Characteristics of MDR E. coli strains isolated from Pet Dogs with clinic diarrhea: A pool of antibiotic resistance genes and virulence-associated genes

The increasing number of multi-drug resistant (MDR) bacteria in companion animals poses a threat to both pet treatment and public health. To investigate the characteristics of MDR Escherichia coli (E. coli) from dogs, we detected the antimicrobial resistance (AMR) of 135 E. coli isolates from diarrheal pet dogs by disc diffusion method (K-B method), and screened antibiotic resistance genes (ARGs), virulence-associated genes (VAGs), and population structure (phylogenetic groups and MLST) by polymerase chain reaction (PCR) for 74 MDR strains, then further analyzed the association between AMRs and ARGs or VAGs. Our results showed that 135 isolates exhibited high resistance to AMP (71.11%, 96/135), TET (62.22%, 84/135), and SXT (59.26%, 80/135). Additionally, 54.81% (74/135) of the isolates were identified as MDR E. coli. In 74 MDR strains, a total of 12 ARGs in 6 categories and 14 VAGs in 4 categories were observed, of which tetA (95.95%, 71/74) and fimC (100%, 74/74) were the most prevalent. Further analysis of associations between ARGs and AMRs or VAGs in MDR strains revealed 23 significant positive associated pairs were observed between ARGs and AMRs, while only 5 associated pairs were observed between ARGs and VAGs (3 positive associated pairs and 2 negative associated pairs). Results of population structure analysis showed that B2 and D groups were the prevalent phylogroups (90.54%, 67/74), and 74 MDR strains belonged to 42 STs (6 clonal complexes and 23 singletons), of which ST10 was the dominant lineage. Our findings indicated that MDR E. coli from pet dogs carry a high diversity of ARGs and VAGs, and were mostly belong to B2/D groups and ST10. Measures should be taken to prevent the transmission of MDR E. coli between companion animals and humans, as the fecal shedding of MDR E. coli from pet dogs may pose a threat to humans.


Introduction
Escherichia coli (E.coli) is one of commensal microbiota in the gut of humans and animals [1].With the widespread use of antimicrobials, the occurrence of multi-drug resistance (MDR) E. coli in humans and animals has posed a major threat to public health [2,3].The presence of MDR E. coli in companion animals, such as pet dogs, undoubtedly raises concerns for both dogs and humans due to the high antimicrobial resistance (AMR) and the capability of carrying various antibiotic resistance genes (ARGs) of MDR strains [4,5].Furthermore, previous studies have shown that there were associations between ARGs and virulence-associated genes (VAGs) existed in E. coli, and ARG-carrying strains may increase the likelihood of carrying VAGs [6].However, only limited studies focused on MDR strains from pets in China, especially in Sichuan province which is considered as a major province for keeping pets in Southwest China [7].
In addition, recent studies have found that E. coli clones can be extensively shared between humans and household animals [8,9].The transmission of high-risk E. coli clones between animals and humans has been recognized as a major public health issue [10,11].For the population structure of E. coli clones, phylogenetic studies have shown that the B2 and D groups were more prevalent than the commensal groups (A and B1 groups) in E. coli isolates from diarrheic pet dogs [12,13].Moreover, the multilocus sequence typing (MLST) was also used to analyze the population structure of E. coli clones [14].Recent studies have identified a high population diversity of STs which related to zoonotic or pathotype in E. coli isolates from pet dogs, such as ST354, ST393, and ST457 E. coli were observed in companion animals from Australia [15].And the high-risk ExPEC clones associated with humans and multidrug resistance, including sequence type (ST) 38, ST131, ST224, ST167, ST354, ST410, ST617 and ST648, have been identified in cats and dogs in Thailand which suggested there may be clonal dissemination between pets and humans [16].
In 2023, the number of domestic pets has exceeded 100 million in China [7].Moreover, the latest annual report shows that the use of veterinary antibiotics for animals in China has exceeded 30,000 tons in 2020, tetracyclines, β-lactamases, and sulfonamides antimicrobial agents were widely used (Veterinary Bulletin of the Ministry of Agriculture and Rural People's Republic of China, 2020) [17].With the widespread use of antimicrobials in animals, the high prevalence of MDR E. coli isolates from pet dogs has raised health concerns for both companion animals and humans [18,19].To better understand the characteristics of MDR E. coli from pet dogs, we analyzed the antimicrobial resistance of E. coli isolates from diarrheal dogs, focusing on ARGs, VAGs, and population structure (ST and phylogenetic groups) for MDR E. coli to evaluate the potential threat.

