binding dyes or fluorescently labeled sequence primers or probes, which are detected during the amplification cycle. The advantage of using real time PCR over other molecular tools is the ability to determine the starting template copy number with a high degree of accuracy with an error rate of about 1 in 9000 nucleotides. Real-time PCR that is quantitative is known as qPCR. Initially the sample fluorescence remains at background levels until enough amplified product is produced to yield a detectable signal in the optical module. The cycle at which this occurs is called the threshold cycle or CT. The CT is determined by the amount of template present at the 31 beginning of the reaction. Therefore qPCR should be optimized to ensure accurate reproducible quantification to the sample. The quality of a qPCR is assayed by developing a linear curve, and calculating the amplification efficiency as well as observing the consistency across replicate reactions. (6) 32 BIBLIOGRAPHY 1. Aarestrup, F.M., Jensen, N.E., Jorsal, S.E. and T.K. Nielsen. 2000. Emergence of resistance to fluoroquinolones among bacteria causing infections in food animals in Denmark. Vet Rec. 146: 76-78. 2. Albrecht T, et al. 1996. “Bacteriology: Normal Flora.” Medical Microbiology, 4th Edition. University of Texas. http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=mmed.section.508. 3. Antibiotic FAQ. McGill University, Canada. Antibiotics and student health. http://www.mcgill.ca/studenthealth/information/generalhealth/antibiotics/ Viewed on 4-13-2008. 4. Barton, M. 2000. Antibiotic use in animal feed and its impact on human health. Nutr Res Rev. 13:279-299. 5. Berg R.D. 1996. The indigenous gastrointestinal microflora. Trends Microbiol. 4:430–5. 6. Bio-Rad Laoratories, Inc. 1999. BIORAD Hand book Bio-Rad Laoratories, Inc. Hercules, CA. 7. Blake, D.P., Hillman, K., Fenlon, D.R. and J.C. Low. 2003. Transfer of antibiotic resistance between commensal and pathogenic members of the Enterobacteriaceae under ileal conditions. J Appl Microbiol. 95: 428-436. 8. Boehme, S., Werner, G., Klare, I., Reissbrodt, R., and W. Witte. 2004. Occurrence of antibiotic-resistant enterobacteria in agricultural foodstuffs. Mol. Nutr. Food Res. 48: 522-531. 9. Center for Disease Control and Prevention. 2007. Get smart: know when antibiotics work. http://www.cdc.gov/narms/faq_pages/11.htm (Viewed September 2007). 10. Champonux JJ, Drew WL, Falkow S, Neidhardt FC, Plorde JJ, and C.G.Ray. 1994 Sherris Medical Microbiology: An Introduction to Infectious Diseases. Connecticut: Appleton & Lange. 33 11. Chee-Sanford J.C., Aminov R.I., Krapac I.J. Garrigures-Jeanjean N., and R.I. Mackie. 2001. Occurrence and diversity of tetracycline resistance gene in lagoons and groundwater underlying two swine production facilities. Appl Environ Microbiol. 67:22-32. 12. 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. 13. Commission on Antimicrobial Feed Additives. 1997. Antimicrobial Feed Additives. Stockholm: Ministry of Agriculture. Chapter 1-4: 1-140. 14. Cooperstock MS, and A.J. Zedd. 1983. Intestinal flora of infants. In: Hentges DJ, ed. Human intestinal microflora in health and disease. New York: Academic Press, p.79–99. 15. Eckurg, P.B., Bik, E.M., Bernstein, C.N., Purdom, E., Dethlefsen, L., Sargent, M., Gill, S.R., Nelson, K.E. and D.A. Relman. 2005. Diversity of the human intestinal microbial flora. Science. 10:1635-1638. 16. Fanaro, S., Chierici, R., Guerrini, P. and V. Vigi. 2003. Intestinal microflora in early infancy: composition and development. Acta Paediatr Suppl. 441:48-55. 17. Gasson, M. J. 1990. In vivo genetic systems in lactic acid bacteria. FEMS Microbiol. Rev. 87:43–60. 18. Geus, Eddes, Gielkens, Gan, Lamers, and Masclee. 1999. Post-prandial intragastric and duodenal acidity are increased in patients with chronic pancreatitis Aliment Pharmacol Ther. 13:937–943. 19. Gilliver, M. A., Bennett, M., Begon, M., Hazel, S. M. and C.A. Hart. 1999. Antibiotic resistance found in wild rodents. Nature 401: 233-234. 20. Gueimonde, M., Salminen, S., and E. Isolauri. 2006. Presence of specific antibiotic (tet) resistance genes in infant faecal microbiota. FEMS. 48: 21-25. 21. Holmes RK, and M.G. Jobling. 1996. Genetics: Exchange of Genetic Information in: Baron’s Medical Microbiology 4th ed., Chapter 5. Univ of Texas Medical Branch. 34 22. Jacobsen, L., Wilcks, A., Hammer, K., Huys, G., Gevers, D., and S. Andersen. 2007. Horizontal transfer of tet (M) and erm(B) resistance plasmids from food strains of Lactobacillus plantarum to Enterococcus faecalis JH2-2 in the gastrointestinal tract of gnotobiotic rats. FEMS Microbiol Ecol. 59:158–166. 23. Levy, S.B., McMurry, V., Burdett, P., Courvalin, P., Hillem, W., Roberts, M.C., and D.E. Taylor. 1989. Nomenclature for tetracycline resistance determinants. Antimicrob Agents Chemother. 33:1373-1374. 24. Lewis, R. 1995. The rise of antibiotic- resistant infections. FDA consumer magazine. September issue. http://www.fda.gov/fdac/features/795_antibio.html 25. Luo, H., Wan, K., H.H. Wang. 2005. High-Frequency Conjugation System Facilitates Biofilm Formation and pAM_1 Transmission by Lactococcus lactis. Appl Environ Microbiol. 71: 2970–2978. 26. Mackie, R.I., Sghir, A. and H.R. Gaskins. 1999. Developmental microbial ecology of the neonatal gastrointestinal tract. Am J Clin Nutr. 69:1035-1045. 27. Madigan, M.T., Martinko J.M., and J. Parker. Brock Biology of Microorganisms, 8th Edition. New Jersey: Prentice Hall, 1997. 28. Mailer, J.S., and B. Mason. Penicillin: Medicine’s wartime wonder drug and its production at Peoria, Illinois. lib.niu.edu. (viewed 4-13-2008). 29. Malinen E., Kassinen A., Rinttila T, and A. Palva. 2003. Comparison of realtime PCR with SYBR Green I or 5’ nuclease assays and dot-blot hybridization with rDNA-targeted oligonucleotide probes in quantification of selected faecal bacteria. Microbiology 149: 269-277. 30. 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. 31. Maton, A, Hopkins, J., McLaughlin, C.W., Johnson, S., Warner, M.Q., LaHart, D., and J.D. Wright. 1993. Human Biology and Health. Englewood Cliffs, New Jersey, USA: Prentice Hall. 32. Marquez, B. 2005. Bacterial efflux systems and efflux pumps inhibitors. Biochimie. 87 1137–1147. 35 33. Nikolich, M.P., Hong, G., Shoemaker, N.B. and A.A. Salyers. 1994. Evidence for natural horizontal transfer of tet Q between bacteria that normally colonize humans and bacteria that normally colonize livestock. Appl Environ Microbiol. 60:3255-3260. 34. Paulsen, I.T., et al. 2003. Role of mobile DNA in the evolution of vancomycinresistant Enterococcus faecalis. Science. 299:2071-2074. 35. Phillips, I., Casewell, M., Cox, T., DeGroot, B., Friis, C., Jones, R., Nightingale, C., Preston, R. and J. Waddell. 2004. Does the use of antibiotics in food animals pose a risk to human health? A critical review. J Antimicrob Chemother. 53: 28-52. 36. Presoctt, J.F. and J.D Baggot. Antimicrobial therapy in veterinary medicine, 2nd edition. Ames, IA: Iowa State University Press 1997. 37. Roberts, M.C. 1994. Epidemiology of tetracycline resistance determinants. Trends Microbiol. 2:353-357. 38. Roberts, M.C. 1996. Tetracycline resistance determinants: mechanisms of action, regulation of expression, genetic mobility and distribution. FEMS Microbiology Review. 19:1-24. 