Antimicrobial resistance and genomic characterization of Escherichia coli from pigs and chickens in Zhejiang, China

Escherichia coli is considered an opportunistic pathogen and an indicator for antimicrobial resistance (AMR) monitoring. Despite many reports on its AMR monitoring, studies based on genome-based analysis of AMR genes are still insufficient. Here, 181 E. coli strains were isolated from anal swab samples collected from pigs and chickens of animal farms located in Eastern China and sequenced through the Illumina platform. The results showed that 87.85% (159/181) of the E. coli isolates were multidrug-resistant (MDR). Ampicillin (AMP)- spectinomycin (SPT)- tetracycline (TET)- florfenicol (FFC)- sulfisoxazole (SF)- trimethoprim/sulfamethoxazole (SXT) was the predominant AMR pattern. By whole-genome sequencing, we found that ST10 (10.49%, 19/181) and ST48 (7.18%, 13/181) were major sequence types. IncFIB and IncX1 were the most prevalent plasmid replicons. The AMR genes blaNDM-5 (1.10%, 2/181), mcr-1 (1.10%, 2/181), tet(X4) (1.10%, 2/181), and cfr (6.08%, 11/181) were also found in these isolates. In addition, among the 169 virulence genes detected, we identified astA (37.02%, 67/181), hlyA (1.66%, 3/181), hlyB (1.66%, 3/181) and hlyD (1.66%, 3/181), which were closely related to heat-stable enterotoxin 1 and α-hemolysin. In addition, there were 33 virulence genes associated with the iron uptake system, and 46 were adhesion-related genes. Our study highlighted the need for routine surveillance of AMR with advanced genomic approaches, providing up-to-date data on the prevalence of AMR for the development and execution of antimicrobial stewardship policy.


Introduction
The recent emergence and rapid increase of multi-drug resistant (MDR, resistance to more than three kinds of antibiotics) bacteria have caused public concern, represented by Escherichia coli resistant to carbapenem, colistin and tigecycline which were recognized as the last line of resort (Li et al., 2017;He et al., 2019;Ma et al., 2022). Due to incorrect use and misuse of antibiotics, the spread of antimicrobial resistance (AMR) is accelerating Tang et al., 2022a,b). The continuous spread of AMR not only increases the difficulty of preventing and controlling livestock and poultry diseases but also seriously threatens livestock products' safety and endangers consumers' health (Xu et al., 2022;Li et al., 2022a).
E. coli is a commonly used AMR indicator in human and food animal (Brisola et al., 2019;Ma et al., 2022). There have been many reports on the study in E. coli and monitoring of the spread of AMR. The emergence of plasmid-mediated carbapenem resistance genes, especially bla NDM , has seriously affected the efficacy of meropenem (0.4%, 1/219; Tang et al., 2019Tang et al., , 2021b. Colistin resistance mediated by a plasmidencoded mcr-1 was first documented in China during routine surveillance of food animals (21%, 166/804; Liu et al., 2016). A retrospective survey showed that mcr-1 was first traced back to 1980 but was not prevalent among bacteria until 2009 (Shen et al., 2016). With the detection rate of mcr-1 in bacteria increasing year by year, a relatively high occurrence rate of the mcr-1 gene (1%) was detected from E. coli in human (Wang et al., 2017). Tigecycline resistance are mediated by two novel genes, tet(X3) and tet(X4), both of which can significantly reduce the efficacy of tigecycline (He et al., 2019;Sun et al., 2019;Guan et al., 2022). Additionally, bacterial strains carrying the cfr gene encoding 23S rRNA methylase, which is resistant to five classes of antimicrobials, including phenols, lincosamides, oxazolidinones, pleuromutilin, and streptomycin A,allowing bacteria to develop MDR (Deng et al., 2014;Tang et al., 2022c).
Whole genome sequencing (WGS) played a vital role in AMR study (Tang et al., 2020b;Li et al., 2022b). We can obtain the tested strains' AMR and virulence genes by combining them with the antimicrobial sensitivity test (AST). It is helpful to better understand AMR's development and transmission (Teng et al., 2022). WGS has become an indispensable and reliable tool for revealing the AMR mechanism in global pathogen surveillance (Boolchandani et al., 2019;Tang et al., 2021a;Yang et al., 2022).
In this study, we investigated the prevalence of AMR E. coli in pigs and chickens from animal farms in Eastern China and evaluated the AMR phenotypes, genotypes, virulence genes, and plasmids replicons. This study helps understand the AMR situation and provides a reference to formulate livestock AMR control policies to better protect food safety in China.