Sample collection
A total of 185 fresh feces samples were collected from pet dogs with clinical diarrhea during August 2021 to June 2022.Samples were excluded if the pet dogs were prescribed antimicrobial therapy or veterinary admission within the previous 3 months.The study was permitted by committee of Sichuan agricultural university (Permission number: DYY-2020303164) and the Sichuan Agricultural University Animal Ethical and Welfare Committee (Permission number: 20210268).
All samples were collected by professional veterinarians in Veterinary Teaching Hospital of Sichuan Agricultural University.Disinfection of hands and changing of disposable gloves were mandatory before sample collection.Samples were taken from the center of fresh feces by using sterile cotton swabs as soon as the pet dogs defecated to avoid cross-contamination from environmental bacteria on the ground.Then, samples were collected in sterile micro centrifuge tubes (Eppendorf) and were sent for processing to the laboratory within 12 h after collection.

Strains isolation
The isolation and identification of E. coli was performed as previous studies described [20,21].Fecal samples were enriched in Luria-Bertani (LB) broth at 37˚C, 120 r/min in a shaking incubator for 24 h.All isolates were identified presumptively by using phenotypic methods, including Gram staining, MacConkey agar growth and Eosin Methylene Blue agar growth.We further used 16S rDNA sequences (Primer: 5'-GAGTTTGATCCTGGCTCAG-3'; 5'-AGAAAGG AGGTGATCCAGCC-3') [22] for final identification of E. coli.The confirmed isolates were stored in Luria-Bertani (LB) broth containing 50% glycerol at −20˚C for further analysis.

DNA extraction and detection of ARGs, VAGs
Total genomic DNA of strains was extracted from isolates by using TIANamp Bacteria DNA kit (Tiangen Biotech, Beijing, China) following the manufacturer's instructions.DNA samples were stored at -20˚C for subsequent polymerase chain reaction (PCR) detection.
Primers of 23 ARGs in 6 categories (including 5 bla CTX-M alleles for group1, 2, 8, 9, and 25) and 25 VAGs in 5 categories were synthesized by Huada Gene Technology Co. Ltd (Shenzhen, China).The PCR primer sequences and conditions for the ARGs and VAGs were showed in S1 and S2 Tables, respectively.PCR products were separated by gel electrophoresis in a 1.0% agarose gel in 1 × TAE buffer (40 mM Tris-acetate, 1 mM EDTA, pH 8.3), stained with Gold-View TM (Sangon Biotech, Shanghai, China), and photographed under ultraviolet light using the Bio-Rad ChemiDoc MP omnipotent imager (Bole, USA).All positive PCR products were sequenced with Sanger sequencing in both directions by Sangon Biotech (Shanghai, China).Sequences of ARGs and VAGs were analyzed online using the BLAST function of NCBI (http://blast.ncbi.nlm.nih.gov).

Phylogenetic grouping and MLST
Phylogenetic grouping (A/B1/B2/D) was determined for MDR E. coli isolates using PCR targeting chuA, yjaA, and TSPE4.C2 following the protocol of Clermont et al. [27].MLST was based on the sequencing results of seven housekeeping genes (adk, fumC, gyrB, icd, mdh, purA and recA) in each strain to obtain a numerical allelic profile which is abbreviated to a unique identifier of each strain: sequence type (ST) [28].Allelic types of all seven housekeeping genes and ST of strains were determined following the protocol of the E. coli MLST database (https://pubmlst.org)[28,29].The primer sequences of genes for MLST and phylogroups were shown in S3 Table.PCR products were separated by gel electrophoresis in a 1.0% agarose gel stained with GoldViewTM and photographed under ultraviolet light.All positive PCR products were sequenced with Sanger sequencing in both directions by Sangon Biotech (Shanghai, China).The sequences of housekeeping gene for MLST were analyzed online using pubMLST database (https://pubmlst.org).
The goeBURST algorithm in phyloviz 2.0 was used to clustering analysis of STs for MDR E. coli isolates, which divided the STs into several clusters, named as clonal complexes (CC), which consist of closely related STs with two allelic differences [30].A clonal complex is typically composed of a single predominant genotype and closely relatives genotype [31].