39. Rolinson G. N. 1998. Forty years of lactam research J Antimicrob Chemother 41:589–603. 40. Salyers, A.A., Gupta, A., and Y. Wang. 2004. Human intestinal bacteria as reservoir for antibiotic resistance. Trends Microbiol. 12:412-416. 41. Salyers, A.A and N. Shoemaker. 2006. Reservoirs of antibiotic resistance genes. Anim Biotechnol, 17:137–146. 42. Schnappinger, D., and W. Hillen. 1996. Tetracycline: antibiotic action, uptake, and resistance mechanisms – A mini review. Arch Microbiol. 165: 359-369. 43. Shoemaker, N.B., Vlamakis, K., Hayes K. and A.A. Salyers. 2001. Evidence for extensive resistance gene transfer among Bacteriodes spp. and among Bacteroides and other genera in the human colon. Appl Environ Microbiol. 67:561-568. 44. Smith D.L., Harris A., Johnson J., Silbergeld E., G. Morris. 2002. Animal antibiotic use has an early but important impact on the emergence of antibiotic resistance in human commensal bacteria. PNAS. 99:6434-6439. 36 45. Snyder, L. and W. Champness. 2003. Molecular genetics of Bacteria. Pgs 197215. ASM Press, Herndon, VA. 46. Stremmel, W. 2005. Retarded release phosphatidylcholine benefits patients with chronic active ulcerative colitis. Gut. 54: 966-971. 47. Taylor, D.E., and A. Chau. 1996. Tetracycline resistance mediated by ribosomal protection. Antimicrob. Agents Chemother. 40: 1-5. 48. Teuber, M. 2001. Veterinary use and antibiotic resistance. Curr Opin Microbiol. 4:493-499. 49. Thomson A, Drozdowski L, Iordache C, Thomson B, Vermeire S, Clandinin M, and G. Wild. 2003. Small bowel review: Normal physiology, part 1. Dig Dis Sci. 48:1546-64. 50. Todar, K. 2002. University of Wisconsin Department of Bacteriology Madison, Wisconsin. www.textbookofbacteriology.net Online text book. 51. US. Department of Health and Human Services. 2007. http://www.niaid.nih.gov/factsheets/antimicro.htm. (Viewed September 2007). 52. Van den Bogaard, A.E., and E.E. Stobberingh. 1999. Antibiotic Usage in Animals- Impact on bacteria resistance and public health. Drugs 58:589-607. 53. Villedieu, A., Diaz-Torres, M.L., Hunt, N., McNab, R., Spratt, D.A., Wilson, M. and P. Mullany. 2003. Prevalence of tetracycline resistance gene in oral bacteria. Antimicrob Agents Chemother. 47: 878-882. 54. Wang H.H., Manuzon M., Lehman M., Wan K., Luo H., Wittum T.E., Yousef A., and L.O. Bakaletz . 2005. Food commensal microbes as a potentially important avenue in transmitting antibiotic resistance genes. FEMS Microbiol lett. 254:226-231. 55. Wassenaar, T. 2005. Use of antimicrobial agents in veterinary medicine and implications for human health. Crit Views Microbiol. 31:1555-169. 37 CHAPTER 3 PROFILES OF TETRACYCLINE RESISTANT BACTERIA IN THE HUMAN INFANT DIGESTIVE SYSTEM 3.1 Objectives The objective of this study was to reveal the impact of the food chain on bacterial antibiotic resistance (AR) profiles in the human gut by analyzing the population of antibiotic resistant (ART) bacteria in infant fecal samples. ART bacterial population of infant subjects at various growth stages were assessed using both conventional culturing method and culture-independent real-time PCR. In addition, the shift of ART bacterial profiles in infant subjects from newborn to solid food consumption was examined. 3.2 Introduction The rapid emergence of antibiotic resistant (ART) pathogens threatens public health and demands for a thorough understanding on the origination, maintenance and dissemination of antibiotic resistance (AR) in the environment and the hosts, in order for the development of targeted control strategies. Natural horizontal gene transfer mechanisms 38 such as conjugation and transformation play a key role in the spread of the AR gene among various strains of bacteria from within the species to cross genera (5, 10). The selective pressure due to the applications of antibiotics in human and veterinary clinical treatments and in food animal, aquacultural and agricultural productions certainly enriched the resistant population in bacteria. However, limiting the use of antibiotics in clinical therapy and in food animal production does not seem to be enough to control the spread of AR (9). Meanwhile, new evidences suggest that AR may be maintained at the absence of the corresponding antibiotic (6, 7, 8, 16, 17). Several recent studies examined the prevalence of ART bacteria in the oral cavity of healthy children that were not administered antibiotics in the past 3 months and found that 100% of the subjects carried ART bacteria, of which 11% on average of the cultivatable microflora was resistant to tetracycline and 7% were resistant to erythromycin (16, 17). Amoung the Tetr bacteria, many were also resistant to amoxicillin and gentamycin (3). A number of studies have also illustrated that human fecal samples are prevalent of ART bacteria (6, 8, 14). Although most of the ART bacteria found in human ecosystems likely are commensal bacteria instead of pathogens, these bacteria may serve as “immobilized” donors constantly supplying AR genes to potential recipients via horizontal gene transfer mechanisms, including pathogens, inside the hosts. Therefore, the internalized AR gene pool represents a potential risk to public health. 39 Recent studies have also illustrated that there is a large load of ART commensal bacteria with AR genes in a variety of retail foods, many of which would normally be consumed without further cooking or processing. Therefore through daily food intake, humans are routinely inoculated with ART bacteria, including opportunistic pathogens, as well as commensals. Commonly identified AR gene carriers include but not limit to Pseudomonas spp., Enterococcus spp., Streptococcus spp., Lactococcus spp., and Staphylococcus spp. (1, 4, 18). Many of these bacteria have the ability to survive low pH conditions comparable to those found in food matrices passing through the stomach (19). The AR genes from the food isolates can be transmitted to human residential and pathogenic organisms via natural gene transformation in the laboratory settings, suggesting their mobility and functionality in the recipients (Wang et al., 2006; Lehman et al, manuscript in preparation). A critical issue yet to be addressed is whether the ART bacteria found in the human digestive microflora, without antibiotic exposure, are originated from food intake. Recently, Gueimonde et al. (2006) found a number of tetr gene pools in fecal samples from breastfed infant subjects who had not been treated with the antibiotic treatment. While this result challenged the empirical belief that selective pressure, being tetracycline in this case, would be essential in the establishment of the corresponding resistant population in the ecosystems, it also suggested that ART bacteria were established during early human development stages. Further studies to reveal the origination, development and persistence of the gut ART bacteria are greatly needed. 