Real-Time Quantitative PCR of tet (C), in 2 Swine Populations: Antibiotic Free versus Conventionally Reared THESIS

Transcription

1 Real-Time Quantitative PCR of tet (C), in 2 Swine Populations: Antibiotic Free versus Conventionally Reared THESIS Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University By James David White, DVM Graduate Program in Veterinary Preventive Medicine The Ohio State University 2015 Master's Examination Committee: Thomas Wittum, MS, PhD, Advisor Paivi J Rajala-Schultz, DVM, PhD, Diplomate ACVPM Armando Hoet, DVM, PhD, Diplomate ACVPM

2 Copyrighted by James David White, DVM 2015

3 Abstract Cultivable bacteria are a fraction of the fecal microbiota, yet serve as the foundation of current antibiotic resistance (AR) research. The small percentage of bacteria that can be cultured may be a biased representation of the AR gene reservoir. As such, our first objective was to perform Real-Time Quantitative PCR (RT-QPCR) of whole fecal DNA extracts for quantification of tet (C), a gene that confers resistance to tetracyclines. A challenge of utilizing community DNA for estimation of gene concentration is selection of the appropriate denominator. In objective 1, we compared tet (C) concentrations based on 3 different denominators: fecal weight, 16S rrna (the nucleic acid responsible for the bulk of the prokaryotic 30S ribosomal subunit) concentration, and community DNA concentration. Approximately 200 mg of feces were collected from 212 pigs. Total fecal DNA was extracted and quantification of tet (C), 16S rrna, and total DNA was performed by Real-Time PCR. To evaluate the correlation of tet (C) concentrations using fecal weight, 16S rrna concentration, and DNA concentration as denominators, Kendall s tau was calculated for each pair of estimates (tet(c)/mg feces: tet(c)/dna concentration; tet (C)/mg feces: tet (C)/16S rrna; tet (C)/DNA concentration: tet (C)/16S rrna). ii

4 There was significant correlation for tet (C) concentrations for all comparisons (tau > 0.69, p<0.05). This may suggest that, at least in comparing tet (C) concentrations in community DNA samples of swine of similar age, the use of the fecal weight as a denominator for concentration estimation is valid as proxy measures for bacterial concentration. In addition, the use of fecal weight represents significant cost savings as compared to the other measures evaluated. As such, these data establish the appropriate denominator for future studies of tet (C) concentrations in swine of similar age. In objective 2, the concentration of tet (C) in fecal community DNA from antibiotic free (ABF) pigs was compared to that of conventionally (CON) reared pigs. Fecal samples from 888 individual pigs from 37 farms (20 farms ABF and 17 farms CON) representing 3 regions (Ohio, Wisconsin, and North Carolina) were obtained using a 200 mg stool tube from pre-market swine. DNA was extracted from these samples and concentration of tet (C) was conducted by Real-Time PCR. Each PCR reaction was conducted in triplicate. For the tet (C) assay, the primer pairs tet (C)-fw and tet (C)-rev were used. All farms and 99% of pigs were found to be tet (C) positive. The mean tet (C) concentrations for ABF and conventional pigs were 4.8x10 4 (sd 2.0x10 5, range x10 6 ) and 1.2x10 6 (sd 9.5x10 6, range 3.8x x10 8 ) copies tet (C) / mg of feces respectively. Statistical analysis with adjustment for clustering suggests that iii

5 conventionally reared pigs had a higher concentration of tet (C) in their feces compared to ABF pigs (p<0.0001). ABF pigs were found to have a lower concentration of tet (C) in their fecal microbiota which may translate into a lower risk for zoonotic transfer of AR. iv

6 To the Mrs., for her endless faith in me when my own wavered. v

7 Acknowledgments To the advisory committee of Thomas Wittum, Paivi J Rajala-Schultz, and Armando Hoet: I remain in your debt. To the mentorship of Julie Funk: you are where it all began. To the graduate students Kate Kleinhenz, Dixie Mollenkopf, Luke Heider, and Billy Walker: Thanks is not a big enough word for your friendship. The love and appreciation in my heart for each of these individuals is so large, it simply cannot be described. vi

8 Vita B.S., Food, Agriculture, and Environmental Science, The Ohio State University Graduate Research Associate, The Ohio State University D.V.M., The Ohio State University Associate Veterinarian, Refugee Canyon Veterinary Services, Hebron, Ohio Relief Veterinarian, Various Practices, California Staff Veterinarian, Carrington College, California Fields of Study Major Field: Veterinary Preventive Medicine vii

9 Table of Contents Abstract... ii Acknowledgments... vi Vita... vii List of Tables... x List of Figures... xi Chapter 1:Review of the Literature Relate of tet (C) Mediated Antimicrobial Resistance Antimicrobials in Public Health Antimicrobials in Swine Health Tetracyclines and Tetracycline Resistance Tetracycline and Other Antimicrobial Resistance Epidemiology Conclusion References viii

10 Chapter 2:Application of Real-Time Quantitative PCR (RT-QPCR) to accurately quantify tet (C) in swine fecal samples Introduction Materials and Methods Results Discussion References Bibliography ix

11 List of Tables Table 1. Comparison of tet (C) concentration as determined by RT-QPCR of whole fecal DNA extracts from 212 fecal samples from pre-market swine on 10 farms in North Carolina using the following 3 denominators: mg of feces, copy 16S rrna, and total community DNA Table ABF pigs on 20 farms in 3 states. tet (C) concentration as determined by RT-QPCR of whole fecal DNA extracts. Mean copies tet (C) / mg of feces 4.8E4 (sd 2.0E5, range E6) Table CON pigs on 17 farms in 3 states. tet (C) concentration as determined by RT-QPCR of whole fecal DNA extracts. Mean copies tet (C) / mg of feces 1.2E6 (sd 9.5E6, range 3.8E1 1.3E8) Table pigs on 37 farms in 3 states. tet (C) concentration as determined by RT- QPCR of whole fecal DNA extracts. Mean copies tet (C) / mg of feces 6.7E5 (sd 6.8E6, range E8) Table ABF and CON pigs on 37 farms in 3 states. tet (C) concentration as determined by RT-QPCR of whole fecal DNA extracts. Distribution of variance of tet (C) concentration in the organizational structure x

