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CHAPTER 4 STUDY POSSIBILITIES The committee considered ways to remedy deficiencies in the information that has been used to support claims that the subther- apeutic use of antimicrobials in animal feeds creates a hazard to the health of humans. The studies suggested in this chapter are not to be interpreted as proposals of work that would provide a sufficient basis for an acceptable quantitative assessment of any risk to human health since the remaining gaps in knowledge would still have to be bridged by conjecture or speculation. ~ ~ Rather, the committee believes these studies to be the most fruitful approaches to quantifying some of the stages in the chain of events from which health hazards might result (see Chapter 2) so that speculations concerning such hazards may be more firmly based. Study 1 should identify relative contributions of subthera- peutic and therapeutic antimicrobial regimens to the emergence of resistant enteric flora in animals. Studies 2 and 3 are designed to assess the extent to which carriage by humans of bacteria having R factors is associated with meat consumption or occupational exposure to bacteria from animals in abattoirs. Study 4 addresses the relationships between carriage of or occupational exposure to Rib organisms and increased morbidity from urinary tract infections. Each proposal points out the limitations of the study and indicates what conclusions can be drawn from the data to be collected. Before these studies commence more detailed protocols should be evaluated by groups of individuals with expertise in the disciplines that are relevant to each study. STUDY 1--THE EFFECTS OF SUBTHERAPEUTIC AND THERAPEUTIC DOSES OF ANTIMICROBIALS ON THE PREVALENCE OF R+ ENTERIC ORGANISMS IN ANIMALS . In this study, the relationship between the appearance of resistant Enterobacteriaceae and the pattern and dose of antimicro- bials used in the feed of food animals or otherwise administered to them could be examined. The proportion of E. colt, salmonellae, and other Enterobacteriaceae carrying R plasmids should be measured in beef cattle, ~ / . . ~. . ~ hogs, and chickens before, during, and after they are red anchor created with various doses of tetracycline at growth pro- motional, prophylactic, and/ or therapeutic levels. This study will clarify the contribution of subtherapeutic and therapeutic dosing regimens to the emergence of resistant enteric flora in animals. 35

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36 Experimental Study Design The committee regards the ability to obtain animals +th a very low, preferably zero, incidence and proportion of R en- teric bacteria in their gut flora as central to the usefulness of this study. Alternative strategies, which could be adopted if it proves impossible to meet this criterion, are discussed under Interpretation of Results. Within each animal species, individual animals not pre- viously exposed to ant~microbials should be allocated randomly to pens, and the pens should be assigned randomly to treatment groups, each of which contains more than one pen of animals. The number of animals in each pen and the number of pens may differ along the three animal species and should be specified so that the samples are of sufficient size for investigators to detect meaningful differences in the proportions of animals carrying tetracycline-resistant organisms among the various treatment groups. Prior to random allocation, several fecal samples should be collected from each animal to establish the baseline for total flora, for the prevalence of R+ organisms, and for the rate at which such pathogens as Salmonella are shed. Fecal specimens should be examined for a short period after penning in order to monitor exchange of bacteria among animals in each pen. Speci- mens should be examined at regular intervals thereafter to moni- tor the changes occurring during the feeding period. Bacterio- logical procedures should be determined on the advice of persons with expertise in the field of veterinary microbiology. Animals should be divided into treatment groups as follows: 1. No antimicrobials. These animals should be given no antimicrobials of any sort during the feeding period. 2. Subtherapeutic levels of tetracyclines. These animals should receive tetracyclines for a period similar to that during which the antimicrobials are administered during typical rearing conditions for that species. Three dose levels should be given to different groups: a. A level no greater than the minimum regarded as necessary to elicit growth promotion or more effi- cient feed conversion. b. The usual level of antimicrobials fed to the particu- lar animal species for growth promotion, improvement of feed conversion, and disease prophylaxis.