Association analysis between ARGs and Antimicrobial resistance phenotypes (AMRs) or VAGs
The statistically analysis of data of AMR, ARGs and VAGs was conducted by using SPSS Statistics (version 26.0).P-value < 0.05 was considered to be statistically significant.Association analysis between ARGs and AMR or VAGs was performed by ggplot2 in RStudio (version 4.2.2;http://www.r-project.org)[32].
Fourteen out of 25 VAGs in 4 categories were detected in our study (

Associations between ARGs and AMRs or VAGs in MDR E. coli strains
Details of the detection rates of ARGs and AMR in 74 MDR E. coli strains were showed in Table 2. High prevalence of the β-lactam antibiotics genes (bla TEM and bla CTX-M , 93.24% and 90.54%, respectively) were detected, and resistance rate of β-lactams antibiotic (AMP) was higher than 90% (71/74, 95.95%).For tetracyclines, tetA (71/74, 95.95%) was detected in MDR strains, and the proportions of strains with tetracyclines-resistant phenotypes were 95.95%.

Discussion
The widespread use of antibiotics has significantly caused the increase of MDR E. coli which were isolated from companion animals [33].To understand the characteristics of MDR strains and evaluate the potential threat, we detected the AMR of 135 E. coli isolates from pet dogs and further screened ARGs, VAGs, phylogenetic grouping and MLST for 74 MDR strains.One hundred and thirty-five E. coli isolates from pet dogs showed high resistance to TET, AMP and SXT, which was consistent with another study in China [34].Notably, TET has been used for treating animal infection disease and has also been used as "growth promoters" or "feed efficiency products" for a long time [35,36], which may activate the resistance of TET in E. coli.Once the antimicrobial-resistant E. coli occurs, it exists for a long time due to hard elimination [37].In addition, TET resistance has been widely observed in E. coli from animals, such as dogs [13,24], yaks [38] and pigs [39], and the antibiotic resistance genes encoding AMR can be transmitted through environment and mobile genetic elements [40][41][42], which may be one of the reasons for high TET-resistant E. coli detected in our present study, even though TET has not been used in the area recently.The similar phenomenon was also found in the resistant strains to 3rd/4th generation cephalosporins, fluoroquinolones and carbapenems antibiotics in our present study, which have been advocated to avoid or restrict use in veterinary antimicrobial selection in China [34,43].The resistance rates of AMP and SXT were consistent with the results from pet dogs in Nigeria, Australia and Brazil [19,23,44].According to previous studies, frequent use of antimicrobial agents in clinics may be responsible for the high resistance to AMP and SXT [45,46], the β-lactam antibiotics have also been widely used in the location of the present study, and SXT is one of the most animal-used antimicrobials in China [45].In our study, we further screened 74 MDR strains from 135 E. coli isolates.Compared with previous studies in Sichuan, the detection rate of MDR strains among E. coli isolates from pet dogs decreased from 100% in 2017 [47] and 70% in 2018 [48] to 54.81% in our study during 2021-2022, which was similar to the trend observed in Northeast China (which decreased from 76.92% in 2012-2013 to 62.42% in 2021) [43].The decrease in the MDR detection rate may be due to that the relevant authorities of China have released several policies to curb the increase in antimicrobial resistance in animal-derived bacteria [43].Although the detection rate of MDR E. coli from pet dogs in Sichuan has decreased, it is still higher than that detected in other countries [44,49].Overall, resistant strains may have been circulating in the dog population long before the antibiotic usage restriction were introduced, and AMR strains are currently difficult to eliminate.The high detection rates of MDR E. coli in our study implied that more effective measures should be taken to control the occurrence and spread of MDR E. coli from companion animals.
