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Prevalence of Escherichia coli and Antibiotic Resistance in Animal-Derived Food Samples — Six Districts, Beijing, China, 2020

What is already known about this topic? Escherichia coli is an important hygiene indicator for animal-derived foods such as pork and chicken, and the contamination of retail meat is associated with the spread of antimicrobial resistance (AMR) and public health. What is added by this report? The prevalence of E. coli in 6 different districts of Beijing was 64.1%. The contamination of chicken was more serious than pork in Beijing. E. coli isolates were highly resistant to sulfonamides (87.4%). TheampC1 and ampC2 genes were the main antibiotic resistance genotype (94.7% and 99.4%). What are the implications for public health practice? This study highlights the need to strengthen the surveillance of antibiotic resistance of E. coli in animal-derived foods. A national or regional multicenter study is required to assess the dissemination and evolution of multidrug resistant (MDR) E. coli in clinical medicine and animal production for food.

Bacterial resistance has become a global problem, among which the resistance of foodborne pathogens has attracted special attention (1). Foodborne pathogens are currently being actively monitored for antimicrobial resistance to analyze the spread and dissemination and support the prevention and control of antimicrobial resistance. Antibiotic-resistant bacteria/genes caused by using antimicrobial drugs in the agricultural industry can spread through the food chain (2). In the animal breeding process, China has launched a foodborne pathogenic bacteria antimicrobial resistance monitoring program and obtained a large amount of basic data. However, we still do not fully understand the antimicrobial resistance of food contaminating bacteria in the circulation link. Escherichia coli (E. coli) bacterial infections have caused a significant increase in morbidity and mortality worldwide, threatening human health (3)(4). Thus, we are using E. coli as a representative to investigate the antibiotic resistance of animal-derived food contaminated bacteria in six districts within Beijing. E. coli is also an important hygiene indicator for animal-derived foods such as pork and chicken. Previous studies have shown that retail meat is associated with the spread of E. coli (5)(6). Therefore, strengthening the monitoring of foodborne pathogenic E. coli is important to protecting human health. In this study, the epidemiological surveillance and typing study of E. coli in retail pork and chicken from six districts (Dongcheng, Xicheng, Haidian, Fengtai, Chaoyang, and Changping) in Beijing, China, was investigated. The results demonstrated that E. coli contamination in chicken is more serious than that in pork, and Chaoyang District displayed the highest E. coli isolation rate. Antimicrobial susceptibility testing showed that foodborne E. coli was highly resistant to sulfonamides with a resistance rate of 87.4%. The prevalence of resistance genes ampC1 and ampC2 occurred in most isolates. Therefore, effective supervision of animal-derived food hygiene to control the dissemination of bacterial resistance is essential to safeguard human health.
A total of 186 E. coli isolates (186/290, 64.1%) were collected from raw chicken and pork samples from Beijing in 2020. The highest rates (73.9%) of E. coli isolates were found in chicken compared to that (42.9%) in pork samples (Supplementary Table S1, available in http://weekly.chinacdc.cn/), which indicated that E. coli contamination in chicken was more serious than in pork. For chicken, Chaoyang District displayed the highest E. coli isolation rate among the six examined regions of Beijing, while Fengtai had the lowest isolation rate (50.0%) (Supplementary Table S1). While regarding pork, Xicheng displayed the highest E. coli isolation rate among the examined regions (66.7%), while Haidian and Chaoyang displayed the lowest isolation rates (33.3%).
The distributions of minimum inhibitory concentrations (MICs) and resistance rates of 186 E. coli isolates against 21 antimicrobial agents were shown in Table 1 and Figure 1A. Antimicrobial susceptibility tests showed that E. coli isolates were highly resistant to SXT (87.4%), followed by DOX (32.57%) and GEN (29.71%). Carbapenems (IMI, MERO, and DOR) had the lowest resistance rate of 4.0% (Table 1). E. coli isolates from six different regions represented high resistance to SXT, and Dongcheng showed 100% resistance to SXT ( Figure 1B).

