EPIDEMIOLOGY

MICROBIAL DRUG RESISTANCE Volume 00, Number 00, 2015 ª Mary Ann Liebert, Inc. DOI: 10.1089/mdr.2015.0061

Antibiotic and Disinfectant Resistance of Escherichia coli Isolated from Retail Meats in Sichuan, China Anyun Zhang,1,2,* Xuemei He,1,3,* Yue Meng,1 Lijuan Guo,1,3 Mei Long,1,3 Hua Yu,4 Bei Li,1 Liangqian Fan,1 Shuliang Liu,5 Hongning Wang,2 and Likou Zou1,3

To demonstrate the resistance of antibiotics and disinfectants to Escherichia coli isolates, 255 E. coli strains were isolated from 328 retail meat samples in this study. Susceptibility testing results showed that 85.5% isolates were resistant to at least one antibiotic drug. The E. coli isolates showed the highest resistance to sulfamethoxazole (61.6%), followed by tetracycline (61.2%), ampicillin (48.2%), cefalotin (29.8%), and kanamycin (22.4%). The minimum inhibitory concentrations of the disinfectants cetyltrimethylammonium bromide, N,N-didecyl-N,N-dimethylammonium chloride, cetyltrimethylammonium bromide, and cetylpyridinium chloride for E. coli were 16–1,024, 4–1,024, 16–512, and 8–512 mg/L, respectively. The emrE, ydgE/ ydgF, mdfA, and sugE(c) genes were commonly present (53.7–83.1%), but the qac and sugE(p) genes were less prevalent (0.0–14.9%). The qac genes were highly associated with antimicrobial resistance. Conjugative transfer experiment indicated that the disinfectant resistance genes, qacF, sugE(p), and qacED1, were located on conjugative plasmids. Pulsed-field gel electrophoresis revealed that the antimicrobial-resistant isolates were associated with the sampling supermarkets or groceries. This study indicated that using quaternary ammonium compounds to decontaminate food processing environments may be ineffective and even provide a selective pressure for strains with acquired resistance to other antimicrobials.

Quaternary ammonium compounds (QACs) are extensively used to control infection and/or microbial contamination in food manufacturing facilities and related environments because these compounds are nonirritating and noncorrosive with slight toxicity and high antimicrobial efficacy over a wide pH range.18 The wide use and misuse of QACs in food environments can impose a selective pressure for bacteria and contribute to the emergence of disinfectant-resistant microbes.15,23,26,32,35 There are five QAC resistance genes, sugE(c), emrE, ydgE/ ydgF, and mdfA, which are known as chromosome-encoded genes conferring QAC resistance.1,8 In addition, other five QAC resistance genes, qacE, qacEA¨1, qacF, qacG, and sugE(p), have been identified on mobile genetic elements in gram-negative organisms.17,24 These genes are generally plasmid and/or integron encoded, conferring efflux-mediated resistance to QACs.39 With the exception of the mdfA gene, which belongs to the major facilitator super family, other genes observed are members of the small multidrug resistance (MDR) family.1,41

Introduction

M

eat serves as a vehicle that transmits food-borne diseases in many countries, particularly in developing countries where hygienic standards are not strictly followed and enforced.10 Escherichia coli is a commensal bacterium in a wide range of hosts, including humans and animals. Most E. coli strains exist in normal intestinal flora, but some strains, such as diarrheagenic E. coli, can cause enteric infections. Food contamination with E. coli may occur in many ways, including production, processing, distribution, and retail marketing. In general, meats are the major hosts contaminated by E. coli, making these products important reservoirs of antimicrobial-resistant E. coli. The level of antimicrobial resistance in E. coli is a useful indicator of resistance dissemination and of selective pressure imposed by antimicrobials used in food for animals and humans.39 The emergence of antimicrobial resistance among E. coli strains that originate from food and animals entails important public health implications. 1

