Characteristics of Extended-Spectrum β-Lactamase-Producing Escherichia coli Derived from Food and Humans in Northern Xinjiang,China

Abstract

This study aimed to investigate the drug resistance, molecular characteristics, and genetic relationship of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli isolated from food and human stool samples in northern Xinjiang. From 2015 to 2016, a total of 431 samples (meats and vegetables) were collected from retail markets and supermarkets located in the regions of Urumqi, Shihezi, and Kuitun in Xinjiang, China, and 20 human stool samples from the Shihezi Hospital. The PCR method was used to detect E. coli, and the presence of ESBL-producing E. coli was confirmed using the K-B disk diffusion confirmatory method. The susceptibility to ESBL-producing E. coli was tested by the microdilution broth method, and the minimum inhibitory concentration was determined. PCR was used to detect the resistance and virulence genes of ESBL-producing E. coli, and phylogenetics, plasmid replicon typing, screening of three integrons, and multilocus sequence typing (MLST) were performed. The results showed that 127 E. coli strains (15 human stool and 112 food samples) were isolated. Out of the 127 E. coli strains, 38 strains (6 human stool and 32 food 34 samples) of ESBL-producing E. coli were identified through screening. These 38 strains showed resistance to cefotaxime (94.74%) and cefepime (94.74%), and were sensitive to meropenem (0.00%). The most detected resistance genes were bla_TEM (47.37%), and the most detected virulence genes were fimH (97.73%), ompA (97.73%), hlyE (97.73%), and crl (97.37%). The isolates belonged to phylogroups B₁ (42.11%), C (23.68%), and A (21.05%). Among the plasmid replicon subtypes, IncFIB was the main type (42.11%). The integrons detected were of the first type (47.37%) and the third type (26.32%). The 38 E. coli strains had 19 different sequence-type (ST) strains. These 38 strains of ESBL-producing E. coli were analyzed using MLST and STs are varied.

Introduction

E scherichia coli is a common commensal in the intestines of humans and animals, and can cause infections under specific conditions (Liu et al., 2018). This bacterium may carry a pool of antimicrobial resistance (AMR) genes and threaten human health by contaminating food and water, causing diseases (Chen et al., 2021; Huja et al., 2015; Lippolis et al., 2018; McNally et al., 2013; Rehman et al., 2017; Xu et al., 2020). Beta-lactam antibiotics are the main drugs for treating infections caused by E. coli. Therefore, the production of the extended-spectrum β-lactamases (ESBLs) is one of the important mechanisms for the clinical emergence of drug resistance in E. coli isolates (Kang et al., 2004; Li et al., 2007). ESBLs are mainly produced by E. coli, Salmonella, and Klebsiella pneumoniae.

The genotypes of ESBLs prevalent in different regions are not the same due to the differences in physicochemical and drug resistance properties (Bush et al., 1995). The genotypes of ESBLs are mainly divided into bla_TEM , bla_SHV , and bla_CTX-M (Shaikh et al., 2015), with bla_CTX-M -type β-lactamase E. coli being the most common in China (Shu et al., 2016; Yang et al., 2019).

Although the number of clinical studies on ESBLs has significantly increased in recent years, here remains a paucity of comparisons between food-derived and human-derived ESBLs, which are usually related to plasmids, also confirming that plasmids are the main factors affecting the multiple resistance of E. coli. Besides, integrons play a vital role in the spread of antibiotic resistance in Gram-negative bacteria, especially E. coli, which is capable of gene transfer and cassette capture (Guerin et al., 2011; Nardelli et al., 2012; van Essen-Zandbergen et al., 2007; Woodford et al., 2009). Plasmids, integrons, and other mobile genetic elements are readily accessible to E. coli, leading to the acquisition of resistance in E. coli (Bennett, 2008; Carattoli, 2009).

The molecular characteristics of ESBL-producing E. coli have been observed and confirmed worldwide for the spread and evolution of drug resistance. However, the data pertaining to the molecular characterization, clustering, and multilocus sequence typing (MLST) analysis of human stool-and food-derived samples for screening ESBL-producing E. coli were scarce. Therefore, this study aimed to determine the prevalence of E. coli and the characteristics of the ESBL-producing E. coli strains in food and human disease samples collected from the northern region of Xinjiang, China, to provide scientific guidance for the rational use of antibiotics in clinical practice in this region.

Materials and Methods

Sample collection, isolation, and identification of E. coli, and screening of ESBL-producing isolates

From 2015 to 2016, a total of 431 food samples (156 samples of meat, 241 samples of vegetables, and 34 samples of raw milk) were collected from different supermarkets and markets located in the northern region of Xinjiang, China, and 20 human stool samples from the Shihezi Hospital. E. coli was isolated and cultured in lauryl sulfate tryptose (LST) broth and EC broth (Qingdao Hope Bio-Technology Co. Ltd.), following which it was purified in an eosin-methylene blue (EMB) agar (Qingdao Hope Bio-Technology Co. Ltd.). Finally, the strain was biochemically identified according to the GB4789.38-2016 standard (China Food and Drug Administration, 2016). Finally, the E. coli β-D-galactosidase gene (uidA) was identified via PCR (Wu et al., 2014), and bacterial DNA (Wen et al., 2019) was obtained using boiled water and stored for subsequent use.

