Retail poultry products are known sources of antibiotic-resistant Escherichia coli , a major human health concern. Consumers have a range of choices for poultry, including conventional, organic, kosher, and raised without antibiotics (RWA) – designations that are perceived to indicate differences in quality and safety. However, whether these categories vary in the frequency of contamination with antibiotic-resistant E. coli is unknown. We examined the occurrence of antibiotic-resistant E. coli on raw chicken marketed as conventional, organic, kosher and RWA. From April – June 2012, we purchased 213 samples of raw chicken from 15 locations in the New York City metropolitan area. We screened E. coli isolates from each sample for resistance to 12 common antibiotics. Although the organic and RWA labels restrict the use of antibiotics, the frequency of antibiotic-resistant E. coli tended to be only slightly lower for RWA, and organic chicken was statistically indistinguishable from conventional products that have no restrictions. Kosher chicken had the highest frequency of antibiotic-resistant E. coli , nearly twice that of conventional products, a result that belies the historical roots of kosher as a means to ensure food safety. These results indicate that production methods influence the frequency of antibiotic-resistant E. coli on poultry products available to consumers. Future research to identify the specific practices that cause the high frequency of antibiotic-resistant E. coli in kosher chicken could promote efforts to reduce consumer exposure to this potential pathogen.

© 2013 Millman JM et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).

Grant information: This work was funded by the Merriam-Powell Center for Environmental Research and the Ecosystem Science & Society Center at Northern Arizona University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Introduction The use of antibiotics in livestock production may pose health risks to humans, as such usage has been correlated with the occurrence of antibiotic-resistant bacteria isolated from human infections1,2. Methods of livestock production differ in antibiotic use, and this can influence the frequency of antibiotic-resistant bacteria on retail meats. For example, antibiotic-resistant Escherichia coli has been shown to be less common on poultry raised without antibiotics (RWA) as compared to poultry raised conventionally3. Likewise, organic poultry can have lower frequencies of antibiotic-resistant bacteria than poultry raised conventionally4–10, although this is not always the case11–13. Organic, RWA, and kosher food products supply a growing market niche14. Consumers perceive that they offer health benefits14–21 and are willing to pay a premium for them22–24. The actual health benefits of organic food are not always clear25, and the health benefits of kosher foods are largely anecdotal. Little is known about the frequency of antibiotic-resistant microorganisms on kosher products. The organic and RWA labels require specific production methods as stipulated in US federal regulations, whereas the kosher label adheres to religious requirements that are regulated privately. The RWA label requires that “livestock have never received antibiotics from birth to harvest”26. The United States Department of Agriculture (USDA) organic standard is only slightly less strict, stipulating that “The producer of an organic livestock operation must not sell, label, or represent as organic any animal or edible product derived from any animal treated with antibiotics”, but also that “Poultry or edible poultry products must be from poultry that has been under continuous organic management beginning no later than the second day of life”26,27. Therefore, injecting antibiotics into eggs or administering them during the first 24 hours of the chick’s life will not violate the letter of the USDA organic standard28,29. Kosher production differs from organic and RWA in that it is inherently predicated on religious requirements. For kosher meat, the major requirements are that it must be from animals that have split hooves and chew their cud, it must not be mixed with dairy products, and all equipment used must be used exclusively for kosher food19. Animals must be slaughtered “humanely”, and meat is typically salted to remove blood rapidly, a practice that has been shown to reduce the microbial load30. Unlike for organic and RWA, kosher poultry is not regulated by Federal laws but rather by private certification organizations, and thus the specific practices vary19. Here, we compared four major types of poultry-conventional, kosher, organic, and RWA-in order to assess the frequency of contamination with antibiotic-resistant E. coli. We focused on poultry products from a major metropolitan center (the greater New York City area) and products available to typical consumers by studying multiple brands of chicken from multiple stores. Our goal was to compare the frequency of antibiotic-resistant E. coli in these four categories of chicken.