The Laboratory of Microbiology, Sichuan Agricultural University, Dujiangyan, People’s Republic of China. Animal Disease Prevention and Food Safety Key Laboratory of Sichuan Province, School of Life Science, Sichuan University, Chengdu, People’s Republic of China. 3 College of Resources, Sichuan Agricultural University, Chengdu, People’s Republic of China. 4 Sichuan Province Entry-Exit Inspection and Quarantine Bureau, Chengdu, People’s Republic of China. 5 College of Food Science, Sichuan Agricultural University, Ya’an, People’s Republic of China. *These two authors contributed equally to this work. 2

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Some reports also indicated that the widespread use of QACs in hospitals, healthcare facilities, food industries, and environment may contribute to the emergence of antimicrobial-resistant microbes.3,19 Previous studies showed that disinfectant resis¨ 1, qacF, qacG, and sugE(p), are lotance genes, qacE, qacEA cated in mobile genetic elements and linked (coexist) with different antibiotic resistance genes.6,24 The mobile genetic elements such as plasmids and integrons are readily transferred among bacteria. Therefore, the widespread use of QACs has raised concerns over their possible involvement in the development of antimicrobial resistance, particularly coresistance to antibiotics.3,19,25 Conjugation experiments were usually carried out to test the horizontal transfer of resistance genes.4 However, insufficient information is available about the occurrence and mechanisms of QAC resistance in gram-negative microorganisms isolated from the food industry.18 The relationships between antibiotic and disinfectant resistance in E. coli isolated from retail meats should also be further clarified. Sichuan province has the biggest number of livestock in China and almost the largest meat consumption due to a population of more than 80 million. Therefore, the current study aims to demonstrate the antibiotic and disinfectant resistance in E. coli isolates and investigate the prevalence of QAC resistance genes. This research also explores the correlation of the presence of these genes and their resistance to other antimicrobials in E. coli isolated from retail meats in Sichuan province, China. Materials and Methods Sampling

A total of 328 meat samples were purchased from supermarkets or grocery stores in Sichuan province between 2012 and 2013. The samples consisted of pork (n = 193), chicken (n = 59), and beef (n = 66). The samples were aseptically collected in sterilized plastic bags and kept cold during transport from the supermarkets/grocery stores to the laboratory. E. coli isolation

E. coli strains were isolated as previously described.39 Briefly, 25-g samples were placed in sterile plastic bags with 225 ml of buffered peptone water, and the bags were vigorously shaken. Up to 50 ml of rinse from each sample and 50 ml of double-strength MacConkey broth were transferred together to sterile flasks. The contents were thoroughly mixed, and then incubated at 37C for 18–24 hr. One loopful of overnight broth culture was streaked onto an eosin methylene blue agar plate (Hangzhou Microbial Reagent Co., Ltd.), and then incubated at 37C for 18–24 hr. One typical E. coli colony was selected and streaked onto trypticase soy agar (TSA) plates. After indole and oxidase tests, presumptive E. coli strains were stored in tryptic soy broth containing 15% glycerol at -80C until use. These isolates were confirmed as E. coli through 16S rDNA gene sequence analysis and Vitek microbial identification. Antimicrobial susceptibility testing

The susceptibilities of the E. coli isolates to 11 antibiotics were determined in accordance with the standard Kirby– Bauer disk diffusion method of the Clinical and Laboratory

ZHANG ET AL.

Standards Institute (CLSI).7 The tested antibiotics were ceftriaxone (CRO; 30 mg), ceftazidime (CAZ; 30 mg), cefalotin (KF; 30 mg), ampicillin (AMP; 10 mg), ampicillin/sulbactam (SAM; 10/10 mg), gentamicin (GM; 10 mg), kanamycin (K; 30 mg), streptomycin (S; 10 mg), tetracycline (TET; 30 mg), ciprofloxacin (CIP; 5 mg), and sulfamethoxazole (SMZ; 300 mg). E. coli ATCC 25922 and E. coli ATCC 35218 were used for quality control. The zone diameter values were used to indicate susceptible, intermediate, and resistant as defined by CLSI.7 Determination of minimum inhibitory concentrations of disinfectants

