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

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.

In this study, a total of 290 raw meat samples (91 pork and 199 chicken) were randomly collected from large and small supermarkets and farmer’s markets in the 6 districts of Beijing. E. coli were isolated using CHROMagar ECC (CHROMagar TM, Paris, France) colored medium after enrichment. The isolated strains were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Detailed materials and methods were provided as supporting information (Supplementary Materials). The gram-negative bacteria drug sensitivity plates Sensititre GNX3F (Thermo Fisher Scientific, Massachusetts, USA) 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) (7) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) standards v10.0 (8). The microbroth dilution method was used to determine their susceptibility to 21 antimicrobial agents from 9 classes, including amikacin, gentamicin, tobramycin, doxycycline (DOX), tigecycline, minocycline, ciprofloxacin, levofloxacin, trimethoprim/sulfamethoxazole (SXT), aztreonam, imipenem, meropenem, doripenem, cefepime, ceftazidime, cefotaxime, colistin, polymyxin B, ampicillin/sulbactam, piperacillin/tazobactam, and ticarcillin/clavulanic acid. Whole genome sequencing (WGS) was conducted using an Illumina Hiseq2500 platform (Annoroad Gene Technology, Beijing, China). Multilocus sequence typing (MLST) was identified using the pMLST 2.0 database (mlst Github http://github.com/tseemann/mlst). Antimicrobial resistance genes were identified by searching the Comprehensive Antibiotic Research Database (CARD, https://card.mcmaster.ca/download). All draft genomes were used for constructing a phylogenetic tree using parsnp software (Supplemetary Materials https://github.com/marbl/) (9), and the trees were finally visualized using the online tool iTOL (http://itol.embl.de/).

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), 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).

Figure 1. 

Phenotypic resistance of Escherichia coli isolates in raw meat samples from six districts, Beijing, China, 2020. (A) Resistant rate of all E. coli isolates against nine different antibiotic classes. (B) Distribution of E. coli resistant phenotypes from different districts in Beijing.

Note: The six districts of Beijing includes Dongcheng, Xicheng, Haidian, Fengtai, Chaoyang, and Changping. Abbreviations: SXT=trimethoprim/sulfamethoxazole; CIP=ciprofloxacin; LEVO=levofloxacin; COL=colistin; POL=polymyxin B; TGC=tigecycline; DOX=doxycycline; MIN=minocycline; AMI=amikacin; GEN=gentamicin; TOB=tobramycin; IMI=imipenem; MERO=meropenem; DOR=doripenem; FEP=cefepime; FOT=cefotaxime; TAZ=ceftazidime; AZT=aztreonam; TIM2=ticarcillin/clavulanic acid constant 2; A/S2=ampicillin/sulbactam 2:1 ratio; P/T4=piperacillin/tazobactam constant 4.

Comprehensive antibiotic resistome analysis using the CARD database in all 166 E. coli isolates indicated that diverse antimicrobial genotypes occurred in chicken and pork samples in Beijing. Among all the resistance genes, ampC1 and ampC2 were highly observed in all isolates (Figure 2), the detection rates were 94.7% and 99.4%, respectively. Extended-spectrum β-lactamase (ESBL)-producing strains of E. coli harboring CTX-M, OXA, CMY, and TEM were detected in this study. The most prevalent ESBL genes were bla_TEM-1D (22.7%) and bla_CTX-M-9 (21.5%), followed by bla_CTX-M-1 (11.0%) and bla_OXA-7 (11.0%). Plasmid-mediated transferrable colistin resistant gene mcr-1 was found in 22 isolates (13.3%). Most mcr-1 genes were located on plasmid (15/22, 68.2%), and IncI2 (11/15, 73.3%) and IncY (4/15, 26.7%) were the main plasmid types. In addition, sulfonamide-resistant genes sul1, sul2, and sul3 were also analyzed, and their detection rates were 16.9% (28 isolates), 32.5% (54 isolates), and 13.9% (23 isolates), respectively. Plasmid typing found that nine plasmid incompatibility (Inc) groups (IncFII, IncFIIp, Inc FIA, IncFIB, IncCol, IncCol156, IncX1, IncI2, and IncY) were common in these isolates. A total of 59 different STs were identified from 166 E. coli isolates. The most prevalent one was ST10 (22 isolates, 13.3%), followed by ST399 (11 isolates, 6.6%), and ST1434 (10 isolates, 6.0%). The remaining 56 STs were all lower than 4.2% abundance (Figure 2). Correlation analysis of antimicrobial resistance genotype and phenotype of E. coli isolates were shown in Supplementary Figure S1. Antimicrobial phenotype was consistent with resistance genotype for aminoglycosides, tetracyclines, fluoroquinolones, and lipopetides, except sulfonamides, penicillins, and carbapenems. Phylogenomic analysis revealed that all the E. coli isolates from animal-derived food and clinic were classified into two lineages, sharing different homologies (Supplementary Figure S2). Typing results showed commonality between human clinical strains, with STs 131 (especially), 297, and 2380 prominent. In addition, there was little crossover between types form humans, chicken, and pork, with ST10 dominated among the most common types from animal-derived foods, which is rarely found in humans (Supplementary Figure S2). Thus, we speculated that the risk of E. coli isolates passing through the food chain was relatively low.

Figure 2. 

Distributions of STs, antimicrobial resistance genes, and plasmid typing among 166 Escherichia coli isolates from chicken and pork across the phylogenetic tree.

Note: The color strips indicate areas corresponding to the isolates. Green colored cells represent the presence of genes and white cells represent the absence of the genes. Brown colored cells represent the presence of plasmid typing and white cells represent the absence of the plasmids.