Genome Characterization of mcr-1–Positive Escherichia coli Isolated From Pigs With Postweaning Diarrhea in China

Diarrheagenic Escherichia coli is the causative agent of diarrhea in infants and animals worldwide. Many isolated strains recovered from pigs with postweaning diarrhea are multidrug resistance (MDR), and hybrids of E. coli are potentially more virulent, as enterotoxigenic E. coli (ETEC)/Shiga-toxigenic E. coli (STEC) hybrids. Here, we used whole-genome sequencing to analyze clinical isolates of the five colistin-resistant E. coli. The E. coli CAU15104, CAU15134, and CAU16060 belonged to ETEC/STEC hybrids, displaying the same serotype O3:H45 and sequence type ST4214. The E. coli CAU16175 and CAU16177 belonged to atypical enteropathogenic E. coli (aEPEC), display O4:H11 and O103:H2, ST29, and ST20, respectively. The E. coli CAU16175 carries six plasmids. An IncHI2-type plasmid, pCAU16175_1, harbors an IS26-enriched MDR region, which includes 16 antimicrobial-resistant genes. An IncFII-type plasmid, pCAU16175_3, harbors mcr-1.1, tet(M), and blaTEM−1B, whereas mcr-1.1 is located within a Tn2 derivative. Our findings indicate that the ETEC/STEC strains of the O3:H45 serotype as well as the aEPEC strains of the O4:H11 and O103:H2 serotypes are associated with postweaning diarrhea in swine and that some of diarrheagenic E. coli contains IS26-enriched MDR region and the mcr-1 gene located within a Tn2 derivative on IncFII plasmid.


INTRODUCTION
Diarrheagenic Escherichia coli (DEC) is a leading cause of infectious diarrhea in humans and animals around the world (1,2). Diarrheagenic E. coli has six well-described pathotypes: enteropathogenic E. coli (EPEC), which is subdivided into typical EPEC (tEPEC) and atypical EPEC (aEPEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli, enteroaggregative E. coli, diffusely adherent E. coli, and enterohemorrhagic E. coli, which is subgroup of Shiga-toxigenic E. coli (STEC) (3,4). All of these DECs possess diverse virulence factors, which are encoded by virulence genes and are responsible for their pathogenicity (5). Enterotoxigenic E. coli strains typically produce one or two toxins, heat-labile enterotoxin (LT) encoded by ltc, and heat-stable enterotoxin (ST) encoded by st (6). In pigs, STEC strains are characterized by producing the Shiga-like toxin variant Stx2e encoded by stx2e (7). Enteropathogenic E. coli strains are defined as forming the attaching and effacing (A/E) lesions mediated by genes located on the locus of enterocyte effacement (LEE) pathogenicity island (including eae) in the intestinal epithelium but not produce Shiga-like toxin (Stx) (8). Moreover, bfpA, which encodes the major subunit of bundle forming pili, is used to subdivide EPEC into tEPEC and aEPEC. Hence, aEPEC strains are defined by eae + , bfpA − , and stx − (9). Three pathotypes (ETEC, STEC, EPEC) are major DEC causing postweaning diarrhea (PWD) in pigs, and many hybrids of E. coli (ETEC/STEC, ETEC/STEC/EPEC) are present during PWD (6,10). The recent data from The Global Enteric Multicenter Study showed that tEPEC and ETEC associated with a higher risk of fatal outcomes in children younger than 24 months with moderate to severe diarrhea (11). Atypical EPEC has outbreaks linked to diarrhea in children (12).
The occurrence of antimicrobial resistance and prevalence of multidrug resistance (MDR) Gram-negative Enterobacteriaceae has been increasing worldwide between humans and animals (13). In the cases of PWD, antimicrobial treatment has been widely used and caused severe drug resistance in DEC (1,6,14). In our previous study, among the 171 E. coli isolates, 94.15% of the strains were MDR, with antimicrobial resistance rates ranging from 2.34% for meropenem to 90.05% for nalidixic acid (6). Among the ETEC strains that cause PWD, resistance to apramycin, neomycin, trimethoprim sulfonamide, and colistin has been increasingly observed (1). Multidrug resistance strains may spread from animals to humans, causing antibiotics ineffective and increasing mortality and morbidity in developing countries (15).
Insertion sequences (ISs) and transposons (Tns), arguably most numerous autonomous transposable elements, are crucial to shape their host genomes, particularly important in the bacterial antimicrobial resistance (24). IS26 and Tn2, which is a member of the IS6 family and Tn3 family, respectively, play a key role in the dissemination of antimicrobial-resistant genes in Gram-negative bacteria (25,26). IS26 is often existed in MDR Gram-negative bacteria, which usually carry large regions containing several antimicrobial-resistant genes that are flanked by and interspersed with copies of IS26 (25). Tn2 is the most abundant in commensal E. coli. (27). ISSwi1-m2 in pNJST258C2 is a derivative of Tn2 that includes IS26 compared with Tn2 Abbreviations: DEC, diarrheagenic Escherichia coli; WGS, whole-genome sequencing; MDR, multidrug resistance; ETEC, enterotoxigenic E. coli; STEC, Shiga-toxigenic E. coli; aEPEC, atypical enteropathogenic E. coli; tEPEC, typical enteropathogenic E. coli; PWD, postweaning diarrhea; IS, insertion sequence; Tn, transposon; PCR, polymerase chain reaction; DR, direct repeat. (28). ISApl1, which is an IS initially identified in Actinobacillus pleuropneumoniae, is a member of the IS30 family and is considered to be an essential element in the mobilization of mcr-1 (29,30).
In the present study, we used whole-genome sequencing (WGS) to analyze virulence and resistant genes of the five clinical colistin-resistant E. coli isolates recovered from pigs with PWD and to characterize the complete sequence of a Tn2 derivative carrying mcr-1.1 on IncFII plasmid, pCAU16175_3, from a swine aEPEC isolate.

