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1 A review of studies submitted to CVM assessing the effects of sub-therapeutic use of
2 antimicrobial drugs on the Salmonella reservoir in food producing animals
3
4 Concern regarding the impact of antimicrobial drug use in animals on the potential
5 development of antimicrobial resistant zoonotic foodborne bacterial pathogens and
6 subsequent transmission to humans as food contaminants was first raised in the 1960’s. In the
7 1970s, the U.S. Food and Drug Administration (FDA) questioned the effect of feeding sub-
8 therapeutic levels of antibiotics to a large number of animals to promote growth. Prominent
9 scientists expected drug resistance to develop as a result of feeding antibiotics to animals
10 under this condition, and expected that these resistant organisms could cause human disease.
11 Scientists were also concerned that the use of certain antibiotics in food-producing animals
12 would promote an increase in the animal reservoir of Salmonella spp. through promotion of
13 cross colonization and infection, prolongation of the carrier state, and relapse of disease. The
14 1970 Antibiotic in Animal Feed Task Force (task force) was formed to address the safety and
15 efficacy issues associated with antibiotics administered in animal feed. The task force report
16 considered the issues of target animal safety, drug effectiveness, and potential public health
17 consequences.
18
19 The task force concluded that therapeutic use presented a small risk to public health and
20 therefore no pre-approval study requirement was imposed on products intended for such use.
21 The conclusion that therapeutic use of antibiotics is safe was based on the following points:
22
23 1. Therapeutic antibiotics used to relieve animal disease were believed to present a small
24 risk
25 because they are typically used at high dose for a short duration in young animals;
26 2. Benefits to the animal outweigh the potential risks to humans;
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27 3. It is necessary to use antibiotics in animals to relieve pain and suffering;
28 4. FDA recognized that healthy animals result in a safer food supply.
29 Scientists of the day argued that sub-therapeutic use created more potential for adverse
30 effects than therapeutic use because of the larger proportion of animals treated, and because
31 of how sub-therapeutic drugs are delivered to animals. A majority of food-producing animals
32 at that time were being fed antibiotics for sub-therapeutic uses such as growth promotion.
33 The antibiotics were given to the animals at levels that exposed bacteria to sub-lethal doses
34 for prolonged periods of time (therapeutic doses are generally high enough to inhibit or kill
35 most bacteria). The task force concluded that a significant effect of therapeutic antibiotic use
36 on pathogen load or antibiotic resistance among pathogens present at time of slaughter would
37 be a low probability event. Additionally, the task force suggested that the benefit to animal
38 health from therapeutic use of antibiotics outweighed the limited risk of adverse
39 microbiological effects on humans that such use might create. Therefore, pre-approval
40 studies were to be required to support the microbiological safety of antibiotics in food-
41 producing animals intended for sub-therapeutic uses only, including growth promotion and
42 feed efficiency.
43
44 21 CFR 558.15 studies
45 As a consequence of the 1970 task force report, requirements for data to address
46 microbiological safety concerns were outlined in the Code of Federal Regulations (21 CFR
47 558.15). Sponsors of antibiotic products were required to submit study results demonstrating
48 that their product did not promote bacterial drug resistance only if their product was intended
49 to be administered for greater than 14 days, for non-prescription use in animals. Sponsors
50 were required to submit all information to the Agency on the impact of their drug(s) on the
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51 “salmonella reservoir” in animals by specific dates depending on the class of drug. As
52 outlined in 21 CFR 558.15 (b)(1) – (b)(3), the following was to be accomplished or submitted
53 to the Agency:
54 1) By July 19, 1973 – records and reports of completed, ongoing, or planned studies,
55 including protocols, on the tetracyclines, streptomycin, dihydrostreptomycin, penicillin,
56 and the sulfonamides;
57 2) By October 17, 1973 - records and reports of completed, ongoing, or planned studies,
58 including protocols, on all other antibiotics;
59 3) By March 4, 1974 - records and reports of completed, ongoing, or planned studies,
60 including protocols, on nitrofurans;
61 4) By April 20, 1974 – data from completed studies on the tetracyclines, streptomycin,
62 dihydrostreptomycin, the sulfonamides, and penicillin assessing the effect of the
63 subtherapeutic use of these drugs in the feed on the salmonella reservoir in the target
64 animal as compared to that in nonmedicated controls;
65 5) By April 20, 1975 – data satisfying all other specified criteria for safety and effectiveness,
66 including the effect on the salmonella reservoir for any antibiotic or sulfonamide drug
67 approved for sub-therapeutic use in animal feeds;
68 6) By September 5, 1975 – data satisfying all other specified criteria for safety and
69 effectiveness, including the effect on the salmonella reservoir for the nitrofuran drugs
70 approved for sub-therapeutic use in animal feeds.