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37 c. A level substantially above the usual feeding level but not above regulatory definition of a maximum subtherapeutic dose. 3. Therapeutic doses of tetracycline. These animals should not receive subtherapeutic antimicrobials. A simulated typical course of tetracycline therapy should be adminis- tered to the group at a specified time during the feeding period in accordance with accepted veterinary practices for the test species. If it is decided that tetracycline should be given to only some animals in the group, this must be recorded. 4. Subtherapeutic plus therapeutic doses of tetracyclines. These animals should be fed the usual subtherapeutic levels (2b) of tetracycline during the entire feeding period and should be treated with a simulated therapeutic course of tetracycline, thus combining 2b and 3 above. In the event of sickness requiring the use of therapeutic doses of antimicrobials in any treatment group, all animals judged (on predetermined criteria) to be sick should be permanently removed from the experiment and from contact with the remaining test animals and should be treated as indicated by prudent veterinary practice. Antimicrobials should be discontinued in all animals prior to slaughter at the time designated by current regulations to en- sure that residual levels do not exceed those permitted in carcasses. Monitoring of R+ bacteria should be continued in some animals for a period after the animals +onmally would have been slaughtered to observe all changes in R prevalence after antimicrobials are withdrawn. Design of data acquisition. The design of the quantitative culture techniques should enable investigators to detect a biologically important difference in the number of tetracy- cline-resistant organisms in fecal specimens from the various treatment groups. These techniques should be designed by per- sons with expertise in microbiology and in biostatistics as it relates to the analysis of microbiological data. The protocols and analytical methods should be specified in advance. Data to be collected: 1. The number of animals in each group carrying Enterobacteria- ceae with R factors mediating tetracycline resistance should

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38 2. be recorded. For each stool specimen, the Enterobacteria- ceae should be enumerated and serotyped, and the percentage carrying R factors mediating resistance to tetracycline should be ascertained by quantitative culture techniques. Salmonellae shed by each animal should be enumerated and serotyped. 3. The weight of each animal should be recorded at the beginning, at the end, and at regular intervals during the experiment. Feed should be analyzed chemically for a range of antimi- crobials prior to use to avoid contamination and to ensure that the desired levels of tetracycline are achieved when supplementation is intended. Contaminated feed should be discarded to avoid the selection (by antimicrobial agents other than tetracycline) of organisms carrying plasmids on which resistance to tetracycline is linked to other resis- tances. The feed consumed in each pen should be recorded, and the consumption of antimicrobials by individual animals should be calculated. Site conditions: One or more sites, such as an agriculture ex- per~ment station or veterinary college, should be selected. They should enable investigators to meet the following criteria: 1. Test animals should be me intained in a controlled experi- mental environment, but they should be handled in a manner that simulates current growing and finishing practices before marketing. 2. The sites should contain isolation facilities to prevent transfer of bacteria among pens. Different personnel will be needed for each pen to prevent cross-infection between pens. 3. Direct veterinary supervision during the course of the experiment should be available. There should be local slaughtering facilities in which the bacteriological characteristics of carcasses produced during this experiment can be monitored.

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39 Observational Studies These studies require the cooperation of commercial operations that use antimicrobials in a pattern similar to some or all of those used in the experimental study design described above. Fecal samples and feed samples should be collected, and the feeding history and weight gains of the animals throughout the feeding period should be recorded. Fecal samples should be analyzed microbiologically with the same methods that were proposed in the experimental study design. The data obtained from these groups should be analyzed in a manner that will enable investigators to compare the appearance of R+ organisms in these groups to that of the groups in the experimental study design. Costs The numbers of animals used in the following calculations are for illustrative purposes only. They should not be interpreted as the committee's recommendation for quantities needed to obtain statisti- cally meaningful results. Investigators should determine the number to be used in accordance with the principles indicated in the study design outlined above. Animals should be observed past the usual mar- keting time in order to assess the full consequences of antimicrobial withdrawal on the microbial flora. The costs of acquiring specimens are included under Veterinary Supervision estimates. Partial costs may be recovered by sale of the animals.