Sample collection and strain isolation
A total of 200 anal swab samples were collected from three cities (Lishui, Jinhua, and Quzhou) located in Zhejiang Province, Eastern China, from March to April 2021. The samples were randomly collected from 110 chickens and 90 pigs in 5 poultry farms and 4 swine farms, respectively (Table 1; Figure 1A). All experiment activities in this study were approved by the Institutional Review Board of Zhejiang Academy of Agricultural Sciences.
Anal swab samples were enriched in 10 ml of buffered peptone solution (BPW, Land Bridge, Beijing, China). After initial pre-enrichment in BPW, 0.

Whole genome sequencing and bioinformatics analysis
The genomic DNA extraction of E. coli was performed using a bacterial DNA extraction kit (Generay, Shanghai, China). The whole genome sequencing was performed on the Novaseq 6,000 (Illumina, SanDiego, CA, United States). Clean reads were assembled using SPAdesv3.12.0 (Bankevich et al., 2012). Genome annotation was performed using the NCBI Prokaryotic Genome Annotation Pipeline (Tatusova et al., 2016). ABRicate 1.0.1 tool 1 and VFDB database were applied to predict the virulence genes and AMR genes. Replicons and sequence type (ST) were determined at the Center for Genomic Epidemiology (CGE). 2 Phylogenetic analysis of the genome and plasmids was performed by kSNP 3.1 software based on the maximum-likelihood method (Gardner et al., 2015). Easyfig 2.2.5 was used for comparative analysis of the plasmids (Sullivan et al., 2011). Geographical distribution of the sampling areas in Zhejiang Provinces, China, and AMR rates of E. coli isolates. (A) Sample sources of 181 strains of E. coli, Zhejiang Province in this study are shaded in green; (B) the number of E. coli isolates from farms in Zhejiang Province resistant to different antibiotics. In this experiment, 14 antibiotics with inflection points were divided into 9 categories; (C) the distribution of MDR strains.

Statistical analysis
TBtools was used for clustering heat map analysis of AMR genes, AMR phenotypes, virulence genes, and plasmid replicons .

Prevalence and AMR of Escherichia coli isolates
A total of 181 E. coli strains were isolated from all 200 anal swab samples with a detection rate of 90.50%. Among them, 96 isolates were from chickens, and 85 isolates were from pigs (Table 1).
The 14 tested antibiotics are grouped into 9 classes ( Figure 1B), including penicillins (AMP and A/C), aminoglycosides (GEM、SPT, and CEF), cephalosporins (CAZ), carbapenems (MEM), sulfonamides (SF and SXT), quinolones (ENR and OFL), tetracyclines (TET), chloramphenicols (FFC) and polypeptides (CL). Carbapenems class of antibiotics, which in this study is MEM had the lowest resistance rate. Meanwhile, the sulfonamides class of antibiotics had the highest resistance rate with 181 resistant isolates, of which 172 were resistant to SF.
The plasmid replicon analysis (Figure 4) showed that a total of 40 types of plasmid replicons were detected in all 181 E. coli isolates, of which 107 (59.12%) carried the IncFIB (AP001918) replicon, followed by IncX1 replicon existing in 86 isolates (47.51%). 94.48% (171/181) of the isolates carried 2 ~ 6 replicons. Similar plasmid replicon types can be found in different cities and animals, suggesting that plasmids carrying AMR genes may be widely spread through horizontal gene transfer.
As shown in Figure 5, 72 acquired AMR genes were detected in this study, among which mdf(A) was carried by all isolates. Notably, two isolates carried carbapenem resistance gene bla NDM-5 , two isolates carried colistin resistance gene mcr-1, six isolates carried tigecycline resistance gene tet(X4), and eleven isolates carried linezolid resistance gene cfr.
Strains from different cities can gather on the same branch. As the last line of drugs, the AMR genes mcr-1, bla NDM , and tet(X4) are distributed in different branches, showing different genetic relationships (Figure 8). In the present study, these AMR genes are more likely to be horizontal gene transfer between E. coli strains. In addition, the cfr gene mainly exists in two branches, which may be the clonal transmission (Figure 8).