Bacterial Isolation and Identification
A total of 455 E. coli strains were obtained from feces samples or small intestinal content from pigs with diarrhea in China between 2014 and 2016 and identified with polymerase chain reaction (PCR) amplification of the uspA gene (6). Of these, 171 E. coli isolates were further screened by antimicrobial susceptibility testing, and the resistance rate of colistin was 20.47% (6). From these 35 colistin-resistant E. coli, we observed ST4214 was a major clone (6/35, 17.14%) based on the multilocus sequence typing and caused severe damage to IPEC-J2 cells (6). Then, ST4214 E. coli CAU15014, CAU15134, and CAU16060 were selected from different swine farms (one isolate per farm), and two MDR aEPEC isolates (E. coli CAU16175 and CAU16177) were randomly chosen for WGS.

Whole-Genome Sequencing
Bacterial isolates were recultured from stock; DNA was extracted using a TIANamp Bacteria DNA kit (Tiangen Biotech Inc., Beijing, China). We used WGS by the Illumina Hiseq platform to get draft genome of the five E. coli isolates and further used WGS by the Oxford Nanopore Technologies MinION platform to get complete genome of the E. coli CAU16175.
The complete genome assembly was constructed from the two sequence data sets using Unicycler (Shanghai Majorbio Biopharm Technology Co., Ltd., Shanghai, China). The serotype, multilocus sequence type, plasmid type, antimicrobial-resistant gene, and virulence gene detection were performed using the Center for Genomic Epidemiology server (https://cge.cbs.dtu. dk). Insertion sequence typing was carried out using the ISFinder database (https://www-is.biotoul.fr/). The complete genome sequences were initially annotated with Rapid Annotation using the Subsystem Technology server (http://rast.nmpdr.org) and curated manually using the BLAST server (https://blast.ncbi.nlm. nih.gov/Blast). The obtained plasmid sequences were aligned with homologous plasmid sequences from NCBI using the BRIG tool (36).