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40 EXPERIMENTAL STUDY: BOVINES - Costs are based on six groups of 20 animals. Purchase of Animals: In order to acquire animals with known antimicrobial expo- sure histories, it may be necessary to pay more than the going market rate, which varies with time. Acquisition of 120 400-lb calves at approximately $1.25/lb will cost: 120 x 400 lb x $1.25/lb Feed and Care Costs: 120 x 240 days x $5.00/day Veterinary Supervision (2 h/day): 2 h x 240 days x $30/h Animal Husbandry: Total Direct Cost = =- $ 60,000 = 144,000 - 14,400 Laboratory Services (based on one specimen/animal every 3 days): 80 specimens/animal x 120 animals x $25/specimen Total Direct Costs Overhead (estimated 50%) $218,400 240,000 458,400 229,200 BOVINES, TOTAL COST $687,600

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41 EXPERIMENTAL STUDY: SWINE - Costs are based on s~x groups of 20 animals. Purchase of Animals: Acquisition of 120 specific pathogen-free animals mately 4 weeks old, will cost: 120 x $180/an~mal Feed and Care Costs: 120 x 180 days x $2.00/day Veterinary Supervision (2 h/day): 180 days x 2 h x $30/h Animal Husbandry: Total Direct Cost = , approxi = $ 21,600 = 43,200 = 10,800 Laboratory Services (based on one specimen/animaI every 3 days): 60 specimens/animal x 120 animals x $25/specimen Total Direct Costs Overhead (estimated 50%) SWINE, TOTAL COST $75, 600 180,000 $255, 600 127,800 $383, 400

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42 EXPERIMENTAL STUDY: CHICKENS Costs are based on six groups of 50 chickens. Purchase of Animals: Acquisition of 300 specific pathogen-free chicks will cost: 300 x $15 Purchase cost may be lower if chicks are reared from eggs rather than obtained already hatched. Feed and Care Costs: 300 chickens x $0.50/day x 100 days Veterinary Supervision (2 h/day): 100 days x 2 in/day x $30/h Anima1 Husbandry: Total Direc t Cost = $ 4,500 = 15,000 = 6,000 = Laboratory Services (based on one specimen/animal every 3 days ): 34 speci~nens/animal x 300 animals x $2 5/specimen Total Direc t Cost Overhead ~ estimated 50%) s CHICKENS, TOTAL COST $ 25,500 255,QOO 280,500 140,250 $420,750

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43 TOTAL COSTS - STUDY PROPOSAL 1: EXPERI~:NTAL STUDY Bovines Swine Chickens $687, 600 383, 400 420, 7 50 $1, 491, 750 Any costs recovered from the sale of the animals could be returned to the contractor. OBSERVATIONAL STUDY Cost for the observational study will be dependent upon the arrange- ments that can be made with those commercial concerns willing to cooperate in the study and the sizes of the groups to be observed.

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44 Interpretation of Results There are a number of possible results from this study: Animals receiving antimicrobials may have no greater preva- lence of R+ organisms and no more Salmonella with pathogenicity for humans than animals not receiving antimicrobials. This is an unlikely result, given data to the contrary. However, if such a result did occur, there would be no evidence to indicate that the subtherapeutic use of antimicrobials might affect human health. The prevalence of R+ organisms or Salmonella with pathogeni- city for humans may be similar in animals on subtherapeutic regi- mens of antimicrobials and those on therapeutic regimens, and these prevalences might be greater than that in the untreated animals. In this instance there would be no evidence to indicate that sub- therapeutic use of antimicrobials increases the possible hazard to humans over that from the therapeutic use of antimicrobials. If the prevalence of either R+ organisms or of pathogenic Salmonella resulting from the subtherapeutic use of antimicro- bials significantly exceeds that from therapeutic use, then one still cannot infer that the subtherapeutic use of antimicrobials represents a definite risk of human disease. Further studies would be required to measure the effects on human health resulting from increased prevalence in animals of R+ organisms or of Salmonella with pathogenicity for humans. If animals with al low initial prevalence of R+ enteric orga ~ obtained, the study ~- nisms cannot be o DEainea ~ Ene scuay snouts De moored to Determine if the feeding of strictly monitored antimicrobial-free feed results in a decline in R+ prevalence. The validity of such conclusions depends upon the degree to which the experimental conditions, antimicrobial regimens, etc., actually parallel production practices. This can be ascertained by including the observational study in the overall design. STUDY 2--STUDIES OF VEGETARIANS AND NONVEGETARIANS This study would measure the extent to which carriage of bacteria with R factors is associated with meat consumption. It begins with the hypothesis that consumption of meat contaminated with antimicrobial-resistant bacteria from animals would result in meat-eaters having a higher prevalence of antimicrobial-resistant enteric bacteria than do vegetarians. Two related studies are described in Chapter 3.