The minimum inhibitory concentrations (MICs) of disinfectants for E. coli were determined by using the agar dilution method as recommended by the CLSI.7 The disinfectants used were cetyltrimethylammonium bromide (CTAB; Amresco), benzalkonium chloride (BC; Chengdu Best-reagent Co., Ltd.), N,N-didecyl-N,N-dimethylammonium chloride (DDAC; Chengdu Best-Reagent Co., Ltd.), and cetylpyridinium chloride (CPC; J&K Chemical Co., Ltd.). Bacterial suspensions were prepared by interpreting three to five well-separated overnight colonies from TSA plates into 3 ml of 0.9% saline, equivalent to the turbidity of a 0.5 McFarland standard. Bacterial suspensions were delivered to the surface of Mueller-Hinton agar (MHA) plates by using a multipoint inoculator (MIT-60P; Sakuma Seisakusho). The final inoculum was *104 cfu/ml. The plates were incubated at 37C for 24 hr. The MICs of QACs were recorded as the lowest concentrations of QACs that completely inhibited bacterial growth on the agar plate. E. coli ATCC 10536 was used as a quality control strain. Detection of QAC resistance genes

All isolates were examined for the presence of qacE, qacED1, qacF, qacG, emrE, sugE(c), sugE(p), mdfA, ydgE, and ydgF resistance genes.41 DNA template was prepared by suspending an overnight culture in 600 ml of reagent grade water. The suspensions were heated at 100C for 10 min and centrifuged at 13,000 rpm for 5 min. PCR reaction was performed in a DNA thermal cycler (Veriti 96-Well Thermal Cycler; Applied Biosystems). Each 25 ml PCR mixture consisted of 2.5 ml of template, 5 ml of 5 · PCR buffer, 1.5 mM MgCl2, 200 mM dNTP, 0.4 mM primers, and 1.25 U Taq DNA polymerase (Tiangen Biotech). The amplified PCR products were analyzed on 2.0% (w/v) agarose gels. The appropriate positive and negative controls for amplification were selected from retail meat E. coli isolates. The positive controls with different disinfectant resistance genes were confirmed by PCR, followed by sequence analysis (Sangon Biotech). All results were confirmed by at least two independent experiments. Conjugation experiments

To investigate whether the disinfectant resistance genes were located on conjugative plasmids, the conjugation experiment was conducted in mixed broth cultures as previously described.4 Sodium azide (NaN3)-resistant E. coli J53 was used as a recipient strain. The mixture prepared with 1,000 ml of donor strain and 100 ml of recipient strain from

ANTIBIOTIC AND DISINFECTANT RESISTANCE OF E. COLI

overnight cultures on TSA plates was mixed with fresh Mueller-Hinton broth. After incubation at 35C for 24 hr, the mixture was inoculated on MHA plates that contained NaN3 (100 mg/L)37,38 and CTAB (128 mg/L) at 35C for 24 hr. The colonies that grew on the selecting medium were collected and identified by PCR. The presence of the resistance genes was also confirmed by PCR. Pulsed-field gel electrophoresis

Up to 96 isolates that were positive for mobile geneticmediated disinfectant resistance genes were selected for pulsed-field gel electrophoresis (PFGE) analysis using the PulseNet protocol (www.cdc.gov/pulsenet/PDF/ecoli-shigellasalmonella-pfge-protocol-508c.pdf). The XbaI-digested DNA fragments were analyzed using 1% agarose gel and a CHEF MAPPER electrophoresis system (Bio-Rad). Salmonella enterica serovar Braenderup H9812 was used as a marker. Run times and pulse times were 6.76–35.38 sec for 18–19 hr with linear ramping at a constant voltage of 6 V per 1 cm. PFGE results were analyzed by BioNumerics software (Applied Maths), and banding patterns were compared by using Dice coefficients with a 1.5% band position tolerance.

