Prevalence of CTX-M types among ESBL-producing pathogenic Escherichia coli isolates from foodborne diarrheal patients in Gyeonggi-do, South Korea

Prevalence and characteristics of extended-spectrum β-lactamase (ESBL)-producing pathogenic Escherichia coli from foodborne diarrheal patients were studied. Analysis of 495 E. coli isolates revealed that 80 isolates were ESBL-producing pathogenic E. coli, and enteroaggregative E. coli and enterotoxigenic E. coli were two of the most prevalent pathotypes. In silico Clermont phylo-typing of the 80 ESBL-producing E. coli showed that phylogroup A (49/80) and D (22/80) were the predominant phylogroups. The average nucleotide identity analysis of ESBL-producing E. coli disclosed that they could be grouped into two phylogenetic groups; 25 A and 55 B groups. All strains, except one, harbored the blaCTX-M gene. All CTX-M-15 type ESBL-producing strains also carried qnrS, a plasmid-mediated quinolone resistance gene (PMQR). These results suggest that the diversity of ESBL-producing E. coli is high and that co-existence of blaCTX-M-15 and qnrS genes is widespread, highlighting their high risk of antibiotic-resistance spreading in infectious disease outbreaks. Supplementary Information The online version contains supplementary material available at 10.1007/s10068-024-01549-5.


Introduction
Even though many countries have food safety guidelines and foodborne disease regulations, large-scale foodborne disease outbreaks continue to increase worldwide (Lee & Yoon, 2021).Foodborne diseases can be divided into two categories, infection or poisoning, which can be further categorized according to etiological agents such as viruses, bacteria, parasites, and chemicals (Bari & Yeasmin, 2018).Among these agents, pathogenic Escherichia coli is one of the most common cause of foodborne disease outbreaks reported in the United States, Japan, and South Korea (Dewey-Mattia et al., 2018;Lee & Yoon, 2021).The Ministry of Food and Drug Safety of South Korea estimates that 5,582 outbreaks of foodborne-related illness occurred in South Korea from 2002 to 2022, resulting in 139,630 patients suffering from foodborne diseases.Norovirus (29.4%) and pathogenic E. coli (22.2%) are two of the most frequently reported etiological agents associated with foodborne disease outbreaks in South Korea (https:// www.foods afety korea.go.kr/).The most serious outbreak of E. coli-caused foodborne illness was reported in Germany in 2011, which subsequently spread to many other countries, resulting in 3,816 identified cases of enterohemorrhagic E. coli infection and 54 associated deaths worldwide (Buchholz et al., 2011;Frank et al., 2011).
In this study, to investigate the distribution of ESBLs and their genomic diversity in ESBL-producing pathogenic E. coli strains isolated from patients with food poisoning, we examined the resistance of 80 ESBL-producing pathogenic E. coli strains to various antibiotics and analyzed their genomic diversities by whole genome sequencing (WGS).This information will help us to better understand the molecular epidemiology of antimicrobial-resistant pathogenic E. coli isolated from foodborne disease outbreaks.

Isolation and identification of ESBL-producing pathogenic E. coli
A total of 495 pathogenic E. coli strains, including 80 ESBL-producing pathogenic E. coli, were isolated from the diarrhea patients (one sample per person) by standard rectal swab sample collection method in Gyeonggi-do, South Korea, by the Research Institute of Health & Environment from 2014 to 2018.General information on 80 ESBL-producing pathogenic E. coli strains was listed in Table S1.For isolation of pathogenic E. coli, each swab sample was incubated at 37 °C for 16 h in tryptone soy broth (TSB; Oxoid, Basingstoke, UK).An aliquot of each TSB enrichment was streaked on MacConkey Agar (Oxoid, Basingstoke,UK) and incubated at 37 °C for 24 h.The pink single colonies were sub-cultures on tryptic soy agar (TSA; Oxoid, Basingstoke,UK) and confirmed using the VITEK 2 system with gram-negative (GN) identification card (bio-Mérieux, Marcy, France).For screening of ESBL-producing pathogenic E. coli strains, identified E. coli strains were cultured on CHROMagar ESBL (CHROMagar, Paris, France) at 37 °C for 24 h.The dark pink to reddish single colonies were sub-cultured on TSA (Oxoid).ESBL production was confirmed using VITEK 2 system with AST-N169 card (bioMérieux).