According to the recommendations of the Clinical and Laboratory Standards Institute (2020), drug susceptibility disks CTX and cefotaxime–clavulanic acid were used for screening ESBL-producing E. coli. This was verified via a phenotypic confirmatory test using the double paper codiffusion method. The difference in the diameter of the bacteriostatic ring between the two drug-sensitive paper sheets ≥5 mm was used to determine an ESBL-producing positive strain (China Food and Drug Administration, 2016). E. coli ATCC25922 was used as the quality control strain to determine the characteristics of ESBL-producing E. coli in food and humans.

Antibiotic-sensitivity experiment

The susceptibility test using E. coli ATCC25922 was conducted using the agar dilution method. A total of 19 antibiotics were administered, representing 7 antimicrobial classes, including cefuroxime, cefotaxime, cefixime, ceftriaxone, cefepime, gentamicin, kanamycin, imipenem, meropenem, chloramphenicol, nalidixic acid, ofloxacin, ciprofloxacin, tetracycline, ampicillin, methicillin, amoxicillin, piperacillin, and polymyxin B. Minimum inhibitory concentration (MIC) and antimicrobial susceptibility were determined according to the CLSI guidelines (Clinical and Laboratory Standards Institute, 2020). The MICs of each strain were determined by the microdilution broth method. The minimum drug concentration for the bacteria-free growth plate was defined as the MIC according to the cutoff point value standards recommended by CLSI: resistant (R), susceptible (S), and intermediate (I) (Clinical and Laboratory Standards Institute, 2020).

Detection of drug-resistant genes and virulence genes

The seven drug-resistant genes (CTX-M-2, CTX-M-9, CTX-M-8/25, bla_TEM , bla_SHV , bla_OXA , and mcr-1) of the obtained isolates were detected using PCR (Dallenne et al., 2010; Kojima et al., 2005; Rebelo et al., 2018) (Supplementary Table S1). Similarly, 15 virulence genes (sfa/foc, afa/dra, kpsMT II, papA, iutA, fimH, hlyE, papC, ompA, crl, stx1, stx2, eae, ipaH, and aggR) of the isolates were detected using conventional PCR (Dias et al., 2010; Iveth Miranda-Estrada et al., 2017; Johnson et al., 2003; Khafipour et al., 2011; Pereira et al., 2011; Silva et al., 2009; Wu et al., 2014) (Supplementary Table S2).

Identification of phylogenetic groups and typing of plasmid replicons

Using four genes (chuA, yjaA, arpA, and TspE4.C2) and two complementary genes (ArpAgpE and trpAgpC) (Supplementary Table S3), E. coli was divided into eight recognized phyla, of which seven belonged to E. coli in the strict sense (A, B₁, B₂, C, D, E, and F) and one corresponded to E. coli branch I (Clermont et al., 2013) (Supplementary Table S4). The presence or absence of the four genes (+ represented positivity) in Supplementary Table S4 allowed the determination of the phylogenic group to which each strain belonged. Moreover, the plasmid replicon typing was performed via PCR analysis involving 18 plasmids (HI1, HI2, I1, X, L/M, N, FIA, FIB, W, Y, P, FIC, A/C, T, FIIS, FrepB, K/B, and B/O) to classify the plasmid-incompatible components in ESBL-producing E. coli isolates (Carattoli et al., 2005) (Supplementary Table S5).

Furthermore, PCR was considered for screening class 1 integron (Int1), class 2 integron (Int2), and class 3 integron (Int3) (Shafiq et al., 2021) (Supplementary Table S6).

Multilocus sequence typing

Seven housekeeping genes (adk, fumC, gyrB, icdA, mdh, purA, and recA) for the MLST test of E. coli were typed according to the details available on the MLST website as the target gene (Wirth et al., 2006). The primers for the seven housekeeping genes are listed in Supplementary Table S7. After PCR and gel electrophoresis, the PCR products were sent to Qingdao Sequencing Company of Ruiboxing Co., Ltd. for phase bidirectional sequencing and splicing. The sequences were submitted to the MLST website to obtain the allele sequence numbers for each housekeeper, and the allele sequence numbers of the seven housekeeping genes formed the allele spectrum of the strain. The allele spectrum was then submitted to the Pasteur database to obtain the sequence-type (ST) model of the strain.