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kanamycin (22.4%), streptomycin (21.2%), ciprofloxacin (14.5%), and gentamicin (11.4%) (Fig. 1). The isolates also showed 2.8–6.7% resistance to ceftazidime, ceftriaxone, and ampicillin/sulbactam. In addition, 49.4% of the isolates demonstrated MDR. The most frequent resistance pattern was SMZ (n = 21, 8.4%), followed by SMZ-TET (n = 18, 7.1%), TET (n = 16, 6.3%), and AMP-SMZ-TET (n = 13, 5.1%). The frequency of antimicrobial resistance varied depending on meat types (Fig. 1). The isolates from pork showed higher resistance to streptomycin, sulfamethoxazole, and tetracycline compared with the isolates from chicken and beef. Likewise, the isolates from chicken showed higher resistance to ampicillin, kanamycin, and cefalotin compared with the isolates from pork and beef. Overall, E. coli from pork showed the highest resistance to antibiotics (n = 141, 87.6%), followed by E. coli from chicken (n = 40, 80.0%), and beef (n = 32, 72.7%). The most frequent resistance patterns in the E. coli isolates from pork were SMZ (n = 20, 14.7%), SMZ-TET (n = 15, 11.0%), TET (n = 11, 8.9%), and AMP-SMZ-TET (n = 10, 7.4%); those from chicken were AMP-TET (n = 4, 10.0%) and AMP-KKF-SMZ-TET (n = 4, 10.0%); and those from beef were KF (n = 5, 15.6%) and TET (n = 4, 12.5%).

Statistical analysis

Susceptibility differences among the strains were analyzed by using SPSS v.12 (1989–2003; SPSS, Inc.), and the chisquare test was used to determine the significance of differences. Statistical significance was considered at p < 0.05. Results Antimicrobial susceptibility

In total, 77.7% (n = 255) of the retail meat samples were positive for E. coli. Among these isolates, E. coli was highly present in chicken (n = 50, 84.8%), followed by pork (n = 161, 79.3%), and beef (n = 44, 66.7%). Overall, 85.5% (n = 218) of the 255 E. coli strains were resistant to at least one antibiotic, 61.6% were resistant to sulfonamides, 61.2% to tetracyclines, 2.8–48.2% to b-lactam, 11.4–22.4% to aminoglycosides, 14.5% to fluoroquinolones, and 5.9% to b-lactam inhibitors. The E. coli isolates showed the highest resistance to sulfamethoxazole (61.6%), followed by tetracycline (61.2%), ampicillin (48.2%), and to a lesser extent cefalotin (29.8%),

MICs of QACs in E. coli

The MICs of CPC, CTAB, BC, and DDAC in the 255 E. coli strains were 8–512, 16–12, 16–1,024, and 4–1,024 mg/ L, respectively (Fig. 2). In general, 67.5% (n = 172), 84.7% (n = 216), 52.6% (n = 134), and 69.8% (n = 178) of the E. coli isolates exhibited MICs of 4–128 mg/L for CPC, 64–256 mg/L for CTAB, 32 mg/L for BC, and 8–16 mg/L for DDAC, respectively. The MIC ranges of the QACs were similar in the different meat types, except that 50% (n = 20) of the E. coli isolates from beef showed a high MIC (512 mg/L) to BC. The MIC50 values of CPC, CTAB, BC, and DDAC were 64, 128, 32, and 32 mg/L, respectively. However, the corresponding MIC90 values were 256, 512, 512, and 512 mg/L. Up to 44.7% (n = 114), 67.5% (n = 172), and 69.8% (n = 178) of the E. coli isolates showed reduced susceptibility to CTAB (MICs, 256–1,024 mg/L), CPC (MICs, 64– 128 mg/L), and DDAC (MICs, 8–16 mg/L) compared with the control strain, E. coli ATCC 10536. Strains for which BC MICs are 50 mg/L are considered

FIG. 1. Antibiotic resistance of Escherichia coli from different meat types. AMP, ampicillin; CAZ, ceftazidime; CIP, ciprofloxacin; CRO, ceftriaxone; GM, gentamicin; K, kanamycin; KF, cefalotin; S, streptomycin; SAM, ampicillin/ sulbactam; SMZ, sulfamethoxazole; TET, tetracycline.