Pathotype determination of pathogenic E. coli
The presence of virulence genes associated with pathogenic properties of E. coli were determined by PCR amplification.The isolates were cultured in TSB at 37 °C for 24 h, and their genomic DNA was extracted from overnight cultures of pathogenic E. coli isolates using the Nextractor NX-48 system and NX-48 bacterial DNA kits (Genolution Inc., Seoul, Korea).PCR was performed using PowerChek™ Diarrheal E. coli 8-plex Detection Kit (Kogenbiotech, Seoul, Korea) according to the manufacturer's instructions.The amplified virulence genes of pathogenic E. coli strains determined ETEC (st and lt encoding heat-stable and heat-labile enterotoxins), EHEC (VT1 and VT2 encoding verocytotoxin 1 and 2), EPEC (eaeA encoding intimin), EAEC (aggR encoding transcription regulator for aggregative adherence fimbria I), and EIEC (ipaH encoding invasion plasmid antigen H).

Whole genome sequencing
All ESBL-producing pathogenic E. coli strains (n = 80) were subjected to whole genome sequencing.Genomic DNA of the isolates was extracted a NucleoSpin Microbial DNA kit (Macherey-Nagel, USA) and TissueLyser II (Qiagen, Germany) by following the manufacturer's instructions.The quality of genomic DNA was determined by NanoDrop spectrophotometer (Thermo-Fisher Scientific, USA), standard agarose gel electrophoresis, and the Qubit 3.0 fluorometer (Thermo-Fisher Scientific).Intact genomic DNA was sheared by a Covaris S220 ultra sonicator (Covaris, USA), and the sequencing library was constructed using the Illumina TruSeq Nano DNA LT library prep kit (Illumina, USA) according to the TruSeq Nano DNA Library Preparation protocol.The quality of the libraries was assessed on a 2100 Bioanalyzer System with DNA 1000 Chip (Agilent Technologies, USA).Sequencing was performed using the Next-Seq 500 Sequencing System (Illumina, USA) to generate 2 × 150 bp read length.The contigs of genomic sequences were de novo assembled using CLC Genomics Workbench v20 (Qiagen, USA) with default parameters.

Isolation of ESBL-producing pathogenic E. coli
A total of 495 pathogenic E. coli strains were isolated from 1,901 clinical specimens obtained from diarrhea patients of foodborne outbreaks in Gyeonggi-do, South Korea from 2014 to 2018.The presence of virulence genes associated with pathogenesis of E. coli was determined by PCR amplification and they could be classified into four pathotypes  1).ETEC and EAEC were identified as the two most frequently isolated E. coli pathotypes, consistent with the previous reports from Iran (Alizade et al., 2019) and Bangladesh (Fahim et al., 2018).The hybrid strains of pathogenic E. coli, which contain multiple virulence genes that may confer higher virulence, have recently been reported worldwide (Santos et al., 2020).However, no hybrid strains were detected in the present study.Antibiotics resistance of the 495 pathogenic E. coli isolates were tested by CHROMagar ESBL selective medium and 80 isolates (16.2%) showed resistance to β-lactam antibiotics.The pathotypes of these 80 β-lactam-resistant isolates were 44 ETEC, 26 EAEC, and 10 EPEC.But none of them were EHEC or EIEC (Table 1).
The prevalence of ESBL-producing pathogenic E. coli in this study was similar to the previously published data from South Korea and China (Song et al., 2009;Xu et al., 2018).These results highlight the diverse distribution of pathogenic E. coli pathotypes in diarrhea patients in Gyeonggi-do, South Korea.Moreover, the emergence of antibiotic resistance, particularly the 16.2% resistance to ESBLs, emphasizes the need for continuous monitoring and surveillance.

Antimicrobial susceptibility of the ESBL-producing E. coli
The antimicrobial susceptibilities of 80 ESBL-producing pathogenic E. coli isolates were tested with 17 antibiotics as described in the Materials and Methods and analyzed the results according to CLSI criteria.All tested isolates showed resistance or intermediate resistance to penicillins (e.g., ampicillin), first-generation cephalosporins (e.g., cefalotin and cefazolin), and third-generation cephalosporins (e.g., cefotaxime and ceftriaxone), whereas only four isolates showed resistance or intermediate resistance to secondgeneration cephalosporins (e.g., cefotetan and cefoxitin).All ESBL-producing pathogenic E. coli isolates were susceptible to carbapenems (e.g., imipenem) (Table S2).While bacteria with resistance to three or more categories of antibiotics were defined as multidrug resistance (MDR) (Nath et al., 2020;Magiorakos et al., 2012), all ESBL-producing pathogenic E. coli isolates examined in this study showed multidrug resistance to five or more antibiotics.Interestingly, 32 out of 44 of the ETEC strains (72.7%, 32/44) showed resistance to five antibiotics with an AMP-CEF-CFZ-CTX-AXO pattern and 12 EAEC strains (46.2%, 12/26) showed resistance to eight antibiotics with an AMP-SAM-CEF-CFZ-CTX-TET-CHL-SXT pattern (Table 2).Remarkably, all isolates displayed multidrug resistance to five or more antibiotics, and EAEC/ETEC pathotype-specific resistance was observed.