The tandem sequences of the seven housekeeping genes were imported into BioNumerics V8.0 software to construct a phylogenetic tree and a minimum spanning tree based on sequence homology.

Results

Isolation, identification, and screening of ESBL-producing strains of E. coli

In this study, 112 strains of foodborne E. coli were isolated from 431 food samples collected from various parts of northern Xinjiang in China, and 15 strains of human E. coli strains were isolated from 20 human stool samples obtained from the Shihezi Hospital. Of 112 strains, 38 E. coli strains producing ESBLs were identified using the phenotypic confirmation test via the double-disk diffusion method (Table 1). Table 1 shows that the screening rate of ESBLs in Urumqi was the highest (41.18%) compared with the three sampling sites. The ESBL screening rate of meat samples was higher than that of human samples and vegetable samples.

Table 1.

Screening of Escherichia coli Producing Extended-Spectrum β-Lactamases from Supermarkets in Urumqi, Shihezi, and Kuitun, Xinjiang

Sampling sites	Sample sources	No. of Escherichia coli isolates	No. (%) of ESBL E. coli isolates
Urumqi	Meats	17	7 (41.18%)
	Vegetables	0	0 (0.00%)
	Human	0	0 (0.00%)
	Subtotal	17	—
Shihezi	Meats	57	16 (28.07%)
	Vegetables	21	6 (28.57%)
	Human	15	6 (40.00%)
	Subtotal	93	—
Kuitun	Meats	9	2 (22.22%)
	Vegetables	8	1 (12.50%)
	Human	0	0 (0.00%)
	Subtotal	17	—

Data of the second type represent the Escherichia coli strains isolated from 127 samples. Shihezi:Shihezi No. 2 Hospital; Long shopping supermarket; Kuitun: Star Supermarket; Urumqi: Good hometown supermarket; Long Tesco supermarket

ESBL, extended-spectrum β-lactamase.

Antibiotic resistance

The resistance rate of 38 ESBL E. coli strains to cefuroxime, cefotaxime, cefixime, ceftriaxone, and cefepime was 78.95% (30/38), 94.74% (36/38), 86.84% (33/38), 86.84% (33/38), and 94.74% (36/38), respectively. However, they were sensitive to meropenem (Table 2). Table 2 shows that the resistance rates of human samples to various antibiotics were higher than those of vegetable and livestock meat samples.

Table 2.

Antibiotic Resistance of Escherichia coli Producing Extended-Spectrum β-Lactamase

Antibiotics		Test range (mg/mL)	MIC (mg/mL)		No. of antibiotic-resistant ESBL-producing Escherichia coli isolates (%)
Classes	Drug names	Test range (mg/mL)	MIC50	MIC90	Meat (n = 25)	Vegetables (n = 7)	Human (n = 6)	Total (n = 38)
Cephalosporin	Cefuroxime	1–128	32	32	22 (88)	3 (42.86)	5 (83.33)	30 (78.95)
	Cefotaxime	0.5–64	4	16	25 (100)	5 (71.43)	6 (100)	36 (94.74)
	Cefixime	0.5–32	4	16	24 (96)	3 (42.86)	6 (100)	33 (86.84)
	Ceftriaxone	1–32	4	16	24 (96)	3 (42.86)	6 (100)	33 (86.84)
	Cefepime	1–64	16	64	25 (100)	5 (71.43)	6 (100)	36 (94.74)
Aminoglycoside	Gentamicin	0.5–32	4	32	7 (28)	2 (28.57)	5 (83.33)	14 (36.84)
Aminoglycoside	Kanamycin	2–256	32	128	9 (36)	1 (14.28)	5 (83.33)	15 (39.47)
Carbapenem	Imipenem	0.125–8	0.25	4	0	0	1 (16.67)	1 (2.63)
Carbapenem	Meropenem	0.125–8	0	0	0	0	0	0
Quinolones	Naphthalic acid	1–128	8	64	8 (32)	0	5 (83.33)	13 (34.21)
	Ofloxacin	0.25–32	2	4	0	0	2 (33.33)	2 (5.26)
	Ciprofloxacin	0.125–8	0.25	1	1 (4)	0	5 (83.33)	6 (15.79)
Aminopenicillin	Ampicillin	1–128	16	128	12 (48)	2 (28.57)	5 (83.33)	19 (50)
	Methicillin	1–128	4	32	8 (32)	0	4 (66.67)	12 (31.58)
	Amoxicillin	2–256	16	256	9 (36)	1 (14.28)	5 (83.33)	15 (39.47)
	Piperacillin	2–256	16	128	8 (32)	0	4 (66.67)	12 (31.58)
Tetracycline	Tetracycline	0.5–64	16	32	17 (68)	2 (28.57)	6 (100)	25 (65.79)
Polymyxin	Polymyxin B	0.125–4	0.25	0.5	2 (8)	0	5 (83.33)	7 (18.42)
Phenicol	Chloramphenicol	2–128	16	64	11 (44)	2 (28.57)	6 (100)	19 (50)

MIC sensitivity standard according to the Clinical and Laboratory Standards Institute (2020). MIC50 and MIC90:MIC50 and MIC90 were the MICs required to inhibit the growth of 50% and 90% of the test cells in a single batch. The range of antibiotics tested according to the Clinical and Laboratory Standards Institute (2020).