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ZHANG ET AL.

a higher frequency in the pork isolates than in the chicken and beef isolates. Association between disinfectant resistance genes and antibiotic resistance

FIG. 2. Distribution of quaternary ammonium compound minimum inhibitory concentrations (MICs) in 255 E. coli isolates. The MICs of E. coli ATCC 10536 of N,N-didecylN,N-dimethylammonium chloride (DDAC), benzalkonium chloride (BC), cetylpyridinium chloride (CPC), and cetyltrimethylammonium bromide (CTAB) were 4, 16, 16, and 128 mg/L, respectively.

sensitive, low-level resistant, and high-level resistant, respectively.28 Thus, 89.2% (n = 227) of the 255 isolates could be considered BC resistant, including 52.6% (n = 134) low-level resistant and 36.5% (n = 93) high-level resistant strains. Prevalence of disinfectant resistance genes

The disinfectant resistance genes ydgE/ydgF (86.7%, n = 221), mdfA (85.5%, n = 218), sugE(c) (83.1%, n = 212), and emrE (78.4%, n = 200) were commonly present in the 255 E. coli isolates. The qacED1 gene was found in 19.6% (n = 50) of the isolates, followed by qacF (18.0%, n = 46), and sugE(p) (2.8%, n = 7). The qacE and qacG genes were not detected in any of the isolates. The top three resistance genotypes were emrE-mdfA-sugE(c)-ydgE-ydgF (24.7%, n = 63), mdfA-sugE(c)-ydgE-ydgF (16.1%, n = 41), and emrE-mdfA-qacF-sugE(c)-ydgE-ydgF (5.5%, n = 14). Regardless of the source, ydgE/ydgF, emrE, sugE(c), and mdfA were commonly found in the retail meat isolates (Fig. 3). The frequency of the qacF and sugE(p) genes was slightly higher in the E. coli isolates from chicken than in those from beef and pork, whereas the qacED1 gene showed

FIG. 3. Presence of disinfectant resistance genes in E. coli from different meat types.

Antibiotic resistance was significantly associated with the presence of qacF in all isolates and qacED1 in the pork isolates ( p < 0.05). Among the 46 qacF-positive isolates, 97.8% (n = 45) were resistant to at least one antibiotic, 93.5% (n = 43) were resistant to sulfamethoxazole, 89.1% (n = 41) to tetracycline, 73.9% (n = 34) to ampicillin, 50.0% (n = 23) to kanamycin, and 36.9% (n = 17) to cefalotin. Up to 82.6% (n = 38) of these isolates were resistant to multiple antibiotics. Twenty-two resistance patterns were found among the qacF-positive isolates. The most frequent resistance pattern was AMP-SMZ-TET (15.2%, n = 7), followed by AMP-KSMZ-TET (8.7%, n = 4), AMP-K-KF-SMZ-TET (6.5%, n = 3), and AMP-CIP-CRO-K-KF-SMZ-TET (6.5%, n = 3). Among the 50 qacED1-positive isolates, 74.0% (n = 37) were resistant to sulfamethoxazole, 70.0% (n = 35) were resistant to tetracycline and ampicillin, 46.0% (n = 23) to ciprofloxacin, 44.0% (n = 22) to cefalotin, and 42.0% (n = 21) to kanamycin. Up to 62.0% (n = 31) of these isolates were resistant to sulfamethoxazole and tetracycline, 36.0% (n = 18) to ampicillin and ciprofloxacin, and 30.0% (n = 15) to streptomycin and sulfamethoxazole. About 70.0% (n = 35) of the isolates were resistant to multiple antibiotics. Twenty-four resistance patterns were found among the qacF-positive isolates. The most frequent resistance patterns were CIPCRO-KF-S-SMZ-TET and K-KF-SMZ-TET (8.0%, n = 4), followed by AMP-CIP-GM-K-S-SMZ-TET (6.0%, n = 3). Similarly, among the 17 sugE(p)-positive isolates, 58.8% (n = 10) were resistant to tetracycline, 52.9% (n = 9) to cefalotin, 47.1% (n = 8) to ampicillin, and 41.2% to sulfamethoxazole. Up to 41.2% (n = 7) of these isolates were resistant to cefalotin and tetracycline and 35.3% (n = 6) to sulfamethoxazole and tetracycline. Approximately 52.9% (n = 9) of the sugE(p)-positive isolates were MDR. Moreover, emrE-mdfA-sugE(c)-ydgE-ydgF and mdfA-sugE(c)ydgE-ydgF gene combinations were the top two genotypes in both resistant and susceptible isolates. Conjugative analysis

