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. Copyright and License information: Editorial Office of CCDCW, Chinese Center for Disease Control and Prevention 2021.

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, available in http://weekly.chinacdc.cn/). 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, available in http://weekly.chinacdc.cn/ 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, 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).

Table 1

Antibiotic resistant phenotype of 186 Escherichia coli isolates against 21 antimicrobial agents obtained from chicken and pork samples from the 6 districts, Beijing, China, 2020.

Antimicrobial classes	Antimicrobial agents	Chicken				Pork			Overall R% §
		S% *	I% †	R% §		S% *	I% †	R% §
Note: The six districts of Beijing includes Dongcheng, Xicheng, Haidian, Fengtai, Chaoyang, and Changping. * S% means the percentage of isolates that were sensitive to a specific antimicrobial. † I% means the percentage of isolates that were intermediate resistant to a specific antimicrobial. § R% means the percent of isolates that were resistant to a specific antimicrobial.
Aminoglycosides	Amikacin (AMI)	94.9	1.5	3.7		100.0	0.0	0.0	2.9
	Gentamicin (GEN)	65.4	1.5	33.1		82.1	0.0	18.0	29.7
	Tobramycin (TOB)	64.7	2.2	33.1		87.2	0.0	12.8	28.6
Tetracyclines	Doxycycline (DOX)	40.4	30.2	29.4		18.0	38.5	43.6	32.6
	Tigecycline (TGC)	98.5	0.0	1.5		94.9	0.0	5.1	2.3
	Minocycline (MIN)	83.8	11.0	5.2		64.1	12.8	23.1	9.1
Fluoroquinolones	Ciprofloxacin (CIP)	69.1	1.5	29.4		79.5	2.6	18.0	26.9
	Levofloxacin (LEVO)	73.5	2.9	23.5		79.5	7.7	12.8	21.1
Sulfonamides	Trimethoprim/sulfamethoxazole (SXT)	14.0	0.0	86.0		7.7	0.0	92.3	87.4
Penicillins	Aztreonam (AZT)	69.1	4.4	26.5		89.7	2.6	7.7	22.3
Carbapenems	Imipenem (IMI)	91.9	3.7	4.4		94.9	2.6	2.6	4.0
	Meropenem (MERO)	94.1	2.9	2.9		97.4	0.0	2.6	2.9
	Doripenem (DOR)	97.8	0.7	1.5		100.0	0.0	0.0	1.1
Cephalosporins	Cefepime (FEP)	80.9	4.4	14.7		97.4	0.0	2.6	12.0
	Ceftazidime (TAZ)	89.7	3.7	6.6		92.3	2.6	5.1	6.3
	Cefotaxime (FOT)	66.2	2.2	31.6		92.3	0.0	7.7	26.3
Lipopeptides	Colistin (COL)	80.9	2.9	16.2		92.3	5.1	2.6	13.1
	Polymixin B (POL)	69.1	15.4	15.4		82.1	15.4	2.6	12.6
β-lactam/β-lactam inhibitors	Ampicillin/sulbactam 2∶1 ratio (A/S2)	66.2	11.8	22.1		74.4	20.5	5.1	18.3
	Piperacillin/tazobactam constant 4 (P/T4)	96.3	1.5	2.2		100.0	0.0	0.0	1.7
	Ticarcillin/clavulanic acid constant 2 (TIM2)	67.7	27.2	5.2		76.9	20.5	2.6	4.6

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 blaTEM-1D (22.7%) and blaCTX-M-9 (21.5%), followed by blaCTX-M-1 (11.0%) and blaOXA-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, available in http://weekly.chinacdc.cn/. 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, available in http://weekly.chinacdc.cn/). 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, available in http://weekly.chinacdc.cn/). 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.

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 β-lactam/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 easily transferred between and within bacterial species. In this study, the resistance gene ampC1 and ampC2 were highly observed in all isolates, and detection rates were 94.7%, 99.4%, respectively. However, the detection rate of E. coli ampC in retail chicken from 2013–2014 (45.0%) dropped to 13.4% in 2018, UK (13). Wu et al. (14) reported the ESBL gene and mcr-1 prevalence of chicken-derived E. coli in many provinces in China and found the detection rate of blaCTX-M was 92.7%. The enrichment and changes of E. coli ampC resistance genes in raw meat sources in China are higher than those in other countries. With the increasing selection of β-lactam drugs, it will continue to mutate and spread in the food supply chain, causing serious public health problems. Among all the ESBL-producing E. coli isolates, the carrying rate of blaCTX-M was 33.1%, blaCTX-M-9 and blaCTX-M-1 were the dominant subtypes. The carrying rate of blaCTX-M-9 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.

Conflicts of interest

No conflicts of interest.