ESBL, extended-spectrum β-lactamase; MIC, minimum inhibitory concentration.

Detection of drug-resistant and virulence genes

Of the 38 ESBL-producing E. coli strains, 17 (44.74%) harbored bla_TEM , 8 (21.05%) harbored bla_CTX-M , and 4 (10.53%) harbored bla_OXA . Although 7 (18.42%) of the 38 E. coli strains were resistant to ofloxacin, only 2 (5.26%) harbored mcr-1, and none of the remaining resistance genes was detected in these 38 E. coli strains. We found that the most common combination of genes was bla_TEM + bla _CTX (15.79%, 6/38), followed by bla_OXA +bla_TEM (5.26%, 2/38) (Fig. 1). Figure 1 shows that bla_TEM was the dominant resistance gene; it was mainly present in meat samples, less in human stool samples, and not detected in vegetable samples. All four resistance genes (mcr-1, bla_OXA , bla_TEM , bla_CTX-M-9 ) were detected in sample 07 (meat sample).

FIG. 1.

Heat map of antibiotic resistance genes, virulence gene, plasmid replicon genes, and integron genes (black squares indicate detection of study gene in these isolates, and gray squares represent absence of genes).

Similarly, out of these 38 strains, 20 (52.63%) harbored fimH, ompA, hlyE, and crl; 9 (23.70%) harbored intA, fimH, crl, hlyE, and ompA (Table 3).

Table 3.

Combination Pattern of Virulence Genes of Extended-Spectrum β-Lactamase Escherichia coli Isolated from Food and Human

No. of virulence genes	Combination patterns of virulence genes	No. of Escherichia coli isolates (%) (n = 38)
Seven	papC-papA-crl-hlyE-ompA-kpsMT II-fimH	1 (2.60%)
Six	iutA-sfa/foc-fimH-crl-hlyE-ompA	2 (5.26%)
Six	iutA-fimH-crl-hlyE-ompA-kpsMT II	1 (2.60%)
Five	fimH-crl-hlyE-ompA-kpsMT II	2 (5.30%)
Five	intA-fimH-crl-hlyE-ompA	9 (23.68%)
Four	fimH-crl-hlyE-ompA	20 (52.63%)
Four	hlyE-crl-ompA-kpsMT II	1 (2.60%)
Three	fimH-hlyE-ompA	1 (2.60%)
Three	fimH-crl-kpsMT II	1 (2.60%)

Molecular characteristics of ESBL-producing E. coli

The phylogenetic analysis was performed on the ESBL-producing E. coli isolates. The isolates were divided into three populations (A, B₁, and C), with B₁ as the dominant bacterium group. The isolates belonged to phylogroups B₁ (n = 16), C (n = 9), A (n = 8), and unknown (n = 5). Only one ESBL human isolate belonging to phylogenetic Group B₁ was found commonly in meat and vegetable samples (Fig. 2). Plasmid replicon typing was also performed on these isolates, and the results obtained were as follows: 16 of 38 strains were of IncFIB type (42.11%), 6 strains were of IncF type (15.79%), 3 strains were of IncP type (7.89%), 1 strain was of IncK/B type, and 1 strain was of IncX type (2.63%).

FIG. 2.

Cluster analysis of genetic relationship among 23 strains of 19 ST food-derived and human-derived Escherichia coli (only 23 of 38 E. coli isolates were successfully sequenced at the time of MLST. Therefore, 23 isolates were studied in MLST; 19 represents that only 19 STs were detected). MLST, multilocus sequence typing; ST, sequence-type.

Figure 1 shows that FIB was the dominant plasmid replicon gene, IncFIB was mainly present in human stool samples (sample nos. 8–13), and the rest were present in pork samples. The results of integron screening showed that 18 of 38 strains belonged to class 1 integrons (47.37%) (IntI1), 10 strains belonged to class 3 integrons (26.32%) (9 strains belonged to ISCR1 and 1 strain belonged to IntI3), and no class 2 integron was found (Fig. 1).

MLST results

The MLST was used to amplify and sequence seven housekeeping genes, and determine whether the strains were related by comparing the differences between sequences. Moreover, MLST helped examine germ line development relations among bacteria from an evolutionary perspective, with the advantage of easy-to-standardize data, and the results of different laboratories could be compared with each other through a database. Only 23 of 38 E. coli isolates were successfully sequenced at the time of MLST, and therefore, 23 isolates were studied in MLST. The ST strains obtained from E. coli were as follows: ST345, ST745, ST2035, ST155, ST58, ST5487, ST6921, ST109, ST13, ST448, ST602, ST197, ST206, ST398, ST48, ST10, ST5422, ST93, and ST38.