E. coli transconjugant strains were obtained for qacF (strain 49m) and sugE(p) (strain N34) genes. Another E. coli

ANTIBIOTIC AND DISINFECTANT RESISTANCE OF E. COLI

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Table 1. MICs of Disinfectants in Recipient, Donor, and Transconjugant Strains MIC (mg/L)

Recipient

Donor Transconjugant

Strain number

Gene

CPC

DDAC

CTAB

BC

J53 N34 J1 49m N34¢ J1¢ 49m¢

— sugE(p) qacED1/sugE(p) qacF sugE(p) qacED1/sugE(p) qacF

16 32 16 64 16 16 64

4 1,024 512 16 8 32 8

256 512 512 256 256 256 256

32 1,024 512 32 32 32 32

BC, benzalkonium chloride; CPC, cetylpyridinium chloride; CTAB, cetyltrimethylammonium bromide; DDAC, N,N-didecyl-N,Ndimethylammonium chloride; MIC, minimum inhibitory concentration.

isolate (strain J1), which harbored the qacED1 and sugE(p) genes, also transferred disinfectant resistance. The transfer rate varied from 5 · 10-4 to 1 · 10-5 per donor. All the E. coli transconjugants exhibited reduced disinfectant susceptibility to QACs (Table 1). PCR indicated that the qacF, sugE(p), and qacED1 genes were detected in the total plasmids from the transconjugants. These results indicated that the disinfectant resistance genes were located on conjugative plasmids and could spread between bacteria under certain conditions. Pulsed-field gel electrophoresis

Up to 31 distinct PFGE types were identified among the 96 E. coli isolates (Supplementary Fig. S1; Supplementary Data are available online at www.liebertpub.com/mdr). To determine whether or not genetically related isolates clustered according to their source (area vs. supermarket), we defined the minimum size of a cluster as one PFGE type. Among these E. coli isolates, different subtypes were associated with sampling places. On the basis of the dendrogram shown in Supplementary Fig. S1, one cluster contained four supermarket A isolates (cluster A, encompassing five PFGE types and containing six strains of E. coli). Cluster B contained five supermarket C isolates from three different areas (WH, JN, and JJ). PFGE types 8 (cluster D) and 10 (cluster F) were the predominating pulsotypes. Clusters C, G, J, L, M, N, and O also showed that the PFGE type was clearly associated with sampling supermarket or grocery regardless of location. By contrast, all the PFGE types showed uniform distribution concerning the different mobile elements that encoded disinfectant resistance genes. The same PFGE types or subtypes were detected in supermarkets located in different areas, indicating the possibility of cross-contamination between farms or processing environment. Thus, the farms or the processing environment of the meat processing plant may be a major source for cross-contamination of meats with antimicrobialresistant E. coli. Discussion

China is one of the countries with the greatest consumption and exportation of animal products. The use of antibiotics and disinfectants in food animals and their role in promoting resistance in food-borne bacteria are important public health issues. However, little is known about resistance to disinfectant and antibiotics and their coresistance in E. coli from retail meats.