12 List of Figures Figure 1. Scatterplot of tet (C) concentrations as determined by RT-QPCR of whole fecal DNA extracts from 212 fecal samples from pre-market swine on 10 farms in North Carolina using the following 2 denominators: mg of feces and total community DNA. All samples were positive for tet (C) with respect to each denominator. (Kendalls tau=0.71, p<0.0001) Figure 2. Scatterplot of tet (C) concentrations as determined by RT-QPCR of whole fecal DNA extracts from 212 fecal samples from pre-market swine on 10 farms in North Carolina using the following 2 denominators: mg of feces and copy 16SrRNA. All samples were positive for tet (C) with respect to each denominator. (Kendalls tau=0.69, p<0.0001) Figure 3. Scatterplot of tet (C) concentrations as determined by RT-QPCR of whole fecal DNA extracts from 212 fecal samples from pre-market swine on 10 farms in North Carolina using the following 2 denominators: copy 16SrRNA and total community DNA. All samples were positive for tet (C) with respect to each denominator. (Kendalls tau=0.69, p<0.0001) xi

13 Figure 4. Illustration describing the side by side boxplot comparison of tet (C) concentration as determined by RT-QPCR of whole fecal DNA extracts originating from 434 ABF pigs on 20 farms in 3 states, mean copies tet (C) / mg of feces 4.8E4 (sd 2.0E5, range E6), median 5.5E3 and 454 CON pigs on 17 farms in 3 states, mean copies tet (C) / mg of feces 1.2E6 (sd 9.5E6, range 3.8E1 1.3E8), median 1.6E xii

14 Chapter 1 Review of the Literature Relate of tet (C) Mediated Antimicrobial Resistance 1.1 ANTIMICROBIALS IN PUBLIC HEALTH The contribution of antimicrobial use in agriculture to increased health burden associated with antimicrobial resistant human infections is a timely issue. There is little argument that when an animal, human or otherwise, is exposed to an antimicrobial (or a population of animals is exposed to an antimicrobial) there is an increase in the proportion of antimicrobial resistant (AR) bacteria. Furthermore, the societal cost of AR is estimated to be from $150 million (without deaths) to $3 billion (with deaths) annually. 1 In 2013, the CDC estimated in the United States, more than two million people are sickened every year with antibiotic-resistant infections, with at least 23,000 dying as a result. 2 The estimates are based on conservative assumptions and are likely minimum estimates. They are the best approximations that can be derived from currently available data. 1

15 Antimicrobials are the most commonly thought of sources of noxious stimuli for bacteria. Even so, prior to the advent of modern antimicrobials, resistant bacteria were evading noxious stimuli by expression of heavy metal efflux pumps. 3 Furthermore, resistant bacteria have been isolated from 30,000 year old permafrost, 4 wild animals, 5 ocean trenches, 6 and reservoirs of ground water. 7 Though antimicrobial resistance predates antimicrobials, the application of antimicrobials has been shown to exert selection pressure 8 to not only pathogenic bacteria of interest but also to commensal bacteria. The resistant bacteria, be it pathogenic bacteria of interest or commensal bacteria, give rise to greater numbers of resistant bacteria, either vertically as in clonal spread or horizontally as in conjugation, transformation, transduction, or by transposons. 9 These resistant bacteria can defeat the antibiotics meant to treat them, increasing morbidity / mortality in the clinical setting. 10,11 According to the CDC in 2002, approximately 2 million people in the United States each year acquire an infection while in hospital (nosocomial), resulting in 90,000 deaths. Additionally, more than 70% of the bacterial etiologies are resistant to at least 1 of the antibiotics commonly used to treat them. At this time, nearly all significant bacterial infections are becoming resistant to the first line antibiotic treatment of choice. 12 2

16 Given the dynamics of microbial ecology, characterized by change, competition, and adaptation, it can be said that managing AR necessitates a multi-disciplinary approach between producers, veterinarians, and medical doctors who are committed to prudent and judicious use of antimicrobials, in hopes to preserve the efficacy of current and newly developing antibiotics, minimize the zoonotic transfer of bacteria harboring AR, and secure a safe, affordable, and abundant food supply. 13 3

17 1.2 ANTIMICROBIALS IN SWINE HEALTH It is helpful to view animal health in terms of swine farms that use antibiotics (conventional or CON) and those who do not (antibiotic free or ABF). CON The results of 9 trials comparing growth rate and feed efficiency between nursery and finishing pigs fed antibiotics and those not fed antibiotics are as follows: nursery pigs fed antibiotics had significantly greater average daily gain (ADG) values and comparable feed efficiency (FE) when compared to nursery pigs reared ABF. In finisher pigs, ADG and FE were nearly identical between the ABF and CON groups. 14 In a summary of over 1,000 experiments conducted in the U.S. over the course of 35 years, it was found young pigs (17-25 kg) reared conventionally had improved growth rate, 16.4% on average and improved FE, 6.9% on average over those reared without antimicrobials. Also, it was found growing pigs (17-49 kg) reared conventionally had improved growth rate, 10.6% on average and improved FE, 4.5% on average over those reared without antimicrobials. Lastly, it was found during the entire growing finishing period (24-89 kg), those pigs reared conventionally had improved growth rate, 4.2% on average and improved FE, 2.2% on average over those reared without antimicrobials. 15 4