It is well-known that the antibiotic resistant phenotype of bacteria is related to ARGs [50].Therefore, we further analyzed the distribution of ARGs in 74 MDR strains.In our study, 12 out of 18 ARGs were detected in 74 MDR strains (detection rates ranged from 16.22% to 95.95%), of which tetA, bla TEM and bla CTX-M were the dominant ARGs.Notably, the detection rate of tetA in our study was higher than previous studies from dogs in China which ranged from 28% to 85.08% [43,51,52].The bla TEM and bla CTX-M were the prevalent β-lactam resistance genes in our study which were consistent with previous study for E. coli from dogs in China [43].We further analyzed the prevalent subtypes of bla CTX-M genes, bla CTX-M-55 (in bla CTX-M-1 group) and bla CTX-M-14 (in bla CTX-M-9 group) were more prevalent.Among variants of bla CTX-M-9 group, bla CTX-M-14 has been considered as a common CTX-M variant worldwide, especially in China, Korea and Japan [53].A high prevalence of bla CTX-M-14 was also observed in our present study (79.10%).Notably, bla CTX-M-14 was usually found on plasmid (such as IncF and IncK) according to previous studies [54,55].The high prevalence of bla CTX-M-14 -positive strains observed in our present study indicated a high risk of potential transmission.For variants of the bla CTX-M-1 group, bla CTX-M-55 has become the second most common CTX-M subtype in Chinese clinical E. coli isolates after bla CTX-M-14 [54], which was also dominant in our present study.Furthermore, Cottell et al. has observed the frequent clonal transmission of bla CTX-M-55 -positive E. coli between different hosts (humans and animals such as duck, chicken, and swine) [54].The high prevalence of bla CTX-M-14 -positive and bla CTX-M-55 -positive strains observed in our present study suggests more studies should focus on the capability of clonal transmission between different hosts in the future.
Increasing studies proposed that the relationship between AMRs and ARGs is not completely consistent [22,56].The detection rates of β-lactam and tetracycline antibiotics resistant phenotypes were generally consistent with the related ARGs.However, for sulfonamides, aminoglycosides, amide alcohols, and quinolones, the detection rates of related ARGs were fluctuated, e.g., the resistant rate of sulfonamides was 90.54% in MDR strains, while the detection rates of sul ranged from 17.57% to 70.27%.Moreover, we further analyzed the statistical associations between ARGs and AMRs, 23 positive association pairs were observed in MDR strains.Only flor and C, bla CTX-M and CTX/CXM/KZ, oqxAB and CIP, aacC2 and TOB/ CN showed consistency in ARG and related AMR.The other 16 association pairs were not completely consistent (Fig 3A).The similar phenomenon was also found in E. coli from captive non-human primates [22] and waterfowl [56] in China which indicated the different expression of ARGs, such as the abnormal expression of ARGs and the expression of ARGs has not reached that level which can activate the antibiotic resistance, may be a possible reason the incomplete consistency between AMR and ARGs [56].
Recent studies have shown that there was an association between ARGs and VAGs, and have proposed that they were linked and interacted with each other [6,56].In order to explore the virulence traits and the association of ARGs and VAGs in MDR E. coli, we analyzed the distribution of VAGs in MDR strains.In our present study, 25 VAGs were selected for detection according to previous studies, among which 12 VAGs (eae, papA, bfpA, aggR, pic, astA, ipaH, stx1, stx2, elt, esta and estb) are related to Diarrhoeagenic Escherichia coli (DEC) [57][58][59], the remaining 13 VAGs (fyuA, iroN, sitA, iucD, Irp2, fimC, tsh, vat, ompT, cvaC, iss, hlyF, hlyA) are related to Extraintestinal Escherichia coli (ExPEC) [60][61][62].Eleven ExPEC-related VAGs and 3 DEC-related VAGs (eae, astA, aggR) were detected in our present study.And the detection rates of VAGs in our study, which ranged from 4.99% to 100%, were higher than previous studies in dogs from Shaanxi and Shandong, China (ranging from 0.6% to 81.6% in Shaanxi and 2.53% to 87.34% in Shandong, respectively) [34,63].Moreover, 75.68% (56/74) of the MDR strains simultaneously combined at least one ExPEC-related and DEC-related VAG, indicating the MDR strains in our present study harbored VAGs related to different pathotypes and could be considered as potentially virulent hybrid pathogenic strains.MDR strains with potential hybrid pathogen detected in pet dogs will pose a threat to other companion animals and their owners.