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45 Study Design Groups of vegetarians and nonvegetarians should be compared for prevalence of resistant Enterobacteriaceae in their fecal flora. The groups must be carefully controlled for factors such as antimicrobial usage, age, socioeconomic status, family size, age of children, and pets. Seventh Day Adventists and Mormons would provide two convenient study groups. In this study the pre- valence of R+ factors is monitored by the use of resistance to tetracycline because of its likely frequency in the population and its therapeutic significance. Moreover, it is the subject of the proposed restrictions by the Food and Drug Administration (FDA). Data to be Collected . The number of persons in each group carrying Enterobacteria- ceae with R factors mediating resistance to tetracycline should be determined. The sample size should be sufficiently large for investigators to identify a meaningful difference, if it exists, in the rate of colonization by organisms with resistance to tetra- cycline. The percentage of Enterobacteriaceae that carry R factors mediating resistance to tetracycline in each fecal specimen should be ascertained by quantitative culture techniques. These techniques should be designed to detect a biologically important difference in the prevalence of tetracycline resistance factors in stool specimens obtained from the groups being compared. Costs A preliminary survey should be conducted to ascertain approx- imate prevalence rates of R+ carriage. A pilot study of 100 vege- tarians and 100 nonvegetarians would cost approximately $100,000 for epidemiological and laboratory services. The size and desirability of the full survey should be decided on the basis of the results of the preliminary survey. Interpretation of Results If the prevalence of organisms with R factors is the same in vegetarians as in meat-eaters, there would be no support from this study for the belief that either the therapeutic or subther+peutic use of antimicrobials in animals affects human health via R orga- nisms on or in meat.

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46 If meat-eaters have a greater prevalence of R+ bacteria than do vegetarians, it is possible to infer that the excess of antimicrobial-resistant bacteria is associated with the ingestion of meat, but it is not possible to differentiate between the effects of subtherapeutic and therapeutic uses of ant~microbials in meat sources. Other confounding variables would include the handling of meat products and contamination of cooking utensils + or work surfaces. If meat-eating is associated with a greater R prevalence, then further studies would be needed to determine if the excess of R+ organisms results in excess morbidity or mor- tality or complicates the treatment of diseases and to determine the influence on R+ prevalence of other aspects of diet. STUDY 3--STUDIES OF ABATTOIR WORKERS, THEIR FAMILIES, AND NEIGHBORHOOD CONTROLS This study would measure the extent to which occupational exposure of humans to bacteria from animals is associated with carriage by humans of bacteria with R factors. Secondarily, it would gauge the spread of these bacteria or R factors among humans who are-in close contact with one another. Abattoir workers are exposed to large numbers of bacteria frog animals but are not exposed to antimicrobial-containing feeds. Consequently, they are exposed to the organisms (which are likely to be resistant in animals fed antimicrobials), but unlike farm workers they are not exposed to dusts containing antimicrobials that might be ingested or inhaled, thereby exerting a selective pressure favoring resistant enteric or respiratory tract bacteria. Thus, abattoir workers can be used productively to evaluate the propensity of bacteria from animals to colonize humans and to study the secondary spread of such bacteria and/or R factors via contact spread to family members. If secondary spread occurs, family members would be expected to have an inter- mediate but increased R factor carriage rate compared to controls. Study Design Groups of abattoir workers involved in the processing of poultry, pork, and cattle, their household contacts, and+neighbor- hood controls should be compared for the prevalence of R Entero- bacteriaceae in their stool flora. The groups must be carefully controlled for factors such as antimicrobial usage, age, socio- economic status, family size, age of children, and household pets. In this study the prevalence of R factors is monitored by the use of resistance to tetracycline as a marker for the same reasons given under Study 2.

