Comparative Genetic Characterization of CTX-M-Producing Escherichia coli Isolated from Humans and Pigs with Diarrhea in Korea Using Next-Generation Sequencing

Pathogenic E. coli causes intra- and extraintestinal diseases in humans and pigs and third-generation cephalosporins are the primary option for the treatment of these diseases. The objective of this study was to investigate the characteristics and correlation between CTX-M-producing E. coli from humans and pigs regarding CTX-M-producing E. coli using next-generation sequencing and bioinformatic tools. Among the 24 CTX-M–producing E. coli, three types of CTX-M genes (CTX-M-12, CTX-M-14, and CTX-M-15) were detected in humans and four types of CTX-M genes (CTX-M-14, CTX-M-15, CTX-M-55, and CTX-M-101) were detected in pigs. A total of 24 CTX-M–producing E. coli isolates also showed the following antimicrobial resistance genes: other B-Lactam resistance gene (75.0%); aminoglycoside resistance genes (75.0%); phenicol resistance genes (70.8%); tetracycline resistance genes (70.8%); sulfonamide resistance genes (66.7%); quinolone resistance genes (62.5%); trimethoprim resistance genes (54.2%); and fosfomycin resistance genes (8.3%). FII (92.3%) and FIB (90.9%) were the most common plasmid replicon in humans and pigs, respectively. A total of thirty-eight different genes associated with virulence 24 CTX-M–producing E. coli and all isolates contained at least more than one virulence gene. A total of 24 CTX-M–producing E. coli isolates showed 15 diverse sequence types (STs): thirteen isolates from human belonged to 6 different STs, and 11 isolates from pig belonged to 9 different STs. The presence of virulence genes in E. coli together with antimicrobial resistance genes (including CTX-M genes) emphasizes the necessity of comprehensive surveillance and persistent monitoring of the food chain to avoid all types of bacterial contamination, regardless of human or pig origin.


Introduction
Escherichia coli is a widespread and abundant bacterium that forms part of the intestinal commensal microbiota of animals and humans. Although many E. coli strains are harmless commensals, the species comprises several zoonotic pathovars that cause intraand extraintestinal diseases in humans and animals, such as diarrhea, urinary tract infections, meningitis, or septicemia [1]. Fluoroquinolones, trimethoprim-sulfamethoxazole, and third-generation cephalosporins are the primary antimicrobial options for treating infections caused by pathogenic E. coli [2].
Third-generation cephalosporin resistance is usually mediated by extended-spectrum β-lactamases (ESBLs), which are generally encoded by genes on mobile genetic elements, such as plasmids and transposons [3]. There are three main types of ESBL (TEM, SHV, or CTX-M) based on their substrate profiles and primary sequences. [4]. Among these, the most prevalent extended-spectrum enzymes correspond to the CTX-M type [5]. Because of the explosive dissemination of CTX-Ms globally, a "CTX-M pandemic" has been reported due to their worldwide increase [6]. CTX-M types have been divided into at least five groups

Whole Genome Sequencing and Analysis
Paired-end DNA libraries were prepared using a Nextera kit (Illumina, San Diego, CA, USA) according to the manufacturer's instructions. Briefly, genomic DNA was tagmented by simultaneously fragmenting and tagging with adapter sequences using the Nextera transposome (Nextera XT DNA Library Preparation Kit, Illumina, San Diego, CA, USA). The tagmented DNA was then amplified using a PCR program. The amplified DNA was purified using with AMPure XP beads. Nextera libraries were then quantified using a Qubit 4 fluorometer, and the size profile was evaluated on an Agilent Technology 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany). Sequencing was performed on the Illumina MiSeq instrument in a 2 × 300 bp format using a MiSeq reagent kit v3 (Illumina). The SPAdes (v3.12) assembler was used for generating assembled genomes and analyzed for antibiotic resistance characteristics (ResFinder v3.1), sequence type (ST) (MLST v2.0), virulence genes (VirulenceFinder v2.0), and incompatibility groups (Inc types) of plasmids (PlasmidFinder v2.0) from the Center for Genomic Epidemiology (Table S1).

Virulence Factors
A total of 38 different genes associated with virulence were identified in 24 CTX-Mproducing E. coli isolates, and all isolates contained at least more than one virulence gene. The genes iha and papA (coding for adhesins), fyuA, and irp2 (coding for iron acquisition systems), kpsMII (coding for adhesins), and sat (coding for toxins) were the most frequent virulence genes in CTX-M-producing E. coli isolates from humans. The genes csgA (coding

Virulence Factors
A total of 38 different genes associated with virulence were identified in 24 CTX-Mproducing E. coli isolates, and all isolates contained at least more than one virulence gene. The genes iha and papA (coding for adhesins), fyuA, and irp2 (coding for iron acquisition systems), kpsMII (coding for adhesins), and sat (coding for toxins) were the most frequent virulence genes in CTX-M-producing E. coli isolates from humans. The genes csgA (coding for adhesins), iucC, iutA, and sitA (coding for iron acquisition systems); traT (coding for adhesins); and hlyE (coding for toxins) were the most frequent virulence genes in CTX-Mproducing E. coli isolates from pigs. The ompT gene was the most predominant virulence gene coding for miscellaneous factors in both humans and pigs.
for adhesins), iucC, iutA, and sitA (coding for iron acquisition systems); traT (coding for adhesins); and hlyE (coding for toxins) were the most frequent virulence genes in CTX-Mproducing E. coli isolates from pigs. The ompT gene was the most predominant virulence gene coding for miscellaneous factors in both humans and pigs.