The types in samples were relatively dispersed. ST48, ST58, ST155, and ST345 were the dominant ST strains, but they accounted for only 8.70% (2/23) of the isolates and were not related to the source and type of samples. No new ST strains were identified in this study.

BioNumerics V8.0 software was used for the cluster analysis of 23 E. coli isolates from different sources (Fig. 2). The genetic similarity of the strains was poor. Of four clusters, three had a genetic similarity of <40% and the remaining one had a genetic similarity of 50% (Fig. 2). However, some of the clustered strains had certain genetic relationships, such as ST155, ST58, and ST5487, and the genetic similarity among the strains of ST48, ST10, and ST5442 clusters was more than 80%.

The gene characteristics of the same ST strain were not necessarily the same. For example, among the two ST345 strains, one strain contained the bla_TEM resistance gene and the other did not contain the gene. However, the genetic profiles of different ST strains might be consistent. For example, ST398 and ST48 (sample no. 39) had identical virulence genes, resistance genes, replicons, and integrons. The phylogenetics corresponding to the same ST strain were consistent. For example, two ST155 strains belonged to Group B₁; however, the sources were different: one from the human sample and the other from the pork sample (Fig. 2). However, the phylogenetics corresponding to different ST strains were also consistent; for example, ST345 and ST155 belonged to Group B₁ (Fig. 2).

Discussion

β-lactamase antibiotics are the most widely used antibacterial drugs in clinics. However, ESBL-producing E. coli is resistant to most β-lactamase antibiotics, negatively impacting clinical treatment. The existence of ESBL-producing E. coli in food and its possible transmission to humans through the food chain are noteworthy public health issues (Jalde and Choi, 2020). Yang et al. (2020) screened ESBL-producing E. coli from animal-derived food samples from the Sichuan Province of China from 2010 to 2016. The results showed that ESBLs were detected in 123 out of the 444 E. coli strains, with a total detection rate of 27.7%, and the detection rate of the bla_TEM genotype was the highest (88.6%).

The results of this study were similar to those of the study conducted by Yang et al., with the detection rate of ESBLs being 31.15% (38/123). Among the four types of β-lactamase enzymes, bla _CTX-M did not become the main genotype of ESBLs until the early 21st century, and bla_TEM and bla _SHV were mainly observed (Bradford, 2001). However, many studies documented that bla_TEM was the most popular genotype in meat samples. In a study conducted in Singapore, bla_TEM was detected in 45.3% (Guo et al., 2021) of livestock and poultry meat. Abdallah et al. (2015) also detected bla_TEM in livestock and poultry meat with a detection rate of 58%. The results of the present study were also consistent with those of Abdallah et al., bla_TEM was the most frequently detected resistance gene.

The detection of drug resistance genes in E. coli can help understand the types, numbers, and rules of drug resistance genes in epidemic strains. The drug resistance genes selected in this study were all β-lactam resistance genes (except mcr-1). In this study, bla_OXA , bla_TEM , and bla_CTX genes were detected, but the bla_SHV gene was not detected. The β-lactam resistance gene bla_TEM was the most prevalent (17/38; 44.74%).

Moreover, the phylogenetic classification of E. coli isolates is important for understanding E. coli populations and elucidating the relationship between strains and diseases (Halaji et al., 2022). According to the phylogenetic analysis, Clermont et al. (2011) divided E. coli isolates into eight phylogenetic groups: B₂, B₁, A, D, F, E, C, and Clade Ι. The commensal E. coli was mainly distributed in Group A, and the ones with more virulence factors were often located in Groups B₂ and D (Picard et al., 1999). Our results for the phylogenetic grouping of E. coli are consistent with those of Ramadan et al. (2020), they were all dominated by Group B₁. Figure 2 shows that the phylogenic grouping is dominated by the B₁ Group with lower toxins, and this may be due to the fact that most of the E. coli isolated from the selected samples in this trial were nonpathogenic.

Table 3 shows that a strain can carry up to six virulence genes, and at least three virulence genes. The detection rates of antiserum survival factor-related gene omp A, hemolysin-related gene hlyE, and colistin-related fimH were as high as 97.37% (37/38). Moreover, these virulence genes are present in both human and two other sources, which is consistent with the findings of Manges et al. (2015) that virulence genes are present in both human and food-animal hosts. The results of this study indicated that Group B₁ was the optimal phylogenetic group, as described earlier, Each strain in Group B₁ contained at least four or more virulence genes,which was consistent with previous findings.