Discussion
The AMR of E. coli has become a worldwide public health problem (Rutuja et al., 2018). Pigs and poultry products may have a cross infection of AMR E.coli during processing and subsequent sale, which increases the risk of transmission and poses a significant threat to the sale of products and people's health (Chang et al., 2020). To understand the current situation of AMR of E. coli, we analyzed AMR, MLST and virulence genes of E. coli isolated from anal swab samples of pigs and chickens in farms in Lishui, Quzhou and Jinhua, Zhejiang Province, Eastern China. In this study, relatively high AMR rates of FFC (79.56%), SXT (85.64%), SF (95.03%), SPT (88.40%) and AMP (84.53%) were detected that were consistent with the previously published results Peng Z. et al., 2022). At the same time, the AMR rates of A/C (3.31%), MEM (2.76%), CL (5.52%) and CAZ (7.18%) were low. CL and MEM are recognized as the last line of defense against gram-negative bacteria Wang et al., 2022) and low AMR rates reveal their resistance's effectiveness is decreasing.
Genomic analysis revealed that the E. coli isolates harbored various AMR genes, which could be consistent with the AMR phenotype. All E. coli isolates contained the AMR gene mdf(A), and most of them also carried tet(A) (68.51%, 124/181), floR (62.98%, 114/181) and sul2 (56.35%, 102/181). These genes Frontiers in Microbiology 06 frontiersin.org mediate resistance to tetracycline, chloramphenicol, and sulfonamide antibiotics. These critical AMR genes in E. coli isolates from food animals present a tremendous public health concern. It's important to mention that the acquired AMR genes in bacterial genomes do not inevitably confer phenotypic resistance and vice versa (Boolchandani et al., 2019;Tang et al., 2022a). Other mechanisms such as SNPs and MDRtransporter also significantly contribute to the phenotypic resistance (Boolchandani et al., 2019). The phenotypic confirmation is still essential for validating of AMR profiles. As we found in this study, Minimum spanning tree of E. coli strains based on MLST and Sankey diagram combining the provinces, STs, farms, and sampling sources based on 181 E. coli isolates. (A) Each node represents a single ST. The size of the nodes is proportional to the number of isolates. The length of branches between each node is proportional to the number of different alleles that differ between two linked nodes; (B) the diameter of the line is directly proportional to the number of isolates, which is also marked with numbers. The lines are colored according to the city and the sampling source.
Frontiers in Microbiology 07 frontiersin.org only the acquired genes of carbapenem, colistin and tetracycline resistance have the highest consistency with the AMR phenotypes. While other acquired AMR genes can not predict the resistance phenotype of the bacteria very well.
Sixty-five different STs were determined in all E. coli isolates. Among them, ST10 accounted for the largest proportion, up to 10.50% (19/181). Next was ST48, and 13 isolates (7.18%) were tested for this ST type. ST10 E. coli has been seen repeatedly in Dendrogram of hierarchical clustering heatmap of the 181 isolates and 40 plasmid replicons. Y-axis is the isolate number, and x-axis is the selected resistance genes identified. Non-expression of resistance is a white background and resistance phenotype is a blue background. Orange indicates isolates were isolated from Quzhou samples, aqua blue indicates isolates were isolated from Lishui, and bright yellow indicates isolates were isolated from Jinhua. Purple indicates that the strain is isolated from pig anal swab samples, and green indicates that the strain is isolated from bird anal swab samples.