In addition, the association between VAGs and ARGs among MDR strains was further analyzed.Five associated pairs were observed, of which 3 pairs were positive and 2 pairs were negative.Compared with other studies of E. coli from giant pandas (46 associated pairs, of which 45 pairs were positive and 1 pair was negative) [6] and waterfowl (43 associated pairs, of which 36 pairs were positive and 7 pairs were negative) [56], the associations observed between ARGs and VAGs in MDR strains from pet dogs were not common in our present study.The possible reasons for drawing associations between VAGs and ARGs may be related to the colocation on the same mobile genetic elements, while negative associations indicated gene incompatibilities [64].Recent studies also showed that many ARGs were inserted into conjugative plasmids carrying VAGs, which may provide conditions for drawing positive associations between ARGs and VAGs [65].In our study, the strongest positive association was observed between ompT and sul2, which is usually detected in plasmids, indicating the positive association between ompT and sul2 may be related to the plasmid [40,66].Similarly, the location of aacC, which usually was detected in the variable region of integron, may be related to the other two positive associations (aacC2/ompT, aacC4/astA) observed in our present study [5].The positive association between ARGs and VAGs implied the high risk of plasmid-mediated co-transmission of ARGs and VAGs in E. coli, which could accelerate the spread of VAGs and ARGs within E. coli populations and enhance the emergence possibility of new pathogens with increased virulence and resistance potential.Negative associations observed in our present study may also be related to mobile genetic elements.For example, the VAG cvaC is usually found in virulent IncF plasmids [66], while bla CTX-M is usually found in integron [67], the different locations on mobile genetic elements may be related to the negative associations between bla CTX-M and cvaC in our present study.While the specific mechanism for the associations observed in our study required more comprehensive studies to validate.
Previous studies have shown that groups A and B1 are considered to be commensal groups, and groups B2 and D are considered to be virulent extraintestinal-related groups [27], which have been classified as potentially pathogenic [5,6].A study from Iran showed that resistant E. coli strains mostly belonged to groups B2 and D [68].Similarly, our present results showed a high prevalence of strains belonging to virulent extraintestinal-related groups (B2 and D) were detected among MDR E. coli from pet dogs.According to previous study, phylogenetic grouping is closely related to virulence genes (group A and B1 strains carry fewer virulence genes, while group B2 and D strains carry more virulence genes) [6], whereas in our present study, no significant difference was observed in the average number of VAGs carried by per strains between commensal groups and virulent extraintestinal-related groups.These phenomena suggest that antimicrobial resistance may increase the possibility of carrying more virulent factors (for example, the number of VAGs carried) in E. coli strains [69], implying that highly antibiotics resistant intestinal E. coli strains may also lead to extraintestinal infections which could be an emergent public health issue for humans, animals and the environment [70,71].
At last, we used MLST to analyze the dominant lineage of MDR strains, and our results showed that 74 MDR strains exhibited a high population diversity of STs (42 STs were identified), of which ST10 was the most prevalent.Another study of E. coli from dogs in China has found that the pandemic clone was ST131 (accounting for 9.8%) [63], which was identified as the most common ST in ExPEC, covering all geographical regions [72].Moreover, studies from Australia and Thailand showed that ST354 and ST410 were the dominant clones in E. coli isolates from dogs, respectively [15,16].In our study, the most common ST was ST10 (occurring in 14.86% of strains) which was different from the above studies, indicating that the prevalent ST lineage of E. coli isolates from pet dogs may vary in different geographical regions.ST10 was widely detected in E. coli from other animals in China, such as pigs [73] and chickens [74], indicating that ST10 may be the prevalent ST in E. coli strains from animals in China.Moreover, ST10 strains mostly belonged to B2 groups in our present study, and this phenomenon was also observed in E. coli isolates from chickens [74] and in the ExPEC isolates from humans [75].In other studies, ST10 strains identified from pigs [73] and humans [76] mostly belonged to group A or D. This phenomenon indicated that the relationship between ST and phylogroups may vary in E. coli isolated from different hosts.