Genetic diversity of ESBL-producing pathogenic E. coli
To analyze genetic diversity of ESBL-producing pathogenic E. coli, whole genome sequencing (WGS) and comparative phylogenetic analysis were performed.For this study, multilocus sequencing typing (MLST), phylogroup analysis, and average nucleotide identity (ANI) analysis based on WGS data were conducted.Bioinformatic analysis of the genomes of 80 ESBL-producing pathogenic E. coli strains revealed that they have an average genome size of 5,346,277 bp, with 1,450 coding sequences (CDSs), seven rRNA genes, and 83 tRNA genes.MLST analysis of 80 ESBL-producing pathogenic E. coli strains using seven house-keeping genes showed that they could be classified  3).The Clermont phylo-typing analysis revealed that phylogroup A (61%, 49/80) and D (28%, 22/80) were the predominant phylogroup in 80 ESBL-producing pathogenic E. coli strains.

Co-harboring of extended-spectrum β-lactamase encoding genes and other antimicrobial resistance genes
It is well known that most of the ESBL-producing pathogenic E. coli carry additional antibiotics resistant genes (Park et al., 2022).Here, all ETEC and EPEC strains harboring the blaCTX-M gene also carried plasmid-mediated quinolone resistance (PMQR), namely qnrS1 (98.0%, 48/49) and qnrS2 (2.0%, 1/49) (Fig. 2).Several previous studies have reported co-existence of PMQR and ESBL genes in E. coli (Azargun et al., 2018;Nazik et al., 2011Viana et al., 2013).The qnr genes were highly specific to ETEC strains in this study (Fig. 2).Previous studies have also reported the co-existence of qnr and blaCTX-M genes in ESBL-producing ETEC, which may be because both genes encoding enterotoxins (LT/ST) and PMQR are plasmid-borne (Gyles et al., 1974;Jiang et al., 2008).However, further research is needed to understand these results better.Likewise, a high prevalence of qnr genes among ESBL-producing E. coli has been previously reported in South Korea (Park et al., 2007).In addition, most of the EAEC strains harboring the blaCTX-M-14 gene also carried the chloramphenicol resistance gene catA1 (92%, 23/25), folate pathway antagonist gene sul1/dfrA5 (88%, 22/25), and tetracycline gene tet(A) (92%, 23/25) (Fig. 2).To confirm the antibiotic resistance attributable to the co-existence of antimicrobial resistance genes, the minimal inhibitory concentrations (MICs) of ciprofloxacin, tetracycline, chloramphenicol, and trimethoprim/ sulfamethocxazole were determined (Table 4).The MICs of tetracycline, chloramphenicol, and trimethoprim/sulfamethoxazole were higher than the breakpoints of CLSI and EUCAST guidelines (Table S3) in most of the CTX-M-14 type ESBL-producing EAEC strains carrying catA1, sul1, dfrA5, and tet(A).This suggests that the phenotype was consistent with the genotype in these strains.Although E. coli is a common commensal gut bacterium, our findings emphasize the high prevalence of ESBL and PMQR gene in pathogenic E. coli strains isolated from diarrhea patients of foodborne disease outbreaks.These results suggest that the prevalence of ESBL-producing pathogenic E. coli strains may become an important public health concern, highlighting the urgent necessity for monitoring the spread of these foodborne pathogens.

Fig. 1
Fig. 1 ANI phylogenetic tree of 80 ESBL-producing pathogenic E. coli clinical isolates from foodborne disease in Gyeonggi-do.ESBLproducing pathogenic E. coli strains are grouped into two major

Fig. 2
Fig. 2 Distribution of antimicrobial resistance (AMR) genes in ESBL-producing pathogenic E. coli isolates.The orange color indicates the presence of β-lactamase genes and blue color indicates the presence of other antimicrobial resistance genes based on ResFinder

Table 3
Distribution of E. coli