18 It has been reported that in addition to performance benefits from antimicrobials, reproductive benefits are also documented. Feeding a high level of a readily absorbed antibiotic a few days before and after breeding is thought to reduce the number of potential pathogens within the sow s reproductive tract, effectively improving overall uterine environment, which likely has a positive effect on embryo implantation. Nine trials with 1931 sows conducted between 1962 and 1994 were summarized and it was found the provision of a readily absorbed antibiotic a few days before and after breeding improved farrowing rate by 6.6% and litter size by 1.5 weaned pigs / litter. A summary of 13 lactation experiments on 2338 sows showed a 1.7% increase in pig survival and a 0.04 kg increase in average weaning weight. 15 ABF In 1986, Sweden saw a ban on antibiotics for growth promotion (AGP). Since then, only therapeutic doses of antibiotics prescribed by a veterinarian are permitted That same year, growing-finishing pigs did not experience any observable reduction in ADG, when compared to However, weaning piglets experienced a 2 fold increase in post-weaning diarrhea, a 1.5% increase in post-weaning mortality, and took 5-6 days longer to reach 25 kg after the 1986 removal of olaquindox. 18 As AGP came to an end in Sweden, the therapeutic use of antibiotics increased to 6% greater than in By 5

19 1989, 75% of all nursery pigs in Sweden were routinely fed antibiotics at therapeutic levels. In 1994, 55 Swedish piglet producing farms were examined and it was concluded that the farms with superior hygiene and segregated rearing systems were prescribed 3 to 4 times less antibiotics than those farms with poorer hygiene, often lacking the facilities to avoid commingling pigs of differing age and immune status. 19 Even though 10 years after the ban on AGP in Sweden, post-weaning mortality declined by 1% and the days to reach 25 kg decreased by days on average, these production results are still short of the pre-agp ban 18 and have come at considerable additional cost to the producer. In January, 1998, Danish swine producers voluntarily discontinued the use of all AGP in pigs greater than 35 kg. By the end of the year, they had decided to discontinue the use of all AGP for pigs in all groups and had accomplished this by the beginning of Like Sweden, Denmark s removal of AGP in grower-finisher diets had no observable effects. Also, removal of AGP in nursery diets equated to an initial 30% increase in pig mortality and a decrease in ADG by 19 g. Post weaning diarrhea also increased, as did the use of therapeutic antibiotics. 21 6

20 It can be said that both countries have discontinued the use of AGP, increased the use of therapeutic antibiotics, yet have managed to decrease the total use of antibiotics. 20 In 1972, the University of Kentucky chose one of its swine herds to withdraw all antibiotics from. Antibiotics have not been used since this time for any purpose. This specific pathogen free herd operates under strict biosecurity and introduces new genetics only by artificial insemination. This herd has experienced a number of reproductive reductions between 1972 and 2002: conception has decreased from 91% to 83%, sows farrowed and weaned fewer pigs/litter, pigs were smaller at weaning, and pig survival decreased from 90% to 81%. 22 In short, there are pros and cons to raising pigs both ABF and CON. The pros of raising CON pigs are increased FE and ADG (effect tends to wane with age), decreased pig mortality, and enhanced reproductive performance. Although, more recent data in rearing systems typical of current production systems (all in / all out over continuous flow), there appears to be no benefit of growth promoting antibiotics. 23 One of the cons of raising CON pigs is the increased prevalence of AR bacteria, which is a relatively consistent finding across all studies. This negative finding can result in treatment failure and/or increased costs of therapy in animals and potentially, in humans. The pro of raising ABF pigs is the decreased prevalence of AR bacteria. The cons of raising ABF pigs are the decreased FE and ADG, increased post-weaning diarrhea and pig mortality, and 7

21 diminished reproductive performance. Through the examples of Sweden, Denmark, and the University of Kentucky, one can appreciate the contributions to animal health made by raising pigs with antibiotics, or at the very least appreciate how important good hygiene and appropriate segregation of pigs of different ages in all pig facilities, especially those with ABF pigs. Antibiotics are used in a number of important ways. The Clinical and Laboratory Standards Institute (CSLI) describes them: therapeutic use for clinically ill animals and non-therapeutic use encompassing prophylaxis to deter or minimize disease onset and growth promotion to enhance an animal s physiologic performance. 24 Our focus will be on high level doses for therapeutic use, intermediate level doses for prophylaxis, and low level doses for growth promotion. These antimicrobials can be administered in feed, water, or by injection. 15 Intermediate level doses for disease prophylaxis help prevent the occurrence of spread of disease within large herds, especially when vaccines are not available or efficacious. Using antibiotics for disease prevention has particular utility during times of high stocking density where the spread of disease has the potential to be rapid and devastating. 25 8

22 Low level doses for growth promotion are thought to act in three ways: 1) nutritionally, 15 thinning intestinal walls to increase nutrient uptake, 26 2) metabolically, 15 decreasing growth depressing microbial metabolites and decreasing the microbial use of nutrients, 27 and 3) the most widely supported theory is that antibiotics encourage growth by suppressing the microorganisms that cause sub-clinical disease. 15 This permits the ingested nutrients to be used more by the animal and less by the gut micro-flora s metabolic processes. 26 Said another way, the sub-clinical disease suppression theory permits the animal to invest more of its energy and resources in FE and daily gain, and less into defending itself against disease. Pig farming without the use of AGP is possible but at a significant cost. 28 The lag between waning passive immunity and waxing acquired immunity creates a susceptible time for young pigs. Also sequential batches of pigs follow one another from the farrowing unit to the nursery to the grower/finisher. This can be problematic for pigs entering a facility, as enough environmental contamination may remain to cause clinical disease. These two events, fledgling immunity and pig flow related problems can be anticipated and treated accordingly with antimicrobial prophylaxis. In countries without AGP, necessity has encouraged innovative production techniques to increase hygiene, develop age and source matched cohorts, and adjust feeding regimens to off-set production deterioration. 18 Other techniques used (both for GP and disease prevention) are pro-biotics, 29 restricted feedings immediately after weaning to decrease the 9