Conclusions and limitations
Seventy-four MDR strains were observed among 135 E. coli isolates from pet dogs, of which 12 ARGs and 14 VAGs were detected, implying that the E. coli isolates can be considered as a pool of ARGs and VAGs.Moreover, groups B2 and D, which are classified as potentially pathogenic, were predominant in the MDR isolates, and ST10 was the prevalent clonal lineage.Our findings have important implications for a preliminary understanding of the characteristics of MDR E. coli from diarrheal dogs and evaluating the potential risk of resistance and virulence in canine E. coli.
In our present study, the number of samples included was limited and the sampling site was only at our Veterinary Teaching Hospital.Future investigations should include more samples and expand sampling areas.Moreover, the use of High-throughput Sequencing or Whole Genome Sequencing could provide more comprehensive information on MDR E. coli strains from diarrheal dogs.

Fig 1 .
Fig 1. Antibiotic resistance patterns of E. coli isolates from pet dogs.(A) The abscissa 0R represents the strains that were sensitive to all antibiotics, and 1-6 R represents strains that were resistant to 1-6 antibiotic categories, respectively.Seventy-four E. coli isolates are MDR, of which 17 strains were resistant to 6 antibiotic categories; (B) Color bars demonstrate the distribution of phenotypic resistance patterns in 74 MDR E. coli isolates, and the Arabic number represent the number of strains.A total of 56 resistance patterns were observed by using disc diffusion assay.The red boxes highlight the prevalent resistant-phenotypes patterns (occurring three times), the other combinations (without red box) occurred only once or twice.https://doi.org/10.1371/journal.pone.0298053.g001 / 135) of isolates were identified as MDR E. coli (Fig 1A).Among the 74 MDR strains, 56 types of resistance phenotypic patterns were observed.The more common patterns were TET/SXT/ AMP, TET/DOX/C/SXT/AMP/AMC, TET/DOX/C/SXT/AMP, TET/C/SXT/AMP/AMC, and CN/TOB/TET/DOX/C/KZ/CXM/CTX/FEP/CIP/SXT/ATM/AMP (Fig 1B).

Fig 2 .
Fig 2. Distribution of antibiotic resistance genes (ARGs) and virulence-associated genes (VAGs) in 74 MDR E. coli strains from pet dogs.(A) The bar graphs show the detection rates of ARGs and VAGs.A total of 12 ARGs and 14 VAGs were detected, of which tetA (95.95%) and fimC (100%) were the most prevalent; (B) The abscissa represents the ID of MDR isolates and ordinate represents ARGs and VAGs.The red and blue regions represent the presence or absence of corresponding ordinate genes in an isolate, respectively.A high diversity of ARGs and VAGs was detected among MDR strains.https://doi.org/10.1371/journal.pone.0298053.g002

Fig 3 .
Fig 3. Heatmap of the correlation-coefficient (r) between ARGs and AMR or VAGs in 74 MDR E. coli strains from pet dogs.Blue indicates positive association (r > 0, P < 0.05) and red indicates negative association (r < 0, P < 0.05).The color scale on the right of figure indicates the r-valve: (A) Heatmap of the correlation coefficient between ARGs and AMRs.The color scale and corresponding r-valve indicate the association between corresponding abscissa AMRs and ordinate ARGs.Twenty-three positive association pairs were observed, of which the strongest association was found between flor and C; (B) Heatmap of correlation coefficient between ARGs and VAGs.The color scale and corresponding r-valve indicate the association between corresponding VAGs (abscissa)and ARGs (ordinate).Five association pairs were observed (3 association pairs were positive and 2 association pairs were negative), of which the strongest positive association was found between ompT and sul2, the strongest negative association was found between bla CTX-M and tsh.https://doi.org/10.1371/journal.pone.0298053.g003

Fig 4 .
Fig 4. Distribution of phylogenetic groups and STs in 74 MDR E. coli strains from pet dogs.(A) The distribution of phylogroups in MDR strains.Commensal groups included groups A and B1, and virulent extraintestinal-related groups included groups B2 and D. Marking * represents a significant difference, *, P < 0.05; **, P < 0.01; ***, P < 0.001.Significantly, the virulent groups were the most prevalent; (B) The average number of VAGs per isolate in each phylogroup.Marking * represents significant difference, *, P < 0.05; **, P < 0.01; ***, P < 0.001.Obviously, the highest value observed was 8.28 in group B2.; (C) Minimum spanning tree of MLST types in MDR strains.The circle size indicates the proportion of isolates belonging to the ST.The color within each circle represents phylogroups and indicates the proportion of isolates belonging to different phylogroups.Each link between circles indicates one mutational event and the distance is scaled as the number of allele differences between STs.The yellow-green outlines of the circles represent the ST is the founder ST of one clonal complex (CC), and the other STs (with purple outlines of the circles) of the CC are derived from the founder ST with two allelic differences.The 74 MDR strains exhibited a high diversity of STs (42 STs were identified) in our present study and ST10 was the most prevalent; (D) The clonal complexes (CCs) among the 42 STs.Forty-two STs were clustered into 6 clonal complexes, and the remaining 23 STs were single.ST-10CC was the most prevalent lineage containing 6 STs; (E) Heatmap demonstrates the distribution of STs and phylogroups.The color scale and corresponding value indicate the number of E. coli isolates belonging to the corresponding phylogroups (abscissa) and STs (ordinate).Blue indicates a low number of strains, white indicates an intermediate value, and red indicates a high number of strains.As the heatmap shows, B2-ST10 was the most prevalent clone.https://doi.org/10.1371/journal.pone.0298053.g004