As an effective method for bacterial genotyping, MLST can reflect the evolutionary relationship within the strain population and is suitable for large-scale epidemiological examinations (Larsen et al., 2012). Hence, the MLST bidirectional sequencing was performed on E. coli. The results showed that no new ST strains appeared, indicating that seven housekeeping genes were relatively stable. It is worth noting that the dominant ST-ST155 is present in both human and livestock meat sources, which is similar to the results of Manges et al. (2015).

The availability of mobile genetic elements, especially plasmids, via horizontal transmission is the main pathway for the emergence and transmission of AMR (von Wintersdorff et al., 2016). Transportable plasmids usually carry multiple resistance genes and facilitate the transmission and evolution of AMR. In view of this, the study tested for the presence of selected 18 plasmid replicons in E. coli. The results showed that E. coli isolates from different sources (e.g., meats, vegetables, and humans) shared the same Inc plasmid.

As a genetic element, integrons play an essential role in the antibiotic resistance of clinical E. coli strains, and they are also associated with various drug-resistant phenotypes (Stokes and Hall, 1989; Vinue et al., 2008). As integrons are known to be important in the development of antibiotic resistance, we examined the presence of three types of integrons in E. coli. Our findings revealed that only the first and third types of integrons were detected. In this test, multiple genetic elements could be detected simultaneously in 38 strains of drug-resistant E. coli.

This result is similar to the results of Sun et al., and further study is needed to determine which antibiotic resistance of E. coli is aggravated by integrons. Also the more resistant strains of E. coli were detected, and the mobile genetic elements were more detected. The multidrug-resistant strains carrying the same mobile elements also showed different degrees of multidrug resistance.

Although the sample size of this study was small, the sample sources were rich and representative, and two mcr-1 containing strains and polymyxin B-resistant strains were detected. I think there are two innovative points in this experiment. First: the drug resistance, genetic detection, clustering, and MLST of strains from different sources were carried out, especially the comparison of the characteristics of human samples and samples from other sources. Second: the abuse of antibiotics, polymyxin is the last line of defense, and we have detected seven strains resistant to polymyxin, the comparison of each characteristic of these strains with other strains is also one of the highlights of our study.

Conclusions

In this study, the prevalence, drug resistance, molecular characteristics, phylogenetic typing, and MLST analysis of ESBL-producing E. coli isolated from food and human stool samples in some parts of northern Xinjiang were monitored. Also, multidrug resistance and virulence genes were observed. The results showed that the drug resistance of E. coli in human stool samples and meat sources was high.

Footnotes

Acknowledgment

The author thanks Shihezi University for providing laboratory and experimental equipment.

Authors' Contributions

H.J., D.Z., Y.W., S.H., Y.N., X.Z., J.D., and B.L., conceived and designed the experiments in this study. Y.W. experiments farce boxingke sequencing company sequencing. Y.W. wrote the article.

Disclosure Statement

No competing financial interests exist.

Funding Information

This work was supported by grants from the National Natural Science Foundation of China (No. 32260621), the Corps Talent Project-Hua Ji, the Hebei Natural Science Foundation (No. C2021411001), and the Hebei University Science and Technology Research Project (No. ZD2021001).

Supplementary Material

Supplementary Table S1

Supplementary Table S2

Supplementary Table S3

Supplementary Table S4

Supplementary Table S5

Supplementary Table S6

Supplementary Table S7

References

Abdallah

, Reuland

, Wintermans

, et al. Extended-spectrum beta-lactamases and/or carbapenemases-producing Enterobacteriaceae isolated from retail chicken meat in zagazig, Egypt. PLos One, 2015; 10(8):e0136052.

Bennett

. Plasmid encoded antibiotic resistance: Acquisition and transfer of antibiotic resistance genes in bacteria. Br J Pharmacol, 2008; 153:S347–S357.

Bradford

. Extended-spectrum beta-lactamases in the 21st century: Characterization, epidemiology, and detection of this important resistance threat. Clin Microbiol Rev, 2001; 14(4):933–951.

Bush

, Jacoby

, Medeiros

. A functional classification scheme for beta-lactamses and its correlation with moleculation-structure. Antimicrob Agents Chemother, 1995; 39(6):1211–1233.

Carattoli

. Resistance plasmid families in Enterobacteriaceae. Antimicrob Agents Chemother, 2009; 53(6):2227–2238.

Carattoli

, Bertini

, Villa

, et al. Identification of plasmids by PCR-based replicon typing. J Microbiol Methods, 2005; 63(3):219–228.

Chen

, Chang

, Shi

, et al. Complete genome sequences and antimicrobia resistance analysis of two multi-drug resistant Escherichia coli isolated from broiler feces. Progr Vet Med, 2021; 42(4):6–11.

China Food and Drug Administration (CFDA). National Standard For Food Safety: Food Microbiology Test: Escherichia coli O157:H7/NM Test. State Health and Family Planning Commission of the People’ s Republic of China;, 2016:16.