23 incidence of scouring, and liquid feeding immediately after weaning to decrease intestinal villi atrophy, therefore, making the pig more resistant to E. coli infections. E. coli is also inhibited in pigs fed fermented feed characterized by a lower ph and a higher yeast concentration. Also, lower protein and lower buffering mineral starter diets decrease the incidence of post weaning diarrhea. Furthermore, high fiber diets tend to decrease gut ph and decrease a pathogen s ability to bind to the lectin ligands on the surface of gut epithelial cells, both of which make a less hospitable environment for E. coli and other pathogens. 20,26 Properly working waterers acidified to a ph of 4 to 5 will contain fewer coliform bacteria and decrease piglet scours. Ideal barn temperatures and proper ventilation decrease lethargy, making for frequent visits to feeders and waterers, and less indigestion. 20 Last but not least, the timely application of vaccines, as determined by sequential serology, field observations, and herd history may negate some of the deleterious effects of a ban or restriction on the use of sub-therapeutic antibiotics. Accurate diagnostics, drug sensitivity testing, and the selection of an appropriate therapeutic regimen are not only good production practices, but they also benefit the producer without sub-therapeutic antibiotics at their dispose. 28 A number of interventions have been named to assist pig producers in a low/no subtherapeutic antimicrobial environment, but each of them comes with considerable cost. On the producer side, it was estimated in 1999 that production costs would increase by $6.05/pig and by $5.24/pig by the end of a 10 year period. The net foregone profit to 10

24 the industry over a 10 year period is billion. 30 The National Research Council estimated the removal of antibiotics for GP and disease prevention would raise the cost of food in the U.S. by $1.2 billion to $2.5 billion. 31 This figure does not take into account the public health consequences of an increased number of pathogens entering the processing plant from animals withheld sub-therapeutic antibiotics, nor the environmental consequences of such a ban. Given that AGP improves production parameters, pigs would have to be fed more to reach market weight. This would increase the amount of land required for an equal amount of food production. In addition, there would also be an increase the waste produced by swine farms, with an increase in the nitrogen, phosphorous and nutrient content of those wastes

25 1.3 TETRACYCLINES AND TETRACYCLINE RESISTANCE In 1948, chlortetracycline was discovered as a product of Streptomyces aureofaciens and oxytetracycline was discovered as a product of S. rimosus. In the years to come, other naturally occurring tetracyclines were discovered. Also synthetic and semi-synthetic analogs were manufactured to improve water solubility for enhanced oral absorption and parenteral administration. The tetracyclines form positively charged coordination complexes with molecules of magnesium or calcium. In this complexed form, they pass through the bacteria s porin channel. Once sufficient concentrations have been reached in the periplasm, the tetracycline dissociates from the metal cation to become a weakly hydrophobic molecule which has little difficulty traversing the lipid bilayer into the cytosol. Once in the cytosol, the tetracycline is again complexed to a magnesium ion 32 and is ready to approach its site of action at the prokaryotic ribosome. At the ribosome, the tetracycline-magnesium coordination complex prevents the bacteria s animoacyl-trna from associating with the ribosomal acceptor (A) site. 32 In this way, the bacteria cannot synthesize the proteins vital to its survival

26 Given that tetracyclines have been used extensively for many years in human medicine, the incidence of bacteria resistant to them has increased and clinically, the use of the tetracyclines have fallen out of favor in most developed countries. 32 Regardless, the tetracyclines are the second most frequent antibiotic used after the penicillins for human therapy worldwide, and they have a role in treating respiratory infections, acne, protozoan parasite infestations, 32 and have become a treatment option consideration (glycyclines such as tigecycline) for serious Gram negative infections that have become resistance to other drug classes. Also, the CDC now recommends only doxycycline or ceftriaxone plus either azithromycin as first line treatment for gonorrhea caused by Neisseria gonorrhoeae, a venereal disease updated to urgent status in 2013, largely due to estimates of the 820,000 gonorrhea cases per year, of which 23% are resistant to tetracycline and 30% are resistant to any antibiotic. 2 Chlortetracycline, oxytetracycline, and tetracycline are approved for use in domestic livestock, honeybees, aquatic species, and certain plants. They are legally used for disease treatment, disease prevention, and growth promotion in the U.S. 37 They remain relatively efficacious in certain applications, are inexpensive, and their consumption among U.S. farm animals (regardless of purpose) increased from 2.6 x 10 6 kg in 1985 to 3.5 x 10 6 kg in to 6.5 x 10 6 kg in