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47 Data to be Collected The number of persons in each group carrying Enterobacteria- ceae with R factors mediating resistance to tetracycline should be determined. The population groups studied should be large enough to provide a high probability for identifying a meaningful differ- ence, if one exists, in the carriage rate for the tetracycline resistance factor. Information on the task performed by the abat- toir workers should be collected in order to determine their potential for exposure to enteric organisms. For each stool specimen, quantitative culture techniques should be used to determine the percentage of Enterobacteriaceae that carry R factors mediating resistance to tetracycline. These techniques should be designed to detect a biologically important difference in prevalence. Costs A preliminary survey should be conducted to ascertain approxi- mate prevalence rate& of R+ carriage. The rates found in such a survey would determine the size and desirability of a full survey. A pilot study of 100 abattoir workers and 100 controls would cost approximately $100,000 for epidemiological and laboratory services. The size and desirability of the full survey should be decided on the basis of the results of the preliminary survey. Interpretation of Results . By comparing rates at which R+ bacteria are carried by abattoir workers with those of their families and neighborhood controls, one can determine if any association exists between occupational exposure to bacteria from animals and an increased prevalence of R factors in the human enteric flora. Such comparisons would also allow one to evaluate spread of R factors among humans in close contact with one another. If the prevalence of organisms with R factors is the same in abattoir workers and controls, this study would provide no support for the belief that resistant bacteria, resulting from either the therapeutic or subtherapeutic use of antimicrobials in animals, significantly affects the flora or health of humans. It would not be possible to attribute a greater prevalence in the abattoir workers solely or in part to the subtherapeutic use of antimicrobials in feeds. Further studies would be needed to determine

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48 if any observed excess of R+ organisms in abattoir workers resulted in excess morbidity or mortality or in complications in the treat- ment of diseases. RATIONALE FOR CONDUCTING STUDIES OF MORBIDITY AND MORTALITY IN HUMANS The three studies described above are intended to serve as indicators of the development and transfer of resistant enteric organisms. If there is no indication of an important association between the exposure of animals to either subtherapeutic or thera- peutic levels of antimicrobials and the development of resistant enteric organisms, or if exposure to bacteria from animals fails to influence the flora of humans, the possibility of detrimental effects on human health would not be sufficiently well established to justify widespread changes in the current use of antimicrobials throughout the meat industry. If either the vegetarian study or the abattoir study indicates that the carriage of resistant orga- nisms by humans is associated with meat consumption or occupational exposure to bacteria from animals, more extensive evaluations of morbidity and mortality would be justified. One possible study is detailed below. STUDY 4--COMPARISON OF CONTROLS WITH SUBJECTS WITH URINARY TRACT INFECTION Laboratory screening services frequently discover urinary tract infections (UTI) by culturing urine samples obtained from women during routine medical examinations. These screenings can be linked with other microbiological tests to determine the existence of an association between R+ Enterobacteriaceae in stool flora and the occurrence of primary UTI and to ascertain the proportion of primary UTI infection that is caused by antimicrobial-resistant Enterobacteriaceae. Brumfitt et al. (1971) have conducted a small study of these topics. Study Design If the results of Studies l, 2, and 3 indicate that more extensive evaluations of morbidity and mortality are necessary, the committee recommends that a screening for primary UTI be con- ducted on 5,000 females who work in the meat processing industry (e.g., in poultry dressing plants) and who thus are exposed to high levels of antimicrobial-resistant bacteria. If the screening