40 Tetracycline and its derivatives are broad spectrum antibiotics with activity against a wide range of Gram-positive and Gram-negative aerobic and anaerobic bacteria. Unlike penicillin, tetracycline is still considered an effective antibiotic option in therapeutic treatments. However, since it has been utilized extensively in the past several decades, Tetr bacteria (both pathogens and commensals) and the corresponding tetr genes are now found in various environmental, animal and human ecosystems (6, 7, 8, 11, 12, 16). Its prevalence exemplified various mechanisms that contributed to the AR maintenance, circulation and dissemination in the microbial ecosystems. Therefore, we choose to investigate the development of tetr bacteria in infant subjects before conventional food consumption to illustrate the contribution of food intake in the AR development in human digestive ecosystems. 3.3 Material and methods 3.3.1 Subjects and sampling Healthy infant subjects between the ages of newborn to 12 months that have not taken antibiotics were recruited. Fecal samples were collected from diapers and delivered within 12 hours of production. Laboratory processing of the samples occurred immediately upon receipt. 41 3.3.2 Culturable microbial population assessment using conventional agar plating Diapers with infant fecal samples were collected by trained parents, saved at 4°C and delivered to research staff members at their earliest convenience. One gram was taken from each fecal sample and serially diluted in 9 mL of 0.85% NaCl solution. Samples were plated in duplication on the following media as indicated in the study to measure the culturable microbial population: Columbia blood agar plate, containing 5% defibrinated sheep blood (Colorado Serum Co., Denver, CO), MRS, Columbia Broth plates supplemented with 5% glucose, Reinforced Clostridial Agar, Enterococcus Agar, and Brain Heart Infusion Agar. The same media containing 16 µg-mL-1 of tetracycline (Fisher Biotech, Fair Lawn, NJ) were used to assess the tetr sub-populations. All of the media used in this study were obtained from Becton Dickinson and Company of Franklin Lakes, NJ. All plates contained 100 µg-mL-1 cycloheximide (Sigma-Aldrich, St. Louis, MO) to minimize the growth of yeasts and molds, and were incubated in anaerobic conditions using BBL Gas Pac containers and anaerobic envelopes (BD BBL, Franklin Lakes, NJ) at 37oC for 48 hours. 3.3.3 DNA template preparation DNA from bacterial isolates used for conventional PCR screening for tetM gene and 16S rDNA identification were prepared using the bead beading and boiling method. Briefly, bacterial isolates were suspended in lysate tubes (BioSpec Products, Bartlesville, OK) 42 containing 120 µL of sterile dH2O (pH 10.5) and 100 mg of 0.1 mm diameter glass beads (Biospec Products Inc., Bartlesville, OK). The samples were homogenized twice using the Mini-Beadbeater™ (BioSpec Products, Bartlesville, OK) for 3 minutes at maximum speed. The lysate tubes were then placed in boiling water for 15-20 minutes followed by an ice bath for another 10 minutes. The samples were centrifuged for 2 minutes at 16,000 x g, and 5µL of the supernatant and its diluents were used as the template in standard 50 µL PCR reactions. For real-time quantitative PCR analysis, the DNA templates from fecal samples were extracted following the procedures described previously (Yu and Morrison, 2004) with slight modification. Briefly, 0.25 gram of fecal sample was suspended in 1 ml of lysis buffer in a 2 ml screw-cap lysate tubes, along with 0.4 g sterile glass beads (0.3g of 0.1 mm and 0.1g of 0.5 mm, Biospec Products Inc.). The sample was homogenized for 3 min using the Mini-Beadbeater™, followed by centrifugation for 5 min. The supernatant was transferred to a 1.5 ml eppendorf tube. The extraction was repeated one more time using 300 µL of fresh lysis buffer to the lysate tube followed by another round of homogenization and centrifugation. The supernatant from two extracts were pooled and proteins were removed by the addition of a 10M ammonium acetate solution. The total DNA was precipitated with a 1:1 volume of isopropanol at –200C for 30 minutes. The pellets were dried under a vacuum desiccator and resuspended in TE buffer. The RNA and remaining protein contaminants were removed by incubating with 2 µL of DNase free RNase (10mg/mL) at 37oC for 15 min, and 15 µl proteinase K with 200µL Buffer 43 AL (from the QIAamp DNA Stool Mini Kit) at 70oC for 10 min. The DNA was further purified using the QIAamp column and centrifuged at 16,000 x g for 1 min. The samples were washed using 500µL of Buffer AW1 and Buffer AW2 (from the QIAamp DNA Stool Mini Kit) and the DNA was eluted in 200µL of Buffer AE (from the QIAamp DNA Stool Mini Kit) and stored at -20°C (20). 3.3.4 Screening for representative tetr gene and identification of AR gene carriers Tetracycline resistant isolates containing the tetM gene were screened by conventional PCR. The pair of primers used to amplify the 974 bp tetM fragment was tetM FP 5’CGAACAAGAGGAAAGCATAAG-3’ and tetM RP 5’- CAATACAATAGGAGCAAGC-3’ (Sigma-Aldrich., St. Louis, MO). The conditions for PCR amplification of the tetM gene were: one cycle at 95°C for 5 min, followed by 35 cycles at 95°C for 30 sec, 53°C for 30 sec, and 68° C for 50 sec and 1 cycle at 68°C for 5 min. PCR products were visualized on 1% agarose gel and products of the expected length were purified using the QIAquick kit (Qiagen, Valencia, CA) following manufacturer’s instructions. The sequences of the PCR fragments with the proper size in 15 % of the samples were determined using a DNA analyzer (ABI PRISMs 3700, Applied Biosystems, Foster City, CA) at the Plant Microbe Genomics Facility, The Ohio State University. The DNA sequences were compared with published tetM resistance gene sequences deposited in the NCBI database. 44 Representative tetM+ isolates were identified using16S rDNA sequence analysis. The primer pair 16S-up 5’-AGAGTTTGATCCTGGCTCCG-3’ and 16S-down 5’-TACCTTGTTACGACTT-3’(Sigma-Aldrich, St. Louis, MO) were used to amplify a 1.5 kb fragment by conventional PCR as describes previously (2). The identities of the AR gene carriers at the genus level were determined by comparing the sequences of the16S rRNA fragments with those deposited in the NCBI database. 3.3.5 A Quantitative assessment of the tetM gene pool by Taqman real-time PCR pair of tetM-specific GAACATCGTAGACACTCAATTG Taqman -3’, PCR primers and (tetMrealFP tetMrealRP 5’5’- CAAACAGGTTCACCGG-3’) franking a 169 bp tetM fragment and a tetM-specific probe (5’-FAM-CGGTGTATTCAAGAATATCGTAGTG-BHQ-3’) were designed following procedures described previously (2). The primers were synthesized by SigmaAldrich (St. Louis, MO) and the probe was made by Biosearch Technology Inc. (Novato, CA). The qPCR thermoprofiles consisted of one cycle at 95°C for 3 min, followed by 40 cycles at 95°C for 30 sec, 53°C for 30 sec, and 68°C for 20 sec and 1 cycle at 68°C for 5 min, using an iCycler (Bio-Rad Laboratories,Hercules, CA). The sequence of this169 bp real-time PCR amplicon is within the region of a 1257 bp tetM fragment flanked by primers tetM FP and tetM realRP. 