27 The practice of adding low concentrations of antibiotics, defined in the United States as < 200 g/ton of feed 31,32,39,40 to animals to improve growth and feed efficiency is referred to as growth promotion or growth enhancement. The labeled dose of chlortetracycline for growth promotion in swine is 50 g / ton of complete feed. 41 A therapeutic dose for an individual pig is 5-10 mg/kg, IM or IV, SID. 42 The NAHMS 2000 Swine Study does not report the total % of sites which used antimicrobials in nursery diets (pigs < 10 wks of age) but does go on to say 82.7 % of sites use them for the purposes of growth promotion. The NAHMS 2000 Swine Study reports 88 % of sites use antimicrobials in grower /finisher diets (pigs weeks of age) and 63.7 % of sites use them for the purposes of growth promotion. 43 The NAHMS 2000 Swine Study reported the average dose of chlortetracycline in feed given to grower/finisher pigs for growth promotion, disease prevention, respiratory disease treatment, and enteric disease treatment was 97.9 g/ton, g/ton, g/ton, and g/ton, respectively. The average dose of oxytetracycline in feed given to grower/finisher pigs for growth promotion, disease prevention, respiratory disease treatment, and enteric disease treatment was N/A, g/ton, g/ton, N/A g/ton, respectively. Also, chlortetracycline was fed to grower/finisher pigs on average 52.1 days for growth promotion, 42.9 days for disease prevention, and 12.5 days for disease 14

28 treatment. Oxytetracycline was fed to grower/finisher pigs on average 25.3 days for disease prevention, and 19.1 days for disease treatment. 43 The 2006 NAHMS Swine Study reports 82.3 % of sites use antimicrobials in nursery diets and 24.5 % of sites use them for the purposes of growth promotion. The 2006 NAHMS Swine Study reports 83.6 % of sites use antimicrobials in grower/finisher diets and 55.1 % of sites use them for the purposes of growth promotion. 44 The 2006 NAHMS Swine Study does not report average doses of chlortetracycline or oxytetracycline in feed given to grower/finisher pigs for growth promotion, disease prevention, respiratory disease treatment, and enteric disease treatment. However, data pertaining to site average number of days given (assumed to be for any reason) is available for chlortetracycline. Chlortetracycline was fed to grower/finisher pigs on average 41.5 days. 44 The 2012 NAHMS Swine Study part III (reference of Management Practices on Smallenterprise Swine Operations) reports nearly 40 % of operations with less than 100 pigs added antibiotics to feed intended for weaned pigs. A higher percentage of operations with 50 to 99 pigs added antibiotics to feed intended for weaned pigs (56.8 %) compared with operations with 1 to 49 pigs (36.1 %). 45 The 2012 NAHMS Swine Study 15

29 part II: Reference of Swine Health and Health Management in the United States is expected summer 2015 and will describe antibiotic usage patterns in pigs post-weaning. When an animal or group of animals is exposed to an antimicrobial there is an increase in the proportion of antimicrobial resistant bacteria. Specifically, when a bacterial population is exposed to some noxious stimuli (tetracycline in this case), some members may die while some members may respond by adapting and proliferating to be better able to survive in these new conditions. These adapting and proliferating members are said to have acquired resistance. 31 In this particular instance, the tetracycline is said to be the selection pressure and the acquisition of genes permitting existence in the face of the tetracycline is said to be the resistance. Generally speaking, bacteria that acquire AR do so by 1) alteration of a previously targeted gene product, such that the antimicrobial no longer has the desired effect, 46 2) enzymatic degradation or inactivation of the antimicrobial, 3) development of an efflux pump system which functions to decrease the antimicrobial concentration within the cell, 47 4) decreased efficiency of the uptake system normally responsible for increasing the antimicrobial concentration within the cell, and 5) some bacteria no longer metabolize the antimicrobial drug into a fully functional inhibitory compound. Once the bacteria have acquired AR, vertical (clonal) transmission may take place, conferring AR to the bacterial progeny. Mobile genetic element (plasmids and transposons) mediated 16

30 horizontal transmission of resistance may take place through 1) conjugation transfer of DNA via a sex pillus, 2) transformation transfer of free or naked DNA fragments, 3) transduction transfer of DNA by a phage vector, and 4) transposons transfer of DNA between plasmids and chromosomal DNA. 31,48-53 Thus far, 11 tet genes coding for ribosomal protection proteins, 23 tet genes coding for efflux pumps, 1 tet gene coding for enzymatic inactivation of tetracycline, and 1 tet gene (tet (U)) coding for tetracycline resistance by an unknown mechanism have been described. Currently, only tet genes coding for ribosomal protection proteins and efflux pumps are found in bacterial isolates from veterinary samples. The 11 tet genes coding for ribosomal protection proteins, primarily found in but not limited to Gram-positive bacteria, create proteins which interact with the ribosome to allosterically disrupt the tetracycline binding site. Given that tetracycline cannot bind, the bacteria s animoacyl-trna associates with the respective ribosomal acceptor (A) site, and bacterial proteins are successfully manufactured. 32,54 The 23 tet genes coding for efflux pumps, tet (K) and tet (L), primarily of gram-positive bacteria, only have their transcripts translated in the presence of tetracycline. 54 A conformational change in their mrna prevents translation in the absence of tetracycline. 32 The genes tet (A), tet (B), tet (C), tet (D), tet (E), and tet (H), primarily of 17

31 Gram-negative bacteria, experience transcription in the presence of tetracycline, but rely on a tet repressor gene to block tet transcription in the absence of tetracycline. 54 In a phenomenon known as co-selection, AR genes in close physical proximity to one another may cross-link, effectively conferring multiple antimicrobial resistances to the bacteria, and delaying loss of resistance to a no longer used antimicrobial In an example of multiple resistances, tetracycline resistant Escherichia coli isolates from healthy pigs throughout Japan underwent antimicrobial susceptibility testing. The pigs were divided into two groups: group #1 was those pigs that had been treated with therapeutic doses of oxytetracycline at some point in time and group #2 was those pigs that had never received therapeutic antibiotics. The region with the greatest use of oxytetracycline naturally had the greatest prevalence of oxytetracycline resistance, but also had the greatest prevalence of kanamycin and trimethoprim resistance, supporting the co-selection phenomena. 59 In an example of delayed loss of resistance, over half of the E.coli organisms obtained from diarrheic swine in a particular study from 2005 were found to be resistant to chloramphenicol, even though the United States banned chloramphenicol for use in food animals in It was suggested the gene responsible for chloramphenicol resistance was maintained not by direct selection pressure, but by gene linkage to resistance genes who themselves are maintained by direct selection pressure. That is to say the chloramphenicol resistance gene persists not because the producers are currently using chloramphenicol, but because the chloramphenicol 18