Clermont

, Christenson

, Denamur

, et al. The clermont Escherichia coli phylo-typing method revisited: Improvement of specificity and detection of new phylo-groups. Environ Microbiol Rep, 2013; 5(1):58–65.

10.

Clermont

, Olier

, Hoede

, et al. Animal and human pathogenic Escherichia coli strains share common genetic backgrounds. Infect Genet Evol, 2011; 11(3):654–662.

11.

Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing. CLSI Supplement M100, 30th Ed. Clinical Laboratory Standards Institute: Wayne, PA; 2020.

12.

Dallenne

, Da Costa

, Decre

, et al. Development of a set of multiplex PCR assays for the detection of genes encoding important beta-lactamases in Enterobacteriaceae. J Antimicrob Chemother, 2010; 65(3):490–495.

13.

Dias

RCS

, Moreira

, Riley

. Use of fimH single-nucleotide polymorphisms for strain typing of clinical isolates of Escherichia coli for epidemiologic investigation. J Clin Microbiol, 2010; 48(2):483–488.

14.

Guerin

, Jove

, Tabesse

, et al. High-level gene cassette transcription prevents integrase expression in Class 1 integrons. J Bacteriol, 2011; 193(20):5675–5682.

15.

Guo

, Aung

, Leekitcharoenphon

, et al. Prevalence and genomic analysis of ESBLs-producing Escherichia coli in retail raw meats in Singapore. J Antimicrob Chemother, 2021; 76(3):601–605.

16.

Halaji

, Fayyazi

, Rajabnia

, et al. Phylogenetic group distribution of uropathogenic Escherichia coli and related antimicrobial resistance pattern: a meta-analysis and systematic review. Front Cell Infect Microbiol, 2022; 12:790184.

17.

Huja

, Oren

, Trost

, et al. Genomic avenue to avian colisepticemia. Mbio, 2015; 6(1): e01681-14.

18.

Iveth Miranda-Estrada

, Ruiz-Rojas

, Molina-Lopez

, et al. Relationship between virulence factors, resistance to antibiotics and phylogenetic groups of uropathogenic Escherichia coli in two locations in Mexico. Enferm Infecc Microbiol Clin, 2017; 35(7):426–433.

19.

Jalde

, Choi

. Recent advances in the development of beta-lactamase inhibitors. J Microbiol, 2020; 58(8):633–647.

20.

Johnson

, Murray

, Gajewski

, et al. Isolation and molecular characterization of nalidixic acid-resistant extraintestinal pathogenic Escherichia coli from retail chicken products. Antimicrob Agents Chemother, 2003; 47(7):2161–2168.

21.

Kang

, Kim

, et al. Risk factors for and clinical outcomes of bloodstream infections caused by extended-spectrum beta-lactamase-producing Klebsiella pneumoniae . Infect Contr Hosp Epidemiol, 2004; 25(10):860–867.

22.

Khafipour

, Plaizier

, Aikman

, et al. Population structure of rumen Escherichia coli associated with subacute ruminal acidosis (SARA) in dairy cattle. J Dairy Sci, 2011; 94(1):351–360.

23.

Kojima

, Ishii

, Ishihara

, et al. Extended-spectrum-beta-lactamase-producing Escherichia coli strains isolated from farm animals from 1999 to 2002: Report from the Japanese veterinary antimicrobial resistance monitoring program. Antimicrob Agents Chemother, 2005; 49:3533–3537.

24.

Larsen

, Cosentino

, Rasmussen

, et al. Multilocus sequence typing of total-genome-sequenced bacteria. J Clin Microbiol, 2012; 50(4):1355–1361.

25.

, Mehrotra

, Ghimire

, et al. Beta-Lactam resistance and beta-lactamases in bacteria of animal origin. Vet Microbiol, 2007; 121(3–4):197–214.

26.

Lippolis

, Holman

, Brunelle

, et al. Genomic and transcriptomic analysis of Escherichia coli strains associated with persistent and transient bovine mastitis and the role of colanic acid. Infect Immun, 2018; 86(1):e00566-17.

27.

Liu

, Ma

, Zhang

, et al. Classification and drug resistance anlysis of pathogenic Escherichia coli in diarrhea piglets. China Anim Husb Vet Med, 2018; 45(10):2876–2884.

28.

McNally

, Cheng

, Harris

, et al. The evolutionary path to extraintestinal pathogenic, drug-resistant Escherichia coli is marked by drastic reduction in detectable recombination within the core genome. Genome Biol Evol, 2013; 5(4):699–710.

29.

Manges

, Harel

, Masson

, et al. Multilocus sequence typing and virulence gene profiles associated with Escherichia coli from human and animal sources. Foodborne Pathog Dis, 2015; 12(4):302–310.

30.