45 The pair of primers (tetM FP 5’-CGAACAAGAGGAAAGCATAAG-3’and realRP 5’CAAACAGGTTCACCGG-3’) and the DNA extract from a tetM+ isolate Enterococcus sp. ACF-R1-1 were used to amplify the 1257 bp tetM fragment by PCR. The DNA concentration was quantified by a pico green assay with a nanodrop (ND 3300 flurospectrometer, Wilmington, DE) and converted to copy numbers using the formula [DNA Mass concentration / Molecular weight] x 6.022 x 1023 copies/mol-1=copies per µL, where MW is the molecular weight of the 1257 bp PCR product. The standard curve used to quantify the tetM gene copy numbers was created based on the Ct values from the real-time PCR using the primer pairs tetMrealFP and tetMrealRP, the tetM-specific probe, as well as a tenfold serial dilutions of the sequenced 1257 bp tetM PCR product with the concentrations between 102 and 108 gene copies as the PCR amplification template. The standards ranged from 1.9 x 108 to 1.9 x 102 gene copies per reaction were always run in triplicate parallel during fecal sample analyses. The potential of the qPCR in assessing AR gene pools was validated by measuring the tetM gene pool in a pure culture of the tetM+ Enterococcus sp. ACF-R1-1. The cells were grown overnight at 37° C in BHI with 16 µg-mL-1 tetracycline, serially diluted in 0.85% NaCl solution, and the DNA was extracted from 1 mL of the diluted culture. The extracted DNA was used for real-time PCR. The bacterial cell numbers of the diluted samples were determined by plating 100µL of diluted culture on BHI agar plates containing 16 µg-mL-1 tetracycline and grown over night at 37°C. 46 The efficiency of the developed procedures in assessing the AR gene pool in fecal samples was validated by artificially spiking a serially diluted pure culture of the tetM+ Enterococcus sp. ACF-R1-1 into a meconium fecal sample which had an undetectable concentration of ART bacteria. Total DNA was extracted following the procedure as described above. The original, 10-1, 10-2 and 10-3 dilutions of the DNA extracts were used as templates for qPCR. The derived tetM copy numbers by real-time qPCR were used to compare with the ART bacteria counts by the conventional plating method. 3.4 3.4.1 Results Assessing culturable gut microflora by conventional plate counting The human gut flora is quite complex, therefore there is no single medium that can be used to recover all the bacteria. To choose the right medium (or media) to properly assess the magnitude of cultivable microflora from human gut, 7 different media were used in the pilot study for both total plate counting and tetracycline resistant bacteria counting. The bacterial counts on Columbia Blood Agar plates with 5% sheep blood were found to sustain the highest or near highest numbers among all types of media, while the bacterial colonies also had the greatest diversity in morphology (Figure 3.1). 47 Log 10 CFU/g 10.00 9.00 TSA 8.00 TSA tetr 7.00 CBA CBA tetr 6.00 MRS 5.00 MRS tetr 4.00 RCA 3.00 RCA tetr 2.00 ENT 1.00 ENT tetr 0.00 Baby A Baby D Baby G Figure 3.1 Variability in microbial recovery on selected bacterial media. TSA-Tryptic Soy Agar, CBA – Columbia Blood Agar with 5% Sheep Blood, MRS Agar – (Lactobacillus Agar) to De Man, Rogosa and Sharpe, CBA – Columbia Blood Agar with 5% Sheep Blood, RCA – Reinforced Clostridial Agar, ENT- Enterococcus Agar. All selective plates contain 16 µg/ml-1 tetracycline. Values represented here are the mean result for replicate plates and the standard deviation was always less then 10%. Therefore Columbia Blood Agar plates with 5% sheep blood at the absence (CBA) and presence of 16 µg-mL-1 tetracycline (CBA T16) were used in further studies to measure the magnitude of total microflora and tetr bacterial population in infant fecal samples. 48 3.4.2 Prevalence of Tetr bacteria in infant fecal samples Tetracycline resistant bacteria were found in all subjects beyond one day of age. The plate counts for all infant fecal samples were summarized in Figure 3.2. There were no detectable counts for meconium samples which were produced at day 1 from two independent infant subjects’ on any of the plates. Months 1, 2, 3, 4, 5, 7, 8, 10 and 11 were represented by one subject and month 6 was replicated by two different subjects and the data seemed to follow a 3rd order polynomial regression curve. The total plate count regression curve was represented by the equation y = 0.0416x3 - 0.8626x2 + 5.0547x + 0.8204, where as the Tetracycline resistant plate count regression curve equation was y = 0.0398x3 - 0.8108x2 + 4.573x + 0.6189. 12 Log10 CFU per g 10 8 6 TPC 4 Tetr 2 0 0 2 4 6 8 10 12 14 Age in Months Figure 3.2 Total and tetracycline resistant plate counts of 11 infant subjects. The meconium samples had no viable colonies on either total plate counts or selective plates and are represented at the lower limits of detection. The relationship between CFU/g and age were tested by polynomial regression TPC R2 = 0.89 (P< 0.001) and Tetr R2 = 0.92(P< 0.000). Values represented here are the mean result for replicate plates and the standard deviation was always less then 10%. 49 The microbial population in these subjects ranged from 1.1 x 108 to 2.1 x 1010 CFU/g of sample, with the lowest count found in the 4 and 6 month-old baby. The tetr population ranged from 5.5 x 105 to 1.8x 108 CFU/g of sample within these subjects. Overall the non-selective plates recovered 1 to 2 logs more bacteria than the tetracycline-containing plates, suggesting approximately 1-10% of the cultivable gut bacteria are tetracycline resistant. 3.4.3 Assessing the tetM gene pool by real-time qPCR Figure 3.3 illustrates the standard curve for qPCR using the developed tetM-specific realtime PCR primer-and-probe set and with an R2 value of 0.9968. The standards ranged from 1.9 x 108 to 1.9 x 102 gene copies per reaction were always run in triplicate parallel during fecal sample analyses. 40 35 y = -4.1114x + 44.819 Threshold cycle (Ct) 2 R = 0.9968 30 25 20 15 10 5 1 2 3 4 5 6 7 8 Log10 tet M gene copy number Figure 3.3 Standard curve. Serial dilution of purified tetM PCR product detected by real-time PCR used for quantification of gene copy numbers. 50 9 The tetM copy numbers from diluted pure culture cells of Enterococcus sp. ACF-R1-1 in Figure 3.4 were approximately 1-2 logs lower than the corresponding bacterial plate Log 10 te t M ge ne c opie s /m l counting numbers from the same samples y = 1.0307x - 1.7374 R2 = 0.9086 7.00 6.00 5.00 4.00 3.00 2.00 1.00 0.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 Log10 CFU / ml Figure 3.4 Extraction efficiency of tetM-specific qPCR. The correlation between the plate counts and real-time PCR detection using a serially diluted pure culture of tetM+ Enterococcus sp. ACF-R1-1. The tetM gene copy numbers of the fecal samples inoculated with serially diluted tetM+ cells were also compared with the plate count results of the same samples, and the copy numbers was about a log lower than the plate count number (Figure 3.5). The results suggest that the efficiency of the described qPCR approach is no more than 10% of the total tetM+ population. 51 Log10 tet M gene copies/g 10.00 y = 1.1209x - 1.9793 9.00 R = 0.9947 2 8.00 7.00 6.00 5.00 4.00 3.00 2.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 r Log 10 tet resistant CFU/g Figure 3.5 Validation of extraction by artificially spiking meconium with pure culture. AR gene pool in fecal samples was validated by artificially spiking a serially diluted pure culture of the tetM+ Enterococcus sp. ACF-R1-1 into a meconium fecal sample which had an undetectable concentration of ART bacteria. The tetM gene was found in each infant fecal sample with the exception of the meconium samples by conventional PCR (data not shown). The tetM gene pool in all infant fecal samples, with the exception of the meconium samples, ranged from 9.0 x 105 to 9. 