32 resistance gene is in close proximity to genes responsible for resistance to antimicrobials the producers actually are currently using. 60 This is particularly problematic: though removing the antimicrobial in question removes the selection pressure, which in turn favors proliferation of susceptible organisms over resistant ones, this process takes a great deal longer than the reverse. In short, the emergence of AR dramatically outpaces the loss of AR. 48,60 Given the variety of ways bacteria can acquire resistance, the presence of multi-drug pumps, and the phenomena of co-selection with its inherent multiple antimicrobial resistances and delayed loss of resistance to a no longer used antimicrobial, there is great interest in novel tetracycline and minocycline analogs such as the glycylcyclines, capable of evading current methods of AR. 40,61,62 Also of interest, are the novel compounds with efflux pump inhibition characteristics, thought to be capable of restoring the efficacy of tetracyclines in those organisms with efflux pump mediated tetracycline resistance. 63 Modified tetracyclines, such as datocycline, 65 deoxytetracycline, 66 and glycylcycline, 67 not subject to ribosomal protection and efflux pump mechanisms of resistance could be hypothesized to prolong the usefulness of therapeutic tetracyclines

33 However, these novel compounds are not immune to reduced efficacy, as organisms exposed to them experimentally, are capable of becoming resistant to them. Should the novel compounds be used imprudently, they too are at risk of becoming obsolete

34 1.4 TETRACYCLINE AND OTHER ANTIMICROBIAL RESISTANCE EPIDEMIOLOGY It is necessary to quantify the scope of AR in a given situation, in order to not only understand its urgency, but also to understand where a greater investment of resources would be warranted. 41 If the level of AR bacteria is high enough to be problematic for a given population, decisions to decrease the use of the antibiotic in question or change the prescription all together can be made in good conscious. Likewise, if the level of AR bacteria does not pose a significant threat, the decision to continue the use of a particular antimicrobial can be made in good conscious. 54 Either way, the first step in addressing a problem is to determine the magnitude of the problem (ranging from no problem at all to severe), so an appropriate solution can be sought. A sufficient number of representative samples taken from the farm to fork continuum (feces from live animals on the farm, feces from live animals in slaughterhouse lairage, swabs of carcasses at the slaughterhouse, and swabs or washes of animal products in the butcher s retail case) are typically chosen to evaluate the level of antimicrobial resistance in bacteria of interest. The goal of antimicrobial susceptibility testing is to determine the zone of inhibition if using the disc diffusion methodology, or the minimum inhibitory concentration (MIC) if using the dilution methodology, to a battery of antimicrobials. A list of antimicrobials to be used in the testing is set forth, for example by National Antimicrobial Resistance Monitoring System (NARMS) 70 in the 21

35 United States, or Danish Integrated Antimicrobial resistance Monitoring and Research Programme (DANMAP) 71 in Denmark. Also in existence are The French Agency for Food Safety (AFSSA) in France, The Ministry of Agriculture, Fisheries and Food (MAFF) in Great Britan, and The Red de Vigilancia de Resistencias Antibioticas en Bacterias de Origen Veterinario in Spain. 69 In this way, one can discriminate between isolates and assess any possible trends towards developing resistance. 69 Furthermore, one can investigate the prevalence of AR within and/or between group(s) over time, the similarities and differences between various geographical locations in terms of AR prevalence, in an attempt to ascertain the risk posed from antimicrobial use. 54 For example, NARMS has recently demonstrated a significant rise in the prevalence of Salmonella resistant to the cephalosporin ceftiofur in human and food animal isolates. For this reason, on January 6, 2012, the Food and Drug Administration s (FDA) Center for Veterinary Medicine (CVM) issued an order prohibiting certain extralabel uses of the cephalosporin class (excluding cephapirin) of antimicrobial drugs in food-producing animals to preserve the efficacy of the cephalosporins in food animals and human patients. 72 Likewise, the DANMAP 2011 report was able to demonstrate a proportional decline in the prevalence of enterococci resistant to the glycopeptide antibiotic vancomycin, due 22

36 to a 1995 ban on the growth promoting glycopeptide antibiotic avoparcin, supporting the value of the ban itself. 71 Epidemiologic research on emergence of antimicrobial resistance from antimicrobial use in animal agriculture arrives at a conclusion respective to its immediate focus, then thru extrapolation attempts to contribute to the much larger focus, namely addressing the question is the use of antimicrobials in animal agriculture a significant source of antibiotic resistance and ultimately human treatment failures? 57 Many examples of epidemiologic studies exist, including antibiotic usage data, molecular subtyping, in-vitro susceptibility characterization of AR genes from animal and human isolates, temporal associations, field studies, case reports, and results obtained from controlled animal experiments. 57,73-75 Antibiotic use data: Antibiotic usage data should be arranged in a useful way that compares like to like, that is to say kg of active substance to kg of active substance after determining potency or monetary value of sales to monetary value of sales after determining price changes and currency exchange rates. Even then, a variety of factors must be considered: duration of use in animals, age of animals, number of animals, dose on a mg/kg basis, seasonality of usage, etc. 57 In terms of kg of active substance, Denmark has seen a decline in avoparcin resistant Enterococcus faecium from broilers, broiler meat, pigs, pork, and healthy humans after the May 1995 ban on avoparcin use. 23