Frontiers in Microbiology 09 frontiersin.org Dendrogram of the hierarchical clustering heat map of isolates and virulence genes. The figure shows the predicted virulence genes factor profile of the studied isolates. Y-axis is the isolate number and x-axis is the selected virulence genes identified. The red color in the small cells indicates virulence genes associated with toxin production, the orange color indicates genes editing other virulence factors, and the white color indicates no virulence expression. Withered grass color indicates isolates isolated from Quzhou samples, yellow indicates isolates from Jinhua, and blue color indicates isolates from Lishui. Incarnadine pink indicates that the isolates were isolated from pig anal swab samples, and bright green indicates that they were isolated from birds.
Frontiers in Microbiology 10 frontiersin.org related pathogenicity studies, with multiple disease outbreaks associated with ST10 in a broiler production unit by testing over 18 months (Bojesen et al., 2022). Nine of the ST10 E. coli isolates detected in our study carried the virulence gene astA, which plays an essential role in causing diarrhea and affecting the survival of animals (Meng et al., 2014;Paixão et al., 2016). In recent years, multiple researchers have found that some virulence genes can promote the AMR of bacteria strains . At the same time, some toxins will be enhanced under the pressure of antibiotics (Wangkheimayum et al., 2022). In our study, the prevalance of astA is up to 36.81%. The astA gene does not sufficiently lead to disease and is also found in healthy pigs, but when it cooperates with F pili or other virulence genes such as sta and stb, it will lead to diarrhea and pose a potential threat to food safety. Hemolysin is an important pathogenic factor of pathogenic E. coli. It not only plays a role in cracking cells but also promotes cell death by activating typical inflammatory bodies in cells. Hemolysin A (HlyA) toxin is important for triggering cell death in human macrophages (Verma et al., 2019;Gu et al., 2021). Our Frontiers in Microbiology 11 frontiersin.org study obtained three isolates carrying encoding genes of three kinds of α-hemolysin, hlyA, hlyB, and hlyD. Here, we would also like to note the proportion of virulence genes associated with the iron uptake system (33/169, 19.53%) and adhesion (46/169, 27.22%). Adhesins are usually a class of glycoprotein or glycolipid biomolecules that are mainly distributed on the surface of bacteria (Berne et al., 2015). The adhesins identified in this study included F1C hairs, K88 hairs, type I hairs, and E. coli common hairs. E. coli isolates harbored several kinds of plasmids, with the dominance of IncFIB (AP001918) followed by IncX1. These plasmids have been verified linked to resistance to several antimicrobial classes, including β-lactams, aminoglycosides, sulfonamides, tetracyclines, etc. Our research also showed that these plasmids were carried by isolates originating from chickens and pigs in Zhejiang provinces, indicating that these plasmids could disseminate widely among different hosts (Hu et al., 2016;Tang et al., 2019Tang et al., , 2020a.

Conclusion
In summary, a comprehensive study of AMR and genomic characterization of E. coli isolated from pigs and chickens anal swab samples in Eastern China was conducted. A high prevalence of MDR E. coli and many virulence determinants in the isolates posed a potential risk to food safety. This is a significant warning for public health safety in Zhejiang Province. It is essential to continue monitoring the MDR E. coli and implement antimicrobial stewardship programs for rational use of essential antimicrobials in animal farms to improve food safety and prevent the emergence of MDR bacteria. Phylogenetic SNP tree of E. coli strains with whole genome sequences. Red and blue dots indicate different virulence genes carrying strains, and yellow, pink, brick and red indicate different AMR genes carrying strains. The strains with different colors represent three different cities.