0 x 108 copies per gram of sample, measured by qPCR using the tetM-specific primer-and-probe set (Figure 3.6). The size of the gene pool varied with subject and age. 52 Log10 tet M gene copies/g 10 9 8 7 6 5 4 3 2 1 0 0 2 4 6 8 10 12 Age in M onths Figure 3.6 The distribution of tetM gene pool in 11 infant subjects. The meconium samples had no detectable concentrations of the tetM gene and were below the limits of detection. The relationship between tetM gene copy/g and age were tested by 3rd order polynomial regression with an R2= 0.72 (P= 0.007). The regression equation was y = 0.033x3 - 0.6602x2 + 3.8274x + 1.1989. Each point is the mean of 2 qPCR runs with 3 technical replicates per run. 3.4.4 The shift of ART bacterial population during infant development The total and tetr bacterial plate counts of fecal samples at various points over the course of a year from two identical twin subjects (Baby E and Baby F) were summarized in Figure 4.7. The subjects were from the same family so the impact of environmental factors on microbial population in these subjects was similar. Baby E had 103 CFU/g-1 of sample on both the selective and nonselective plates for the meconium sample produced within 24 hrs, and Baby F had no viable counts on either selective or non-selective plates. The twins were further sampled on day 1, 2 weeks, and months 1, 2, 3, 4, 7, 8, 10 and 11. At 2 weeks of age the resistant counts for both infants were approximately 1.50 x 105 53 CFU/g of sample, which was almost half of the counts of the non-selective plate counts. By the beginning first month, the non-selective plates yielded around 7.00 x 109 CFU/g and the tetr counts were approximately two logs less. After one month of age the nonselective bacterial counts in the twin subjects were 1 to 3 logs higher than the counts from the tet plates. Based on the polynomial regression curves in Figure 3.7, the difference between the two subjects throughout the examination period suggests that the Tetr bacteria did not vary greatly. A Wilcoxon test was conducted to determine if there was a statistical difference between the subjects which resulted in a P value of 0.002 suggesting that there is not a statistical difference between the two subjects. r Total Plate Counts Tet Plate Counts 12 9 8 8 6 4 Baby E 2 Baby F L o g 10 C F U p e r g L o g 10 C F U p e r g 10 7 6 5 4 Baby E 3 Baby F 2 1 0 Poly. 0 0 2 4 6 8 10 12 0 Age in Months 2 4 6 8 10 12 Age in Months Figure 3.7 Total and resistant plate counts of twin over the course of a year. The Baby F meconium sample had no viable colonies on either total plate counts or selective plates and is represented by the lower limits of detection in both graphs. The relationship between CFU/g and age were tested by polynomial regression, TPC for Baby E R2=0.48, (P= 0.008) and Baby F R2=0.54, (P=0.003), the Tetr plate counts Baby E R2=0.62, (P< 0.000) and Baby F R2=0.66, (P=0.014). Values represented here are the mean result for replicate plates and the standard deviation was always less then 10%. 54 The tetM gene pool in every sample was analyzed, with the exception of the day 1 and week 2 samples from the twins. These samples had a low volume and/or excessive mixing with the diaper matrix making them not suitable for adequate analysis. As illustrated in Figure 3.8, the tetM gene pool for Baby E between months 1 to11 ranged between 3.6 x 105 to 4.0 x 107 copies per gram of sample, where as Baby E tetM gene pool ranged from 1.1 x 106 to 3.7 x 107 copies per gram of sample during the same period of time. There were no detectable tetM gene pools found in the meconium samples Log10 gene copies per uL within the limits of detection. 9 8 7 6 5 4 3 2 1 0 Baby E Baby F 0 2 4 6 8 10 12 Age in Months Figure 3.8 The distribution of the tetM gene pool in twin subjects. Twins tetM development from similar environmental factors detected by qPCR over the course of a year. The meconium samples had no detectable concentrations of the tetM gene and are below the limits of detection. The relationship between tetM gene copy/g and age were tested by polynomial regression Baby E R2= 0.68 (P=0.10) and Baby F R2=0.75, (P =0.056). Each point is the mean of 2 qPCR runs with 3 technical replicates per run. 55 The difference between the two subjects at the same growth stage throughout the study remained within a log, suggesting a reasonably consistency of the data. A Wilcoxon test was conducted on the twin real-time sample results supporting there was not a significant variation between the sampling periods based on the P- value of 0.007. 3.5 Discussion The impact of foodborne microbes, particularly non-pathogenic bacteria, on both the development of human gastrointestinal microflora and the circulation of the AR genes among the hosts and environment remains poorly understood. It is worth noting that this study was conducted using limited incubation conditions and antibiotic concentrations to screen for resistant bacteria, which likely are only optimal for a small percentage of the microbial population. The sample collection procedures did not offer special protection for strict anaerobes from the damaging effects of atmospheric oxygen. In addition, the initial sample collection procedures were handled by the guardians of the infant subjects instead of research staff members, therefore additional variability may be introduced. Overall, it is anticipated that the detected culturable microbes only measured a very small percentage of the total gut bacterial load in these subjects. Regardless of this fact, the data reported here still demonstrated that the infant gut is rapidly colonized by tetracycline resistant bacteria. Of the tetr isolates from the CBA plates containing 56 tetracycline, 28 isolates were identified using 16S rDNA sequence analysis and most were found to be Enterococcus spp. and Streptococcus spp. With the exception of the newborn infant’s first bowel movement, Tetr bacteria and tetM gene pool were detected in each subject in this study. Baby E had low counts on both the selective and nonselective plates for the meconium sample produced within 24 hrs, while Baby F had no viable counts on either selective or non-selective plates. However, the presence of a sanitary napkin in the Baby E meconium sample suggested this sample might have been contaminated. Regardless, since conventional foods have not been introduced into the infants’ diet, the results suggest that they are not the direct cause of the resistant bacteria in the infant gut. Other pathway(s) likely are responsible for the transmission of the AR gene-containing bacteria. The total plate counts yielded expected microbial counts. However the rapid emergence of Tetr bacteria in infant gut flora without the exposure to tetracycline was a surprise. This data indicates that the spread and maintenance of resistant populations occur regardless of corresponding antibiotic selective pressure. Based on results from conventional culture plating, the Tetr bacteria in infant subjects rapidly reached 5.5 x 105 to 1.9 x 108 CFU per gram of samples shortly after birth. Because this method only measures those recoverable under the limited experimental conditions, the real magnitude of Tetr bacteria in these subjects is higher than these numbers. 