DISCUSSION
In this study, we determined the prevalence and characteristics of foodborne pathogen E. coli in retail meat in six districts of Beijing, China. The prevalence of E. coli was 73.9% in chicken samples and 42.86% in pork. Greater contamination of chicken than pork concurs with previous findings (10). These results indicated that E. coli contamination of raw chicken in Beijing was a serious public health problem. Thus, better measures should be taken to control E. coli contamination in chicken. Antimicrobial susceptibility test in this study demonstrated that all the 186 E. coli isolates were highly resistant to at least one tested antibiotic class (sulfonamides, aminoglycosides, tetracyclines, fluoroquinolones, penicillins, carbapenems, cephalosporins, lipopeptides, and βlactam/β-lactam inhibitors). The highest resistance to trimethoprim/sulfamethoxazole (87.4%) was observed, which might be due to sulfonamides being used widely in animal husbandry in China (11). Nowadays, the average resistance rates in E. coli to representatives of these antibiotic classes were higher than 40% (12). Eating retail chicken and pork without strict hygiene supervision should be avoided. These results highlight the contamination status of antibiotic resistant E. coli and provide an important reference value for the risk assessment and control of multidrug-resistant bacteria.
ESBL genes were located on plasmids that can be was 48.7%. The ST10 is a well-known clonal lineage (mainly harboring CTX-M gene); it is also known that some of the Inc plasmids were implicated in the spread of beta-lactamases genes and other genes encoding resistance to antibiotics. Our WGS results indicated that 76 of the ESBL-producing E. coli isolates had 31 distinct STs, and ST10 was the most prevalent (9/76, 11.8%). Colistin serves as the "last line of defense" for the clinical treatment of gram-negative bacterial infections (15). With the discovery of mcr family, China has banned the use of colistin as a feed additive to prevent the dissemination of the gene. However, we found high levels of mcr-1 in chicken-derived E. coli, which suggests that we should strengthen strict monitoring of food-borne pathogens in animal-derived foods that carry such drug-resistant genes. This study has several limitations. The geographical distribution of the samples in this study was mainly concentrated in the central urban area, not representative of the entirety Beijing. The number of isolated strains of E. coli in retail pork samples was small, and strengthening the supervision of the strains in pork samples is necessary in future studies.
This study was a typical survey of the prevalence of E. coli in animal-derived foods in six districts of Beijing, China. E. coli contamination in chicken was more serious than that in pork. The isolates showed multi-drug resistance phenotypes, especially sulfonamides and tetracyclines. The β-lactamase genes ampC1 and ampC2 were the main drug resistance genes, and the colistin resistance gene mcr-1 was found at a high level in chicken-derived E. coli. Considering the multidrug resistance of E. coli in animal-derived foods, especially ESBL-producing E. coli, continuous monitoring the emergence and spread of MDR E. coli would facilitate disease control and treatment. Further national or regional multicenter studies are necessary to assess the dissemination and evolution of MDR E. coli in both clinical medicine and food animal production in China.

Sample Collection, Isolation and Identification
From July to October 2020, a total of 290 animal-derived food samples (91 pork and 199 chicken samples) were collected from 16 large supermarket chains and farmers' markets in 6 districts of Beijing Municipality. The specific collected samples commercially available cuts of pork meat and commercially available chicken cuts. All samples were placed in Labplas TWIRL'EM sterile homogeneous bags (Labplas, Canada) after being collected and brought to the laboratory for testing. A single sample was placed in a sterile sampling bag to prevent cross-contamination during sample collection. The target strains were isolated as previously described (1), and the brief description was shown as following: 10 mL brain heart infusion (BHI) broth (Land Bridge, Beijing, China) was used to wash the sample surface, transfer to a 10 mL EP tube, and incubation at 37 °C for 24 h. 0.5-1 mL of the enrichment culture obtained was cultured on CHROMagar TM ECC medium (CHROMagar, France) and inoculated at 37 °C for 24 h. The blue colonies on the plate were further purified using Luria agar (LA) and identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry analysis.

Antimicrobial Susceptibility Testing
The gram-negative bacteria drug sensitivity plates Sensititre GNX3F were used for the antibiotic susceptibility test of the isolated strains according to the recommendations of the Clinical and Laboratory Standards Institute guidelines (CLSI, M100-S30) (2) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) standard v10.0 (3). E. coli ATCC25922 was used as the quality control strain.

Whole Genome Sequencing
Genomic DNA from E. coli isolates was extracted for whole genome sequencing using the Wizard Genomic DNA Purification Kit (Promega, Madison, WI, US) following the manufacturer's instructions. The DNA libraries were constructed using a KAPA Hyper Prep Kit (Roche, Basel, Switzerland). Sequencing was carried out with Illumina Novaseq 6000 platform (Illumina, San Diego, CA), which generated 150-bp paired-end reads from a library with an average insert size of 350 bp. Raw sequence data of E. coli isolates were assembled using SPAdes v3.13.1 (4) via the