Despite the widespread use of chemical interventions, E. coli is commonly present in retail meats.39 In the current study, E. coli was commonly present in retail meats. The highest contamination was found in chicken (84.8%), followed by pork (79.3%), and beef (66.7%) products. Similar results were observed in the survey on the prevalence of E. coli in retail meats in Germany and the United States.9,14 The National Antimicrobial Resistance Monitoring System (NARMS) retail meat program also indicated that most poultry meats (chicken, 83.5%; turkey, 82.0%) are contaminated with this organism, followed by beef (68.9%), and pork (44.0%).39 Varying recovery rates are reported in other studies from the United States and other countries.20,27 Antimicrobial resistance has become a global problem.30 In the current study, E. coli strains were more frequently resistant to sulfamethoxazole, tetracycline, ampicillin, and cefalotin (29.8– 61.6%). Similarly, E. coli isolates from chicken breast, ground turkey, ground beef, and pork chop samples by the NARMS retail meat program are most commonly resistant to tetracycline (50.3%), followed by streptomycin (34.6%), sulfamethoxazole/ sulfisoxazole (31.6%), and ampicillin (22.5%).39 Similar findings on common resistance to sulfamethoxazole, tetracycline, and ampicillin in E. coli isolated from retail meats were also reported in China and other countries.9,13,21,34 The frequency of antimicrobial resistance varied depending on meat type. The E. coli isolates from pork showed higher resistance than those from chicken and beef. The isolates from pork showed significantly greater resistance to ampicillin, kanamycin, streptomycin, sulfamethoxazole, and tetracycline than those from beef and chicken. Furthermore, the high levels of resistance in pork may be partly attributed to the common use of these antibiotics as treatment in swine in Sichuan Province. China is the largest antimicrobial producer and consumer in the world. Estimates from China indicated that the annual antimicrobial production in China was 210 million kilograms, and 46.1% was used in livestock industries,12 at least four times the amount used in the US livestock industry in 1999.5 In China, the use of antimicrobials both for animal disease treatment and growth promotion is unmonitored.40 Sichuan province has the biggest number of swine in China, which may lead to high use of antimicrobials causing high antimicrobial resistance in bacteria from swine and pork. Bacterial adaptation to QACs is mainly documented for BC, and limited data are available for resistance to other QACs.28,31 Chuanchuen et al. reported that S. enterica isolates exhibit MICs of BC ranging from 8 to 256 mg/L.