71
72
73 Study design
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74 The studies were conducted as a set that included a bacterial shedding study and a
75 bacterial resistance study. Protocols for these experiments were clearly defined and
76 controlled to avoid introducing bias. Both studies included a negative control group, and a
77 treated group. The negative control and treated groups were inoculated with a laboratory
78 strain of Salmonella typhimurium that possessed an identifying characteristic, commonly
79 nalidixic acid resistance. Additionally, the laboratory strain of S. typhimurium needed to be
80 free of transferable resistance elements.
81
82 Fecal and tissue Salmonella spp. were enumerated and susceptibility tested in the
83 shedding studies, and fecal Escherichia coli were tested for susceptibility in the resistance
84 studies. The studies generally lasted 8 weeks. The test animal was generally the same
85 species as that intended for the marketed product and the test animals were not required to be
86 near normal market or slaughter age.
87
88 The parameters used to determine whether antimicrobial drugs increased consumer
89 exposure to bacterial resistance or pathogen load were 1) drug effect on pathogen quantity,
90 prevalence, and duration of infection, and 2) drug effect on Salmonella spp. antibiotic
91 susceptibility. A second study was conducted to determine drug effect on resident E. coli
92 resistance. These parameters are described in the Center for Veterinary Medicine (CVM)
93 guidelines 18 and 19 titled Human Health Safety Criteria, and Animal Health Safety Criteria,
94 respectively. These guidelines are the product of the task force effort, and can be accessed
95 via the CVM internet site ( www.fda.gov/cvm ).
96 The determination of passing or failing was generally based upon statistical
97 differences
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98 between the treated group and the control group. Aside from measuring the drug effects on
99 shedding, tissue invasion, and resistance, other measurements and/or information were
100 collected to ensure the integrity of the study. These measurements included information
101 relevant to the following questions:
102
103 1. Did the product have antibiotic properties?
104 2. Did the study address cross-contamination and did it occur?
105 3. Were there sufficient numbers of animals in the shedding and resistance studies?
106 4. Did the animals consume enough drug to test the highest intended dose?
107 5. Was any other drug intentionally or unintentionally given to the animals during the study?
108 6. Was a natural strain of Salmonella present in animals, water, or feed to confound the test?
109 7. Did the test strain of Salmonella carry a distinct stable marker?
110 8. Was the strain capable of receiving resistance factors, and could it colonize in the animal?
111 9. Was the microbiology methodology appropriate and standardized?
112 10. What tissues were examined for Salmonella spp.?
113 11. How often were samples taken?
114 12. Were the studies performed long enough to detect an effect if an effect occurred?
115 13. What level of resistance in coliforms was present in the animals in the resistance study
116 prior to starting the study?
117
118 These integrity measurements were the basis for study rejection rather than failure. Minor
119 faults in the integrity measurements were usually justified as not affecting the outcome.
120 Results from pre-approval studies intended to address the 558.15 requirements
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121 CVM accumulated data from the shedding and resistance studies described above over
122 a 20-year period. A retrospective analysis of all the submitted data follows. The following
123 table describes results from the analysis according to the following pharmacological
124 categories. Since the results of individual products are proprietary information, the results are
125 summarized according to broad drug classes.
126
127
Drug class Total Drug:Animalb Passc Repeat Fail Reject
Macrolides/Lincosamides 9 4:3 4 3 2 3
Ionophores 13 7:3 8 3 2 4
Unclassified Gram+ drug 15 6:3 7 5 4 4
Streptogramins 1 1:1 1 0 0 0
Glycopeptides 2 2:1 1 0 0 1
Bambermycins 2 1:2 2 0 0 0
Broad spectrum drug 2 2:2 1 0 1 0
TOTALa 44 24 11 9 12
a
128 Total refers to total number of studies submitted
b
129 Drug:animal refers to the number of drugs tested in a drug class: number of animals species in which the drug
130 was tested
c
131 Pass, repeat, or fail refers to how FDA classified their conclusion of the study results
132
133 A total of 44 sets of resistance and shedding studies were submitted. Eleven of the
134 studies were repeats of previously submitted studies. In nine of the sets, one of the two
135 studies in the set failed to satisfy the safety criteria (statistically significant difference
136 between treatment and control groups with respect to numbers of bacteria shed). Two of the
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137 nine studies failed because of increased resistance in the resistance study, and the remainder
138 failed because of increased shedding in the shedding study.
139
140 Of the three studies where the spectrum of the drug matched the test organism's Gram
141 stain response, two of them failed because of increased resistance (one of the drugs was a
142 broad spectrum product tested in coliforms; the other drug was a Gm+ product, tested against
143 a Gm+ organism).
144
145 Two of the applications were never approved based on failure to meet the microbiological
146 safety criteria. Seven succeeded in satisfying the criteria by altering the conditions of use:
147
148 • One application was approved after the duration of treatment was shortened to 2 weeks.