37 Likewise, Denmark has experienced a near linear relationship between the increased use of tetracycline in pigs and the recovery of tetracycline resistant E. faecium from pigs, pork, and healthy humans. 71 Molecular subtyping: The purpose of molecular subtyping is to demonstrate congruity between the AR bacteria in animals (or their resistance determinants), animal products, and those found in humans, to infer the transmission or potential for transmission of AR. Six Danish volunteers ingested a 10^9 CFU suspension of a vancomycin susceptible E. faecuim of human origin. 3 hours later, the volunteers ingested a 10^7 CFU suspension of a vancomycin resistant E. faecium of chicken origin. Fecal samples were collected from the volunteers at -48 hours, every day for 7 days, day 14, and day 35. Transconjugants were recovered from 3 of the 6 volunteers, demonstrating the transmissibility of an AR genetic determinant from bacteria of animal origin to the same respective bacteria of human origin. 71 Invitro characterization of AR genes of animal and human isolates: Vancomycin resistant E. faecium from humans, pigs, and poultry were examined for the Tn1546 transposon and the resistance gene VanX. Tn1546 transposon was found to exhibit only minor variations outside of the region coding for vancomycin resistance in human and animal isolates. In addition, a base pair variation at position 8234 corresponding to a G for poultry and a T for swine were found to partially align with the respective countries 24

38 dietary custom. 76 For example, the VanX gene from human isolates in Saudi Arabia, where no pigs are produced or consumed, had nucleotide G, not T, at position Temporal associations: Another modality trying to assess the association between agricultural and veterinary use of antimicrobials and human treatment failures is one that seeks to establish antimicrobial sensitivity before the use of a particular antimicrobial and increased resistance thereafter. For example, ciprofloxacin resistant Campylobacter increased from 0% to 11% in man and from 0% to 14% in poultry products between 1982 and 1989 in the Netherlands. This is thought to be due to the introduction of norfloxacin in 85, enrofloxacin in 87, ciprofloxacin in 88, perfloxacin and ofloxacin in Epidemiological data: A number of epidemiological studies aim to demonstrate an increased likelihood of transmission of bacteria, AR and otherwise, from livestock to those in close proximity to them. It has been shown that Escherichia coli obtained from bovine and porcine species can be given an engineered plasmid psl222 (for marker purposes), fed back to the animal from which it came, and then be recovered from flies, mice, poultry, and the humans caring for the cattle and pigs in its original form (as porcine or bovine E. coli with marker plasmid psl222)

39 Field Studies: The use of nourseothricin, a novel antibiotic, neither used in human or veterinary medicine, was fed to pigs as a growth promotant in former East Germany. Two years after the initiation of its use, plasmids conferring nourseothricin resistance were discovered in E. coli from the pigs, the farm s employees and their families, and members of the nearby community. The same sample populations from a distant community not using nourseothricin revealed E. coli free of plasmids conferring nourseothricin resistance. 79 In another example, the feces of ABF chickens, farm employees, and neighbors were found to contain low numbers of tetracycline resistant organisms. The chickens were then fed a tetracycline supplemented feed and within 1 week, their intestinal flora contained almost entirely tetracycline resistant organisms. Interestingly, within 6 months, greater than 31 % of employees of the farm submitted fecal samples characterized by greater than 80% tetracycline resistant bacteria while less than 7 % of their non-farm employee neighbors had sample submissions of greater than 80% tetracycline resistant bacteria. 80 Case studies: In March 1998, a veterinarian treated calves for diarrhea in his own and 3 other herds. In April 1998, his son became ill. Stool samples from both the calves and the boy were positive for Salmonella enterica serovar typhimurium that was later found to be remarkably similar by pulse field gel electrophoresis

40 Controlled animal experiments: Often researchers obtain the proportion of some AR indicator bacteria from medicated animals (conventional or CON) and compare them to non-medicated animals (antibiotic free or ABF) after culturing them on some selective media and exposing them to a battery of commonly used antibiotics of varying concentration to ascertain susceptibility profiles. 57 The increasing popularity of organic and natural food production systems has created an opportunity to study certain features of them; of particular interest is antimicrobial susceptibility of foodborne pathogens, and how they compare to those found in conventional food production systems. Though antimicrobial use is only 1 of several features making organic and natural food productions systems different from conventional food production systems, it will be our focus here. For poultry, it was found the Campylobacter coli isolated from cecal cultures of conventionally reared broilers ( standard and export ) had 81% and 90% tetracycline resistance respectively whereas 51% of Campylobacter coli from free range broilers were found to be tetracycline resistant. 82 Also, the Campylobacter spp isolated from conventionally reared broilers and turkeys were found to be 46% and 67% fluoroquinolone resistant, respectively, as compared to < 2 % in those poultry raised organically. 83 It was also found when selective media for ciprofloxacin resistant campylobacter was inoculated with poultry carcass swabs, the 2 conventional brands yielded 43% and 96% samples resistant while antibiotic free brands yielded 13% and 5% 27