Discussion
Third-generation cephalosporin-resistant E. coli may have been selected and maintained in humans and pigs owing to the use of third-generation cephalosporins or coselection after the use of other antimicrobials, such as aminoglycosides, β-lactams, phenicol, quinolone, sulfonamides, trimethoprim, and tetracyclines [3]. The majority of isolates described in this study also contained resistance genes to these classes of antimicrobials. The tetA gene was one of the most frequently observed resistant genes in the CTX-Mproducing E. coli isolates, which is consistent with the findings of Girlich et al. [24]. The sul1 and sul2 genes, which encode a sulfonamide-resistant dihydropteroate synthase, were identified in 11 (45.8%) CTX-M-producing E. coli isolates. In addition, we found that 12 (50.0%) CTX-M-producing E. coli isolates carried aph(3 )-Ib and aph(6)-Id genes, which encode aminoglycoside adenylyltransferases. These two genes have already been reported as major determinants of sulfonamide and gentamicin resistance in pathogenic E. coli, respectively [25,26]. Although chloramphenicol was banned in food-producing animals in Korea in 1990 because of its suspected carcinogenicity, catB3 and cmlA1 genes, which encode a specific chloramphenicol transporter, were detected in nine (37.5.0%) and five (20.8%) CTX-M-producing E. coli isolates, respectively. The plasmid-mediated quinolone resistance (PMQR) genes aac(6 )-Ib-cr, qacE, and qnrS1 were identified in both pigs and humans. PMQR genes may be significantly associated with the β-lactamase gene and are detected in CTX-M-producing E. coli at high levels [27]. In addition, the most common plasmid replicons were IncF plasmids, including FIB (79.2%), FII (70.8%), and FIA (62.5%). IncF plasmids play an important role in the spread of antimicrobial (including ESBLs) and virulence and resistant determinants among pathogenic E. coli [28].
Some virulence-associated genes have been described as important genes because they cause intestinal/extraintestinal infections in humans and animals [29]. The air, astA, and eilA genes, which were detected in pig isolates, have been related with enteroaggregative E. coli in humans [30]. These virulence genes can play a role in the pathogenic potential in humans [30]. The iss and lpfA, associated with the extraintestinal pathogenic E. coli reported in a humans and pigs worldwide, were also found in both humans and pigs in this study [31][32][33]. In particular, the iss gene is recognized as being liable for E. coli immune evasion by increasing serum survival and also for its role in extraintestinal pathogenic Escherichia coli (ExPEC) for enhanced survival of bacteria in serum [30]. In addition, the iha gene, dominating the ExPEC adherence virulence genes, has been identified in both humans and pigs [34,35]. We also found several toxins that belong to the ExPEC group, such as ireA, cnf1, vat, sat, senB, and pic, in animals, humans, or both.
In this study, CTX-M-15-producing E. coli was mainly related with the ST131, which was not found in the E. coli from pigs in our study. CTX-M-15 was detected in E. coli ST327 and ST1642 in pigs, which were also found in human patients [36,37] but less frequently than ST131. The most prevalent STs in pigs were ST100 and ST10, which are the predominant enterotoxigenic E. coli types, and which are important pig pathogens in Canada, Germany, the United States, and Thailand (http://mlst.warwick.ac.uk/mlst/dbs/Ecoli, accessed on 30 April 2023). Because diverse STs are related to pathogenic and antimicrobial resistant strains, the emergence of a pathogenic E. coli showing diverse STs may pose a risk.

Conclusions
In this study, we genetically characterized and investigated the correlation of CTX-M-producing E. coli isolated from humans and pigs suffering from diarrhea in Korea. Although diseased pigs are less likely to be a source of antimicrobial-resistant bacteria compared to healthy pigs that enter food chain, the emergence of antimicrobial-resistant bacteria can be a public health problem as they are transmitted between pigs. This is the first study to genetically characterize and investigate the prevalence and interrelation of CTX-M-producing E. coli isolated from humans and pigs with diarrhea in Korea. The presence of virulence genes in E. coli together with antimicrobial resistance genes (including CTX-M genes) emphasizes the necessity for comprehensive surveillance and persistent monitoring of the food chain to avoid all types of bacterial contamination, irrespective of human or pig origin.