57 The real-time qPCR method is culture-independent, therefore it measures the total AR gene pool in live and dead, both culturable and unculturable cells. Furthermore, it measures the sum of AR gene copies from ART bacteria, without distinguish the situation where certain cells may carry multiple copies of AR genes. Therefore, theoretically the size of the gene pool as measured by qPCR should be higher than that by conventional culture plating method. However, because of the procedures involved in the qPCR method, especially the lack of efficiencies in extracting DNA from cells and in removing of potential PCR inhibitory compounds from fecal samples, the results by qPCR as performed in this study are much lower than its full potential. By comparing the size of the tetM gene pool, one of the dominant tetr genes commonly found in natural and host environment, with the Tetr bacterial counts from both validation studies of using pure culture in the bacterial medium and artificial spiked fecal samples (Fig. 3.4 and Fig. 3.5), we concluded that the efficiency of the qPCR as performed is no more than 10% of its full potential. It is worth noting that all numbers in this study are obtained by analyzing ART bacteria population or AR gene pool from the fecal samples, instead of directly sampling the microbial flora from the surface of the gut. While these numbers are a good indication of the AR status in the microflora associated with the human digestive ecosystems, it is important to recognize that these numbers reflect not only bacterial population attached to the gut, but also those entered the digestive track from food or other contacts, and survived and possible amplified before they released with the feces. Enterococcus sp. and 58 Streptococcus sp. are among the dominant groups of bacteria recovered in this study, suggesting their outstanding capabilities in surviving the human digestive environment and possibly colonizing the gut surface (8). In summary, several conclusions still can be drawn from this study. Among those, the tetM gene appears to be prevalent in infant subjects, the gene pools rapidly reach to a relatively high level and are maintained over time, though varied by subject and age. The colonization, amplification and maintenance of ART bacteria occur regardless of the absence of the corresponding selective pressure. The human gastrointestinal tract is a dynamic environment and the data reported here may represent transient as well as gut microflora. A larger subject size along with more frequent sampling would provide a better understanding as to the origination, maintenance and dissemination of AR in the environment and the hosts. Also, tetM is only one of 38 tetracycline resistant genes known to date and this study provides quantification of only a small part of the total picture (13). There is need to examine the other tet resistant genes, which may provide more information on the transmission, maintenance and circulation of ART bacteria and AR genes between the environment and the hosts. 59 BIBLOGRAPHY 1 Boehme, S., Werner, G., Klare, I., Reissbrodt, R., and W. Witte. 2004. Occurrence of antibiotic-resistant enterobacteria in agricultural foodstuffs. Mol. Nutr. Food Res. 48: 522-531. 2 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. 3 Diaz-Torres, M.L., Villedieu, A., Hunt, N., McNab, R., Spratt, D.A., Allan, E., Mullany, P., and M. Wilson. 2006. Determining the antibiotic resistance potential of the indigenous oral microbiota of humans using a metagenomic approach. FEMS Microbiol Lett. 258(2): 257-262. 4 Durán, G. M., and D.L. Marshall. 2005. Ready-to-eat shrimp as an international vehicle of antibiotic-resistant bacteria. J Food Prot. 68:23952401. 5 Gilliver, M.A., M. Bennett, M. Begon, S.M. Hazel, and C.A. Hart. 1999. Antibiotic resistance found in wild rodents. Nature 401:233–234. 6 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. 7 Lancaster, H., Ready, D., Mullany, P., Spratt, D., Bedi, R. and M. Wilson. 2003. Prevalence and identification of tetracycline-resistant oral bacteria in children not receiving antibiotic therapy. FEMS Microbiol Lett. 228:99-104. 8 Lindberg, E., Adlerberth, I., and A.E. Wold. 2004. Antibiotic resistance in Staphyloccocus aureus colonizing the intestines of Swedish infants. Clin Microbiol Infect 10:890-894. 9 Manson, J.M., Smith, J.M. and G. M. Cook. 2004. Persistence of vancomycin-resistant Enterococci in New Zealand broilers after discontinuation of avoparcin use. Appl Environ Microbiol. 70:5764–5768. 60 10 Molbak, K. 2004. Spread of resistant bacteria and resistance genes from animals to humans- The public health consequences. J. Vet. Med. 51:364369. 11 Nikolich, M.P., Hong, G., Shoemaker, N.B. and A.A. Salyers. 1994. Evidence for natural horizontal transfer of tet Q between bacteria that normally colonize humans and bacteria that normally colonize livestock. Appl Environ Microbiol. 60:3255-3260. 12 Teuber, M. 2001. Veterinary use and antibiotic resistance. Curr Opin Microbiol. 4:493-499. 13 Roberts, M. 2005. Update on acquired tetracycline resistance genes. FEMS Microbiol Lett. 245:195-203. 14 Salyers, A.A., Gupta, A., and Y. Wang. 2004. Human intestinal bacteria as reservoir for antibiotic resistance. Trends in Microbiology. 12: 412-416. 15 Smith D.L., Harris, A.D., Johnson, J.A., Silbergeld, E.K. and G. Morris. 2002. Animal antibiotic use has an early but important impact on the emergence of antibiotic resistance in human commensal bacteria. PNAS. 99:6434-6439. 16 Villedieu, A., Diaz-Torres, M.L., Hunt, N., McNab, R., Spratt, D.A., Wilson, M. and P. Mullany. 2003. Prevalence of tetracycline resistance gene in oral bacteria. Antimicrob Agents Chemother. 47: 878-882. 17 Villedieu, A., Diaz-Torres, M.L., Roberts, A. P., Hunt, N., McNab, R., Spratt, D.A., Wilson, M. and P. Mullany. 2004. Genetic Basis of Erythromycin Resistance in Oral Bacteria. Antimicrob Agents Chemother. 48(6): 2298–2301. 18 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. 19 Waterman, S., and P. Small. 1998. Acid-sensitive enteric pathogens are protected from killing under extremely acidic conditions of pH 2.5 when they are inoculated onto certain solid food sources. Appl. Environ. Microbiol. 64:3882– 3886. 61 20 Yu, Z. and M. Morrison. 2004. Improved extraction of PCR-quality community DNA from digesta and fecal samples. Biotechniques. 36:808-12. 62 CHAPTER 4 CONCLUSIONS AND FUTURE DEVELOPMENT 4.1 Summary In summary this study illustrated that the infant gut is rapidly colonized by tetracycline resistant bacteria after birth. Tetr bacteria and tetM gene were detected in 100% of the infant subjects without prior exposure to tetracycline, indicating ART bacteria colonization in the gut is independent from the corresponding selective pressure. Since ART bacteria were detected and its development was monitored in breast-fed and formula-fed babies way before they started conventional food consumption, the results also suggest that pathways other than conventional food intake likely are responsible for transmitting the ART bacteria to the subjects. For instance, in our preliminary study, ART bacteria were detected from a mother’s hand. Therefore human and environmental contact could play a role in transmitting ART bacteria. On the other hand, despite the fact that we had sampled infant formula and not ART bacteria were detected, theoretically these products are not sterile. Although mother’s milk should be fairy clean, infants can still get exposed to ART bacteria through contacting mother’s skin during feeding. Therefore we cannot rule out the contribution of the food chain in the establishment of 63 the ART population in infant digestive track. It is inevitable that the constant supply of ART bacteria from conventional food consumption, coupled with occasional colonizationand horizontal gene transmission, will likely shape the development gut flora. But its overall impact is yet to be revealed. 