41 samples resistant. 84 Similarly, 67% of the salmonella isolates cultured from fecal samples on conventional poultry facilities demonstrated multidrug resistance (3 or more classes of antimicrobials) compared to 11% of the salmonella isolates from fecal samples on pastured poultry. 85 For dairy, Campylobacter spp. cultured from the environment, feed and water, bulk tank milk, lagoon, and bird droppings on conventional dairies yielded 58.3% of isolates resistant to tetracycline as compared to 49.3% resistance from the campylobacter isolated from the same sources on organic dairies. 86 Furthermore, it was found conventional dairies were 7.5 times more likely than their organic counterparts to have at least 1 streptomycin resistant salmonella isolated from fecal samples. For swine, the frequency of tetracycline resistant Campylobacter coli recovered from feces and swabs of conventionally raised pigs was 83.4% while the frequency of erythromycin resistant C.coli recovered from conventional feces and swabs was 77%. By comparison, the frequency of tetracycline resistant C.coli recovered from antibiotic free feces and swabs was 56.2% while the frequency of erythromycin resistant C.coli recovered from antibiotic free feces and swabs was 34.5%. 87 Also, the percentage of resistant E.coil isolates was consistently lower in antibiotic free herds as compared to those herd raised conventionally : ampicillin resistance ABF 4.7%, con 30%, gentamicin resistance ABF 0%, CON 1.4%, oxytetracycline resistance ABF 41.6%, CON 87%, 28

42 sulfamethizine resistance ABF 61.2%, CON 77.5%. 88 Lastly, the Salmonella spp. cultured from conventional feces were found to have a greater percentage of isolates resistant to 9 of 10 antimicrobials as compared to the Salmonella spp. cultured from ABF feces. 89 Recently, 35 ABF swine farms were compared to 60 CON swine farms in terms of proportion of E. coli resistant to a panel of 14 commonly used antibiotics. It was found that CON farms had significantly greater proportions of E. coli resistant to ampicillin, chloramphenicol, tetracycline, and sulfamethoxazole that the corresponding ABF farms. 69 The 4 previous examples have been comparing the prevalence of antimicrobial resistance of some cultured indicator bacterial species of interest. The next 3 examples will discuss the prevalence of antimicrobial resistance by employing, either fully or in part, molecular techniques such as PCR and real time PCR as non-culture methodologies of evaluating samples. After isolation of tetracycline resistant E.coli on TBX agar supplemented with 8 9 incremental tetracycline concentrations, it was found by PCR the isolates derived from the feces of a conventionally reared sow had greater tetracycline resistance at 1.6 ug / ml (85.1%) compared to those of an antibiotic free sow (62%). Furthermore, the 29

43 greatest tetracycline concentration at which a resistant isolate from a conventional sample could grow (25.6 ug / ml) was greater than that of a resistant isolate from the antibiotic free sample (19.2 ug / ml). 90 When total DNA extracts from lagoon and groundwater samples at swine facility (A) and swine facility (C), each using chlortetracycline for treatment, prophylaxis, and growth promotion, were compared to one another by real-time qpcr it was found the tetracycline resistance gene tet (W) had 99.8% sequence homology, thus demonstrating transmission, either by antibiotic resistant bacterial migration or horizontal resistance gene transfer, from lagoons to groundwater. 91 DNA was extracted from 6 different swine facilities, 1 of their corresponding lagoons, 1 of their corresponding Ekokan upflow biofilter systems, and 1 of their high-rise hog manure handling systems. After 3 real-time PCRs were performed on the extracted DNA, though it was found the tet (G) abundance was lower yet not significant in composted manures when compared to those from fecal samples, house effluent, and lagoons, the abundance of tet (A), tet (C), tet (M), tet (O), tetb (P), tet (Q), tet (S), tet (T), and tet (W) were significantly lower than that of fecal samples, house effluent, and lagoons

44 Similarly, DNA was extracted from 10 pigs on 6 different swine facilities, 13 composted manure samples, 10 samples from 1 of their corresponding Ekokan upflow biofilter systems, and 6 samples from 1 of lagoons. After 6 real-time PCRs were performed on the extracted DNA, it was found that the abundance of erm (A), erm (B), erm (C), erm (F), erm (T), and erm (X), (genes conferring resistance to erythromycin and tylosin) were significantly lower in composted manure than that of fecal samples, house effluent, and lagoons. 93 When E.coli was cultured from the 1,263 rectums and cloacae of non-clinical humans, pigs, chickens, turkeys, sheep, cows, goats, cats, dogs, horses, geese, ducks, and deer, tetracycline resistance was found to range between 3% and 78%. Isolates from samples originating from domestic livestock and humans tended to have a greater percentage of resistance than isolates from samples originating from wild animals. This could be due to the fact that domestic livestock and humans tend to have more exposure to tetracyclines than wild animals. A multiplex PCR for 14 tetracycline resistance genes was performed on whole fecal DNA extracts from a cohort of calves raised conventionally and another cohort of calves raised without antimicrobials and it was found the calves with continuous exposure to tetracyclines carried a greater diversity of tetracycline resistance genes

45 It has been shown that resistance determinants have been shared between non pathogenic commensals and pathogenic bacteria 69 even as one may belong to a Gram negative genera and the other a Gram positive genera. 95 Not only do sentinel bacteria (pathogens) of interest harbor the ability to exhibit resistance and confer it to previously sensitive bacterial species, but so to do non pathogenic bacterial commensals. Often, antimicrobial resistance is reported as percentage of isolates exhibiting resistance, the number of resistant bacteria per gram of sample, or the proportion of individuals harboring resistant organisms. 96 Bacterial features such sample size, bacterial species, populations size, mutation rate, horizontal transfer rate, and relative survival of resistant strains can be variables associated with resistance. 97 In addition, host features such as signalment, contact rates, population size, management practices, and exposure to antimicrobials can also be variables associated with resistance. 98 The numerous sources of variability owe to the complexity of quantifying the amount of antimicrobial resistance in a given situation. In attempting to quantify the amount of antimicrobial resistance in a given situation, numerous considerations exist. Selection of samples from defined populations requires a framework to estimate resistance in the target population accurately and to enable statistical inferences to be made. 96 The complexity of measuring resistance has been recognized before 99,100 but few studies have attempted to quantify the effects of 32