149 • One application was approved after successfully repeating the study using a lower dose
150 (the application was approved with the lower dose).
151 • Two studies were repeated and the results were acceptable on the second attempt.
152 • Three studies were approved based on further information submitted by the sponsor.
153
154 Twelve of the studies were rejected for one or more of the following critical reasons (the
155 numbers in parentheses refers to the number of times this problem was identified):
156
157 • Salmonella susceptibility test results were not submitted (2);
158 • Coliform susceptibility test results were not submitted (3);
159 • Susceptibility test quality control was inadequate (3);
160 • Shedding lasted too long to measure prevalence/duration (2);
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161 • Environmental control animals were contaminated (4);
162 • Environmental control animals were not included (1);
163 • Animals used in the study failed to meet the 20% resistance baseline for inclusion (1);
164 • The data were too disorganized to interpret(2);
165 • The study contained too few animals(1).
166
167 Problems identified
168 Although information gained over 20 years experience was useful for determining how
169 many of the drugs tested impacted pathogen shedding, problems were identified during the
170 review of the studies, and included:
171 1. Of the studies submitted, 42 out of 44 were for Gm+ spectrum drugs. The spectrum of the
172 test drug matched Salmonella spp. and coliform bacteria in two study sets (they are Gm-
173 and as such are inherently resistant to the test drugs). For similar reasons, the study
174 design prevented the detection of transfer of resistance from E. coli to Salmonella spp.
175 Most studies demonstrated the transfer of resistance to test Salmonella from the enteric
176 flora, but the transfer occurred equally often in the control group without drug pressure.
177 None of the studies showed transfer of resistance to Salmonella due to a drug effect.
178 CVM believes this drug effect was not detected because Salmonella was inherently
179 resistant to the drugs being tested, i.e. there was no selective advantage to acquired
180 resistance.
181 2. Susceptibility test results provided limited information regarding a drug’s effect on the
182 naturally-occurring intestinal flora. Susceptibility testing was performed on isolated
183 species and may not have reflected changes in the susceptibility of the majority of
184 organisms in the gut population. Studies like these should ultimately seek to capture a
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185 picture of changes in the entire population regarding antibiotic resistance, before, during
186 and after a study for a particular antibiotic. By selecting a few isolates of a species for
187 testing there is a chance for “selective” reporting of desired MIC results, while ignoring
188 trends in changing susceptibility to the tested drug(s).
189 3. In order to measure drug effects, artificially high inocula were used. Differences detected
190 in the results may be artifacts created by the high numbers of organisms placed in the test
191 animals.
192 4. A laboratory strain of bacteria may not be representative of how naturally-occurring
193 Salmonella spp. respond when challenged by an antimicrobial drug.
194 5. Strict research conditions may not represent the actual “drug:bug” interaction that would
195 occur under normal animal management conditions. Animals in the studies are unusually
196 stressed because they must be maintained in isolation to avoid cross contamination, and
197 stress is recognized as an important factor in the response to disease challenge.
198 6. The studies typically tested a small number of animals, particularly in comparison with
199 the large numbers of animals in studies that assess effectiveness. The studies have too
200 few animals to be sure that the effect seen is really due to the drug, or even more
201 importantly, that an effect that is not seen is not one that will eventually have a substantial
202 public health impact.
203 7. The small size of the study creates environmental biases. Drug effects can be masked by
204 cross contamination when the sample size is small, and the animals in these studies were
205 housed separately in small groups to prevent cross-contamination. Consequently,
206 differences noted between treatment groups reflect environmental effects as well as drug
207 effects.
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208 8. Retrospective analysis suggests that antibiotic drugs affect pathogen load more than
209 resistance. Recall that 7/9 studies that failed did so because of increased shedding.
210
211 Summary
212 Twenty years of experience reviewing the “558.15 studies” described above revealed
213 that in most cases, failures could be attributed to apparent pathogen shedding effects. The
214 studies conducted on some drugs clearly demonstrated an increase in the shedding of bacteria
215 of public health concern. The retrospective analysis of these studies also served to highlight
216 the various problems associated with the design and conduct of such studies and the
217 intepretation of their results. Although questions have arisen regarding these studies, they
218 were designed and conducted within the constraints of policy and regulation at the time of
219 inception, and did yield some useful information. A consideration of the potential
220 microbiological effects of antimicrobial new animal drugs has been identified as a significant
221 component of the animal drug safety evaluation process. The lessons learned from the
222 “558.15 studies” along with 20 years of advancement in scientific knowledge, are important
223 factors for helping to develop appropriate methodologies for evaluating the relevant
224 microbiological effects associated with the use of antimicrobial drugs in food-producing
225 animals.
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