Nardelli

, Marina Scalzo

, Soledad Ramirez

, et al. Class 1 integrons in environments with different degrees of urbanization. PLoS One, 2012; 7(6):e39223.

31.

Pereira

RVV

, Santos

TMA

, Bicalho

, et al. Antimicrobial resistance and prevalence of virulence factor genes in fecal Escherichia coli of holstein calves fed milk with and without antimicrobials. J Dairy Sci, 2011; 94(9):4556–4565.

32.

Picard

, Garcia

, Gouriou

, et al. The link between phylogeny and virulence in Escherichia coli extraintestinal infection. Infect Immun, 1999; 67(2):546–553.

33.

Ramadan

, Jackson

, Frye

, et al. Antimicrobial resistance, genetic diversity and multilocus sequence typing of Escherichia coli from humans, retail chicken and ground beef in Egypt. Pathogens, 2020; 9(5):357.

34.

Rebelo

, Bortolaia

, Kjeldgaard

, et al. Multiplex PCR for detection of plasmid-mediated colistin resistance determinants, mcr-1, mcr-2, mcr-3, mcr-4 and mcr-5 for surveillance purposes. Eurosurveillance, 2018; 23:29–39.

35.

Rehman

, Zhang

, Wang

, et al. Experimental mouse lethality of Escherichia coli strains isolated from free ranging tibetan yaks. Microb Pathog, 2017; 109:15–19.

36.

Shafiq

, Huang

, Shah

, et al. Characterization and resistant determinants linked to mobile elements of ESBLs-producing and mcr-1-positive Escherichia coli recovered from the chicken origin. Microb Pathog, 2021; 150:104722.

37.

Shaikh

, Fatima

, Shakil

, et al. Antibiotic resistance and extended spectrum beta-lactamases: Types, epidemiology and treatment. Saudi J Biol Sci, 2015; 22(1):90–101.

38.

Shu

, Hou

, Lin

, et al. Detection of Escherichia coli plasmid-mediated β-lactamase from livestock and poultry and characterization of antimicrobial resistance. J China Agric Univ, 2016; 21(8):92–97.

39.

Silva

, Leitao

, Tenreiro

, et al. Genomic and phenotypic characterization of Escherichia coli isolates recovered from the uterus of puerperal dairy cows. J Dairy Sci, 2009; 92(12):6000–6010.

40.

Stokes

, Hall

. A novel family of potentially mobile DNA elements encoding site-specific gene-integration functions: integrons. Mol Microbiol, 1989; 3(12):1669–1683.

41.

van Essen-Zandbergen

, Smith

, Veldman

, et al. Occurrence and characteristics of class 1, 2 and 3 integrons in Escherichia coli, Salmonella and Campylobacter spp in the Netherlands. J Antimicrob Chemother, 2007; 59(4):746–750.

42.

Vinue

, Saenz

, Somalo

, et al. Prevalence and diversity of integrons and associated resistance genes in faecal Escherichia coli isolates of healthy humans in Spain. J Antimicrob Chemother, 2008; 62(5):934–937.

43.

von Wintersdorff

CJH

, Penders

, van Niekerk

, et al. Dissemination of antimicrobial resistance in microbial ecosystems through horizontal gene transfer. Front Microbiol, 2016; 7:173.

44.

Wen

, Liang

, Gu

, et al. Comparison of methods of DNA extraction for real time PCR analysis. J Pathogen Biol, 2019; 14(6):621–624.

45.

Wirth

, Falush

, Lan

, et al. Sex and virulence in Escherichia coli: An evolutionary perspective. Mol Microbiol, 2006; 60(5):1136–1151.

46.

Woodford

, Carattoli

, Karisik

, et al. Complete nucleotide sequences of plasmids pEK204, pEK499, and pEK516, encoding CTX-M enzymes in three major Escherichia coli lineages from the United Kingdom, all belonging to the international O25:H4-ST131 clone. Antimicrob Agents Chemother, 2009; 53(10):4472–4482.

47.

, Xi

, Lv

, et al. Presence and antimicrobial susceptibility of Escherichia coli recovered from retail chicken in China. J Food Prot, 2014; 77(10):1773–1777.

48.

, Lv

, Li

, et al. In vitro bacteriostatic activity of 38 traditional Chinese medicines against a swine E.coli . Chin J Vet Med, 2020; 56(5):66–71.

49.

Yang

, Shu

, Zhao

, et al. Drug resistance of Escherichia coli isolates from food-antimals obtained from 2010 to 2016 in Sichuan. J Northwest A&F Univ (Nat Sci Ed), 2020; 48(9):24–30+36.

50.

Yang

, Zeng

, Lin

, et al. Antimicrobial resistance analysis and extended-spectrum-β-lactamaeses genes detetion in Escherichia coli isolated from swine. Chin J Zoonoses, 2019; 35(1):45–50.

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