Methods Sample collection During April–June 2012, raw chicken was purchased from supermarkets, butcher shops, specialty stores, and food distributors in the greater New York City area. A variety of widely available brands were procured in four categories: conventional, kosher, organic and RWA. Within each category of chicken purchased, we collected at least four samples of each brand. Some samples included more than one category (e.g., kosher and organic). Five collections occurred resulting in 213 total samples. Samples were drumsticks or samples from which drumsticks were removed for analysis (all with skin). After purchase, each chicken sample was placed in a labeled, ziplock bag, and placed in a cooler with ice packs. Three coolers with ice packs were shipped overnight to T-Gen North within two days of collection. Laboratory analyses Chicken samples arrived at the laboratory in their original packaging and were refrigerated at 4°C until processed. One putative E. coli strain was isolated and screened from each sample using standard methods for assaying for antimicrobial resistance described by the Clinical and Laboratory Standards Institute (CLSI)31. The use of one strain per sample enabled efficient testing among a population of chicken samples for differences in the frequency of antibiotic resistance. One whole drumstick was selected from each package or removed from each whole chicken sample using a sterilized knife. Each sample was transferred aseptically to a Stomacher Bag (VWR, Radon, PA, USA, catalog number 11216–902) containing 250 ml MacConkey broth (Alpha Biosciences, Baltimore, MD) and agitated at speed 7 for 3 min on a rocking platform shaker (VWR, Radon, PA, USA, model no. 40000–302) and incubated overnight at 44°C. A 10 μl loop was used to inoculate a VRBA+MUG (Teknova, Hollister, CA) plate with the enriched broth. The plate was incubated at 37°C for 2 h and then at 44°C for 22 h, along with QA/QC strains ATCC E. coli 35218, Klebsiella pneumoniae, Hafnia alvei, Citrobacter freundii and Serratia plymuthica. QA/QC strains not listed as ATCC were isolated and identified using the BD Phoenix at Flagstaff Medical Center. From each VRBA+MUG plate, four putative E. coli colonies were streaked to CHROMagar (Hardy Diagnostics, Santa Maria, CA) and incubated 20 to 24 h at 37°C. One putative E. coli colony, appearing pink to rose, was streaked to a second CHROMagar plate and incubated 20 to 24 h at 37°C. For each sample, a putative E. coli isolate was inoculated into an assigned well of a 96-well plate containing 75 µl of Tris EDTA (TE) buffer. DNA was released from cell suspension with a thermal cycler (Bio-Rad, Hercules, CA) using the following parameters: heated lid, 95°C; block temperature, 90°C for 15 min. To confirm the identity of putative E. coli isolates, a uidA qPCR assay and a universal bacterial qPCR (BactQuant32) were used. For each reaction, 2 μl of DNA was added into 8 μl of master mix, with the final reaction containing 1.8 μM of each forward and reverse uidA primer, 0.25 μM uidA- VIC probe, 0.90 μM of each forward and reverse Pan16S primer, 0.25 μM Pan16S-FAM probe, 1X QuantaPerfeCTa® Multiplex qPCR SuperMix w⁄ROX (Quanta Biosciences, Gaithersburg, MD) and molecular-grade water. All samples were run in triplicate and each experiment included a standard curve and no-template controls. The 7900HT Real-Time PCR System (Applied Biosystems, Carlsbad, CA) was used to run the reactions with following conditions: 3 min at 50°C for UNG treatment, 10 min at 95°C for Taq activation, 15 s at 95°C for denaturation and 1 min at 60°C for annealing and extension × 40 cycles. Six isolates were excluded from further analysis because they were not confirmed as E. coli using the qPCR assay. Guidelines from the Clinical and Laboratory Standards Institute (CLSI) for disk diffusion methods31 were used to test each strain for resistance to antibiotics. Some strains did not grow under assay conditions (n=23) and were excluded from further analysis. Twelve antibiotics were tested, representing seven classes of drugs: tetracycline (class, tetracyclines); ampicillin and ampicillin sulbactam (class, penicillins); cefazolin, cefoxitin, and ceftriaxone (class, cephalosporins); gentamicin and amikacin (class, aminoglycosides); nalidixic acid and ciprofloxacin (class, quinolones); trimethoprim sulfamethoxazole (class, folate pathway inhibitors); and imipenem (class, carbapenems) (VWR, Radon, PA). Breakpoint guidelines from the CLSI M100 Tables 2A through 2J for E. coli31 were used to classify strains into “resistant”, “intermediate” or “susceptible”; designations of “intermediate” were lumped with “resistant” for purposes of statistics and inference, a conservative approach with respect to consumer safety. Statistical analyses Analysis of variance (ANOVA) was used to test whether antibiotic resistance varied among the brands of chicken sampled, using SYSTAT 13.1. Effects of brand within each category were tested (i.e., using all the data within conventional, organic, kosher, RWA). For each drug, Microsoft Excel for Mac Version 14.1.0 was used to conduct chi-square tests to determine whether the frequency of resistance varied among categories of chicken: conventional, organic, kosher and RWA. The total number of drugs and drug classes to which each strain was resistant were enumerated. One-way ANOVA was used to compare the average number of drugs to which strains were resistant among categories, using samples with only one category designation (n=120). This test captures the effect of a consumer’s choice whether to purchase chicken in one category over another on the likelihood of exposure to antibiotic-resistant E. coli. Multi-factor ANOVA was used to test whether trends held across the broader dataset (n=184), including samples with multiple category designations. The collection of samples included adequate replication (>14) for every possible two-way combination of labels (organic & kosher, RWA & organic, and RWA & kosher). Replication for the three-way combination (organic, kosher & RWA) was low (n=5), and all samples were from one brand. To avoid bias, these samples were excluded from the ANOVA. Each of the three labeling categories was included as a factor in three-way ANOVAs (organic, RWA, and kosher, each with two levels), with the number of drugs and drug classes exhibiting resistance as response variables. This tests for the effect of each category and for interactive effects of combining categories.