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49 can be conducted in a processing plant that can provide reliable data concerning the antimicrobials received by the birds that are processed, the antimicrobial-resistant profiles of the flora of the poultry carcasses should be compared with those of the isolates from humans. Such additional sampling would add to the costs of the study but would provide exceedingly useful corrobor- ative information. In conjunction with the screening of meat processors, an equally large group of controls should be studied, e.g., the 9,000 women in East Boston, Massachusetts first studied by Kass (1978) could be resurveyed. These surveys would enable investigators to compare female meat processors who have bacteri- uria with similarly infected women in an urban setting far removed from contact with livestock and to match cases and controls for age, race, parity, and antimicrobial history. Participating screening services should use uniform procedures and criteria to detect UTI's. New cases should be asked to return for a confirmatory urinary culture, at which time a rectal swab should be obtained to determine the carriage of R+ Enterobacteria- ceae. Women who have not received antimicrobials during the preced- ing calendar year should then be matched with control (non-UTI) women of comparable age, race, parity, and negative antimicrobial history as determined by the screening service. For each stool specimen from a case or control, all Enterobacteriaceae should be serotyped and the percentage with R factors should be ascertained by quantitative culture techniques. The number of cases and con- trols required to discriminate between no association and a meaning- ful difference at acceptably low probabilities for error must be specified in a detailed protocol. The control population of 9,000 should yield approximately 150 cases of bacteriuria, approximately 100-120 of which will be infected with E. cold (Kass, 1978~. The role of resistance selected by the therapeutic use of antimicrobials in humans should be rigorously investigated and controlled in this study. Even a negative result with a large group would enable investigators to estimate the upper bound of the risk to human health. All confirmed cases of UTI should be studied in the following manner. 1. Each isolated infecting strain should be tested for antimicrobial susceptibility patterns, type distribution, and plasmid DNA sequence homology.

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mine: 50 2. Each patient should be sent a questionnaire to deter Basic demographic data Basic household composition Identification of infection as a sporadic case or part of an outbreak Occupation of patient, spouse, and household contacts Outcome of illness--expense in terms of workdays lost, medical expenses, etc. Present health status of subject Antimicrobial history of subject and household members and pets during past 2 years Hospitalization history of subject during past 2 years Major illness of subject and household members during past 2 years Patients with UTI characterized by bacteria with resistance to antimicrobials should be compared with patients with UTI characterized only by antimicrobial-sensitive bacteria to deters mine whether there are differences in antimicrobial history, in exposure to animals or carcasses that had been in contact with antimicrobials, or in exposure to feeds containing antimicrobials. Interpretation of Results If women with resistant enteric flora have a relatively high risk of a UTI compared to those without resistant fecal flora, the prevalence of R+ fecal flora among new cases should be greater than that among comparable controls. The observation of such an + association would favor the interpretation that the carriage of R organisms, from whatever source, is indicative of an elevated risk of UTI. An insignificant association would indicate that the pre- sence of R+ enteric flora does not make an important contribution to this form of morbidity. This study would be sensitive to a differential virulence between resistant and susceptible enteric flora causing UTI's, but it would not elucidate the contribution of

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51 the subtherapeutic use of antimicrobials in animal feeds to this cause of illness. Whatever the results of this study, they would not provide justification for drawing general conclusions about the likely changes in the virulence of other pathogens gaining resistance to antimicrobials. The comparison of UTI prevalence between groups with high and low exposure to R+ enteric organisms would provide some indication of the amount of primary UTI that is attributable to an occupational exposure. With a sufficiently large control group and high risk + groups in proximity to animals or carcasses with high levels of R organisms, a positive result would enable some conclusions to be drawn about total antimicrobial use in animals and UTI in humans caused by antimicrobial-resistant bacteria. No conclusion could be drawn about the relative contribution of subtherapeutic use to the increased morbidity. Costs If such a study were conducted over 2 years, which would allow time for training of personnel and analyzing results, an estimated $150,000 per year would be required. If the study were spread over 3 years, the total annual cost would be reduced by approximately 15%. If the information concerning the ant~microbials received by poultry passing through the processing plant can be obtained and the flora of the carcass is subsequently sampled, then add~tional~costs will be incurred.