4.2 Future studies This data in this study suggests that the spread of resistant populations occurs regardless of selective pressure from the presence of the antibiotic and that merely limiting the use of antibiotics will not likely slow down the future developments of AR in bacteria, at least not in the near future. Further studies are needed to be conducted to have a better understanding to reveal the origination, development and persistence of the gut ART bacteria. In order to determine a more comprehensive picture the adult gut ecosystem needs to be further characterized. A larger sample size is needs to be examined as well as other tetracycline resistance genes to better understand the contribution that foodborne ART bacteria have on the AR development in human digestive ecosystems. 64 BIBLIOGRAPHY 1. Aarestrup, F.M., Jensen, N.E., Jorsal, S.E. and T.K. Nielsen. 2000. Emergence of resistance to fluoroquinolones among bacteria causing infections in food animals in Denmark. Vet Rec. 146: 76-78. 2. Albrecht T, et al. 1996. “Bacteriology: Normal Flora.” Medical Microbiology, 4th Edition. University of Texas. http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=mmed.section.508. 3. Anderson, A.D., J. McClellan, S. Rossiter, and F.J. Angulo. 2003. Public health consequences of use of antimicrobial agents in agriculture. In: Knobler, S.L., Lemon, S.M., Najafi, M., Burroughs, T. (Eds.), Forum on Emerging Infections: The Resistance Phenomenon in Microbes and Infectious Disease Vectors. Implications for Human Health and Strategies for Containment—Workshop Summary. Board on Global Health, Institute of Medicine, Appendix A, pp. 231–243. 4. Antibiotic FAQ. McGill University, Canada. Antibiotics and student health. http://www.mcgill.ca/studenthealth/information/generalhealth/antibiotics/ Viewed on 4-13-2008. Barton, M. 2000. Antibiotic use in animal feed and its impact on human health. Nutr Res Rev. 13:279-299. 5. Berg R.D. 1996. The indigenous gastrointestinal microflora. Trends Microbiol. 4:430–5. 6. Bio-Rad Laoratories, Inc. 1999. BIORAD Hand book Bio-Rad Laoratories, Inc. Hercules, CA. 7. Blake, D.P., Hillman, K., Fenlon, D.R. and J.C. Low. 2003. Transfer of antibiotic resistance between commensal and pathogenic members of the Enterobacteriaceae under ileal conditions. J Appl Microbiol. 95: 428-436. 8. Boehme, S., Werner, G., Klare, I., Reissbrodt, R., and W. Witte. 2004. Occurrence of antibiotic-resistant enterobacteria in agricultural foodstuffs. Mol. Nutr. Food Res. 48: 522-531. 65 9. Center for Disease Control and Prevention. 2007. Get smart: know when antibiotics work. http://www.cdc.gov/narms/faq_pages/11.htm (Viewed September 2007). 10. Champonux JJ, Drew WL, Falkow S, Neidhardt FC, Plorde JJ, and C.G.Ray. 1994 Sherris Medical Microbiology: An Introduction to Infectious Diseases. Connecticut: Appleton & Lange. 11. Chee-Sanford J.C., Aminov R.I., Krapac I.J. Garrigures-Jeanjean N., and R.I. Mackie. 2001. Occurrence and diversity of tetracycline resistance gene in lagoons and groundwater underlying two swine production facilities. Appl Environ Microbiol. 67:22-32. 12. 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. 13. Commission on Antimicrobial Feed Additives. 1997. Antimicrobial Feed Additives. Stockholm: Ministry of Agriculture. Chapter 1-4: 1-140. 14. 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. 15. Cooperstock MS, and A.J. Zedd. 1983. Intestinal flora of infants. In: Hentges DJ, ed. Human intestinal microflora in health and disease. New York: Academic Press, p.79–99. 16. Davidson DJ. In the matter of enrofloxacin for poultry: withdrawal of approval of Bayer Corporation's new animal drug application 1 (NADA) 140-828 (Baytril). In: FDA Docket No. 00N-1571; 2004. 17. Diaz-Torres, M.L., Villedieu, A., Hunt, N., McNab, R., Spratt, D.A., Allan, E., Mullany, P., and M. Wilson. 2006. Determining the antibiotic resistance potential of the indigenous oral microbiota of humans using a metagenomic approach. FEMS Microbiol Lett. 258(2): 257-262. 18. 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 19. Eckurg, P.B., Bik, E.M., Bernstein, C.N., Purdom, E., Dethlefsen, L., Sargent, M., Gill, S.R., Nelson, K.E. and D.A. Relman. 2005. Diversity of the human intestinal microbial flora. Science. 10:1635-1638. 20. Environmental Defense Fund. 2001. http://www.edf.org/documents/619_abr_general_factsheet_rev2.pdf (viewed March 2008). 21. Fanaro, S., Chierici, R., Guerrini, P. and V. Vigi. 2003. Intestinal microflora in early infancy: composition and development. Acta Paediatr Suppl. 441:48-55. 22. Food and Drug Administration, Center for Veterinary Medicine, April 28, 2000 HHS Response to House Report 106-157- Agriculture, Rural Development, Food and Drug Administration, and Related Agencies, Appropriations Bill. Executive Summary. 23. Fries, R.. 2004. Conclusions and activities of previous expert groups: the Scientific Steering Committee of the EU. J Vet Med B Infect Dis Vet Public Health. 51:403-7. 24. Gasson, M. J. 1990. In vivo genetic systems in lactic acid bacteria. FEMS Microbiol. Rev. 87:43–60. 25. Geus, Eddes, Gielkens, Gan, Lamers, and Masclee. 1999. Post-prandial intragastric and duodenal acidity are increased in patients with chronic pancreatitis Aliment Pharmacol Ther. 13:937–943. 26. Gilliver, M. A., Bennett, M., Begon, M., Hazel, S. M. and C.A. Hart. 1999. Antibiotic resistance found in wild rodents. Nature 401: 233-234. 27. 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. 28. Holmes RK, and M.G. Jobling. 1996. Genetics: Exchange of Genetic Information in: Baron’s Medical Microbiology 4th ed., Chapter 5. Univ of Texas Medical Branch. 29. Jacobsen, L., Wilcks, A., Hammer, K., Huys, G., Gevers, D., and S. Andersen. 2007. Horizontal transfer of tet (M) and erm(B) resistance plasmids from food strains of Lactobacillus plantarum to Enterococcus faecalis JH2-2 in the gastrointestinal tract of gnotobiotic rats. FEMS Microbiol Ecol. 59:158–166. 67 30. Kolar., M., Pantcek, R, Bardon. J, Vagnerova,I., Typovska, H., Doskar, J. and I. Valka. 2002. Occurrence of antibiotic-resistant bacterial strains isolated in poultry. Vet. Med. 47:52-59. 31. Lancaster, H., Ready, D., Mullany, P., Spratt, D., Bedi, R. and M. Wilson. 2003. Prevalence and identification of tetracycline-resistant oral bacteria in children not receiving antibiotic therapy. FEMS Microbiol Lett. 228: 99-104. 32. Levy, S.B., McMurry, V., Burdett, P., Courvalin, P., Hillem, W., Roberts, M.C., and D.E. Taylor. 1989. Nomenclature for tetracycline resistance determinants. Antimicrob Agents Chemother. 33:1373-1374. 