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Moreover, 55% of the tested isolates show MICs of 128– 256 mg/L.6 In the present study, 67.5%, 84.7%, 52.6%, and 69.8% of the E. coli isolates had MICs of 4–128 mg/L for CPC, 64–256 mg/L for CTAB, 32 mg/L for BC, and 8– 16 mg/L for DDAC, respectively. The MIC of DDAC for 153 E. coli isolates ranges from 2 to 64 mg/L.2 Interestingly, cross-resistance between disinfectants in E. coli was also observed. Among the 227 BC-resistant (‡32 mg/L) strains, 218 are also resistant to DDAC, 175 resistant to CPC (‡32 mg/L), and 107 resistant to CTAB (‡256 mg/L), respectively. Reduced susceptibilities to disinfectants have been reported among bacteria isolated from food or food production environments.11,28,33 Compared with previous studies,30 the present results showed that the resistance to QACs in E. coli from retail meats in Sichuan province is highly frequent. Disinfectants may be used to a wide extent in slaughterhouses and meat production facilities. According to the disinfection requirements for a slaughterhouse (SB/T10660-2012) established by China, the QACs were allowed to be used in the range of 0.015–0.1% in slaughterhouses and meat production facilities. However, the use of disinfectants is also unmonitored in China. There are some studies that reported the increase in use of high concentration of QACs (10%) without any restriction.22 The high prevalence of disinfectant-resistant E. coli in retail meats revealed that E. coli can serve as an important reservoir for the dissemination of QAC resistance. The present study demonstrated that various disinfectant resistance genes were present in E. coli. Chromosome-encoded efflux pump genes, such as ydgE, ydgF, mdfA, emrE, and sugE(c), were commonly present in 53.7–83.1% of the E. coli isolates. However, the frequency of mobile element-encoded disinfectant resistance genes was relatively low in this study (0.0– 19.6%). Similar findings were reported in E. coli and Salmonella from food animals and retail meats.6,17,39 The qacE gene was found in only 1 of 37 Pseudomonas aeruginosa strains.17 In another study, the qacED1 gene was detected in 60.3% of clinical E. coli isolates.36 The widespread use of QACs in Sichuan province may contribute to the emergence of antimicrobial-resistant bacteria and spread of resistance genes. Therefore, using QACs for the decontamination of the environment may not be as effective as expected. In recent years, concerns have increased for QAC exposure that may facilitate the selection of antibiotic-resistant bacteria. Susceptibilities to amoxicillin and cotrimoxazole are significantly associated with a high MIC of DDAC in clinical E. coli strains.2 BC-resistant isolates also show resistance to gentamicin and chlorhexidine.29 In the current study, the qacED1 and qacF genes were highly associated ( p < 0.05) with antibiotic resistance. Up to 20 sulfamethoxazoleresistant isolates among 50 qacED1-positive isolates had a high MIC of BC (512 mg/L). Similarly, 13 sulfamethoxazoleresistant isolates among 24 qacF-positive isolates showed high resistance to CTAB (512 mg/L). Studies have shown that qacED1 is common in enteric bacteria and is located at the 3¢-conserved segment of class 1 integrons that carry sul 1 (sulfonamide resistance determinant).16 The qacED1 and sul genes usually coexist in integrons, which could explain that 74.0% (n = 37) of the qacED1-positive isolates showed coresistance to sulfamethoxazole. Moreover, the qacF gene is located in class 1 integrons,19,24 leading to 93.5% (n = 43)

ZHANG ET AL.

resistance to sulfamethoxazole. Therefore, the use of QACs in the food processing environment may facilitate the selection of strains that exhibit acquired QAC resistance and that carry genes encoding resistance to medically important antibiotics.28 PFGE results revealed that the antimicrobial-resistant isolates were associated with samples from supermarkets or groceries. E. coli from the same supermarkets in different areas showed the same PFGE types, which provided evidence of cross-contamination in farms or processing environment. Conjugative analysis also showed that QAC resistance may transfer between different strains by plasmids, which could be a serious risk for human health. This study demonstrated that antimicrobial resistance was common among E. coli isolates from retail meats. The farms or the processing environment may be a major source for cross-contamination with antimicrobial-resistant E. coli. The QAC resistance genes were commonly present among E. coli isolates. The qac genes were highly associated with antimicrobial resistance phenotypes. Therefore, the abuse and widespread use of QACs, especially the use of QACs under sub-MIC, to decontaminate food processing environments may be ineffective and can even provide a selective pressure for strains with acquired resistance to other antimicrobials. The use of QAC disinfectants may influence the types of antimicrobial-resistant organisms that reach consumers through food. However, if the disinfection conditions are well applied, the risk for selecting antimicrobialresistant bacteria might be lower. Acknowledgments

This research was supported by the National Natural Science Foundation of China (31400066), the Program for Changjiang Scholars and Innovative Research Team in University (PCSIRT, IRT13083), and the State General Administration of the People’s Republic of China for Quality Supervision and Inspection and Quarantine (AQSIQ, 2014IK242). Disclosure Statement

No competing financial interests exist. References

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Address correspondence to: Likou Zou, PhD College of Resources and Environment Sichuan Agricultural University Jianshe Road 288 Chengdu 611130 People’s Republic of China E-mail: [email protected]