33. Lewis, R. 1995. The rise of antibiotic- resistant infections. FDA consumer magazine. September issue. http://www.fda.gov/fdac/features/795_antibio.html 34. Lindberg, E., Adlerberth, I., and A.E. Wold. 2004. Antibiotic resistance in Staphyloccocus aureus colonizing the intestines of Swedish infants. Clin Microbiol Infect 10:890-894. 35. Luo, H., Wan, K., H.H. Wang. 2005. High-Frequency Conjugation System Facilitates Biofilm Formation and pAM_1 Transmission by Lactococcus lactis. Appl Environ Microbiol. 71: 2970–2978. 36. Mackie, R.I., Sghir, A. and H.R. Gaskins. 1999. Developmental microbial ecology of the neonatal gastrointestinal tract. Am J Clin Nutr. 69:1035-1045. 37. Madigan, M.T., Martinko J.M., and J. Parker. Brock Biology of Microorganisms, 8th Edition. New Jersey: Prentice Hall, 1997. 38. Mailer, J.S., and B. Mason. Penicillin: Medicine’s wartime wonder drug and its production at Peoria, Illinois. lib.niu.edu. (viewed 4-13-2008). 39. Malinen E., Kassinen A., Rinttila T, and A. Palva. 2003. Comparison of realtime PCR with SYBR Green I or 5’ nuclease assays and dot-blot hybridization with rDNA-targeted oligonucleotide probes in quantification of selected faecal bacteria. Microbiology 149: 269-277. 40. Manson, J.M., Smith, J.M. and G. M. Cook. 2004. Persistence of vancomycinresistant Enterococci in New Zealand broilers after discontinuation of avoparcin use. Appl Environ Microbiol. 70:5764–5768. 68 41. 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. 42. Marquez, B. 2005. Bacterial efflux systems and efflux pumps inhibitors. Biochimie. 87 1137–1147. 43. Maton, A, Hopkins, J., McLaughlin, C.W., Johnson, S., Warner, M.Q., LaHart, D., and J.D. Wright. 1993. Human Biology and Health. Englewood Cliffs, New Jersey, USA: Prentice Hall. 44. Molbak, K. 2004. Spread of resistant bacteria and resistance genes from animal to human- The public consequences. J of Vet Med. 51: 364-369. 45. Nikolich, M.P., Hong, G., Shoemaker, N.B. and A.A. Salyers. 1994. Evidence for natural horizontal transfer of tet Q between bacteria that normally colonize humans and bacteria that normally colonize livestock. Appl Environ Microbiol. 60:3255-3260. 46. Paulsen, I.T., et al. 2003. Role of mobile DNA in the evolution of vancomycinresistant Enterococcus faecalis. Science. 299:2071-2074. 47. Phillips, I., Casewell, M., Cox, T., DeGroot, B., Friis, C., Jones, R., Nightingale, C., Preston, R. and J. Waddell. 2004. Does the use of antibiotics in food animals pose a risk to human health? A critical review. J Antimicrob Chemother. 53: 28-52. 48. Presoctt, J.F. and J.D Baggot. Antimicrobial therapy in veterinary medicine, 2nd edition. Ames, IA: Iowa State University Press 1997. 49. Randerson, J. 2003. Ban on growth promoters has not increased bacteria. New Scientist. 183:13-15. 50. Roberts, M.C. 1994. Epidemiology of tetracycline resistance determinants. Trends Microbiol. 2:353-357. 51. Roberts, M.C. 1996. Tetracycline resistance determinants: mechanisms of action, regulation of expression, genetic mobility and distribution. FEMS Microbiology Review. 19:1-24. 52. Rolinson G. N. 1998. Forty years of lactam research J Antimicrob Chemother 41:589–603. 69 53. Salyers, A.A., Gupta, A., and Y. Wang. 2004. Human intestinal bacteria as reservoir for antibiotic resistance. Trends Microbiol. 12:412-416. 54. Salyers, A.A and N. Shoemaker. 2006. Reservoirs of antibiotic resistance genes. Anim Biotechnol, 17:137–146. 55. Schnappinger, D., and W. Hillen. 1996. Tetracycline: antibiotic action, uptake, and resistance mechanisms – A mini review. Arch Microbiol. 165: 359-369. 56. Shoemaker, N.B., Vlamakis, K., Hayes K. and A.A. Salyers. 2001. Evidence for extensive resistance gene transfer among Bacteriodes spp. and among Bacteroides and other genera in the human colon. Appl Environ Microbiol. 67:561-568. 57. Smith D.L., Harris A., Johnson J., Silbergeld E., G. Morris. 2002. Animal antibiotic use has an early but important impact on the emergence of antibiotic resistance in human commensal bacteria. PNAS. 99:6434-6439. 58. Snyder, L. and W. Champness. 2003. Molecular genetics of Bacteria. Pgs 197215. ASM Press, Herndon, VA. 59. Stremmel, W. 2005. Retarded release phosphatidylcholine benefits patients with chronic active ulcerative colitis. Gut. 54: 966-971. 60. Taylor, D.E., and A. Chau. 1996. Tetracycline resistance mediated by ribosomal protection. Antimicrob. Agents Chemother. 40: 1-5. 61. Teuber, M. 2001. Veterinary use and antibiotic resistance. Curr Opin Microbiol. 4:493-499. 62. Thomson A, Drozdowski L, Iordache C, Thomson B, Vermeire S, Clandinin M, and G. Wild. 2003. Small bowel review: Normal physiology, part 1. Dig Dis Sci. 48:1546-64. 63. Todar, K. 2002. University of Wisconsin Department of Bacteriology Madison, Wisconsin. www.textbookofbacteriology.net Online text book. 64. US. Department of Health and Human Services. 2007. http://www.niaid.nih.gov/factsheets/antimicro.htm. (Viewed September 2007). 65. Van, T.T.H., Moutafis,G., Tran, L.T.,and P. J. Coloe. 2007. AR in Food-Borne Bacterial Contaminants in Vietnam. Appl Environ Microbiol. 73: 7906-7911. 70 66. Van den Bogaard, A.E., and E.E. Stobberingh. 1999. Antibiotic Usage in Animals- Impact on bacteria resistance and public health. Drugs 58:589-607. 67. Van Looveren, M., Daube, G., De Zutter, L., Dumont, J., Lammens, C., Wijdooghe, M., Vandamme, P., Jouret, M., Cornelis, M. and H. Goossens. 2001. Antimicrobial susceptibilities of Campylobacter strains isolated from food animals in Belgium. J. Anti Chemother. 48, 235-240. 68. Villedieu, A., Diaz-Torres, M.L., Hunt, N., McNab, R., Spratt, D.A., Wilson, M. and P. Mullany. 2003. Prevalence of tetracycline resistance gene in oral bacteria. Antimicrob Agents Chemother. 47: 878-882. 69. Villedieu, A., Diaz-Torres, M.L., Roberts, A. P., Hunt, N., McNab, R., Spratt, D.A., Wilson, M. and P. Mullany. 2004. Genetic Basis of Erythromycin Resistance in Oral Bacteria. Antimicrob Agents Chemother. 48(6): 2298–2301. 70. Wang, H., Manuzon, M., Lehman, M., Wan, K., Luo, H., Wittum, T., Yousef, A., and L. Bakaletz. 2006. Food commensal microbes as a potentially important avenue in transmitting antibiotic resistance genes. FEMS Microbiol Lett. 254:226231. 71. Wassenaar, T. 2005. Use of antimicrobial agents in veterinary medicine and implications for human health. Crit Views Microbiol. 31: 1555-169. 72. Waterman, S., and P. Small. 1998. Acid-sensitive enteric pathogens are protected from killing under extremely acidic conditions of pH 2.5 when they are inoculated onto certain solid food sources. Appl. Environ. Microbiol. 64:3882– 3886. 73. Werner, G., Klare, I., Reissbrodt, R., and W. Witte. 2004. Occurrence of antibiotic-resistant enterobacteria in agricultural foodstuffs. Mol. Nutr. Food Res. 48: 522-531. 74. WHO. 2000. WHO Global Principless for the Containment of Antimicrobial Resistance in Animals Treated for Food: Report of a WHO Consultation, Geneva, Switzerland, June 5-9, 2000. 75. WHO. 2003. World Health Organization, Impacts of antimicrobial growth promoter termination in Denmark, The WHO international review panel’s evaluation of the termination of the use of antimicrobial growth promoters in Denmark. http://www.who.int/salmsurv/en/Expertsreportgrowthpromoterdenmark.pdf 76. Yu, Z. and M. Morrison. 2004. Improved extraction of PCR-quality community. DNA from digesta and fecal samples. Biotechniques. 36:808-12. 71