Conjugation Assay
The conjugation experiment was carried out using the E. coli CAU16175 as the donor and the E. coli J53 (resistant to sodium azide) as the recipient. The transconjugant was screened on BHI agar plates containing sodium azide (150 mg/L) and colistin (4 mg/L). The presence of mcr-1 in the transconjugant was confirmed by PCR, and the MICs of antimicrobial agents for the transconjugant were determined using the agar dilution method. The conjugation experiment was repeated three times, and the conjugation frequencies were calculated as the number of transconjugants per recipient. The transconjugant strains were distinguished from donor occurring natural mutants of sodium azide-resistant E. coli by verifying eae, which is marker gene for E. coli CAU16175 donor.

GenBank Accession Number
The draft genome sequences of the E. coli CAU15104, CAU15134, CAU16060, and CAU16177 isolates have been deposited at GenBank under SRA accession no. SRR10828049, SRR10828048, SRR10828047, and SRR10828046, respectively. The complete genome sequences of the E. coli CAU16175 have been deposited at GenBank under SRA accession no. SRR10813965.

Antimicrobial Susceptibility Testing of the Five E. coli Isolates
Antimicrobial susceptibility testing of the five isolates showed that they were MDR exhibiting resistant to at least three different classes of antimicrobials ( Table 1). All the isolates showed MDR to KAN, STR, TE, SXT, NAL, and PB.

Conjugation Assay
The susceptibility testing of polymyxin B and E showed that the MIC values for the transconjugants (J53-mcr-1) increased to 8 and 16 mg/L, respectively (  (Figure 1).  Table 2). We chose five most similar IncFII-type plasmids and reported IncFII-type plasmid-mcr-1 pKP81-BE (KU994859) for comparison analysis (Figure 2). Although the pCAU16175_3 demonstrates highly sequence homology with the other five IncFII-type plasmids, the mcr-1.1 gene only exists in pCAU16175_3 and pKP81-BE. Besides the mcr-1.1 gene, the pCAU16175_3 also carries the antimicrobial-resistant gene bla TEM−1B and tet(M), which are close to the mcr-1.1 gene. Noticeably, the nucleotide location of the pCAU16175_3 from 2,079 to 12,231 has high homology with the Tn2 derivative ( Figure 3A). This structure is a unique identification compared with the NCBI database and was confirmed by conventional PCR FIGURE 2 | Comparative analysis of seven IncFII-type plasmids pCAU16175_3, pEC1515-3, pEC974-3, pH1038-142, pFORC_081_1, plasmid R1, and pKP81-BE (carrying mcr-1) using the BLAST Ring Image Generator. The concentric rings show similarity compared the pCAU16175_3 as reference plasmid in inner ring with the other plasmids in the outer rings. The varying color levels indicate a BLAST result with matched degree of shared regions, as shown to the right of the ring. The outer circle with red arrows and green arrows denotes antimicrobial resistance gene and transposable elements, respectively. Detailed information of the complete sequences of IncFII plasmids is described in Supplementary Table 2. Figure 1A). The genetic content of the structure analysis showed that the structure is located within mcmM (encoded microcin M). A five-nucleotide direct duplication (TATTT) was identified flanking the structure. Unlike the Tn2, this structure harbors mcr-1-pap2-ISApl1 and IS1X2-hptet(M)-IS15DI, deletes the resolvase (tnpR), and truncates the transposase (tnpA). Besides, the striking features of the insertion sites of ISApl1 were found, which includes high AT content and 2-bp direct repeats (DRs) (AA) (Figure 3B). From the result of conjugation experiment and PCR, the whole structure gene did not occur transposition (Table 4, Supplementary Figure 1B). The mcr-1 gene was transferred to J53 without the pap2 gene, whereas colistin resistance was transferred to J53. IS15DI was also transferred to J53.

DISCUSSION
Postweaning diarrhea caused by DEC is an economically important disease for the swine industry around the world. In our previous study, we collected 455 E. coli isolates recovered from feces samples or small intestinal content from pigs with diarrhea in China between 2014 and 2016 to know the E. coli pathotype and the antimicrobial susceptibility of the isolates (6). Most of the isolates belonged to ETEC, followed by aEPEC, which is similar to the report in Spain; that is, most cases of PWD are significantly associated with ETEC (67%) and aEPEC (21.7%) (37). Our previous study showed that 95.91% of 171 E. coli isolates were MDR exhibiting resistance to at least three different classes of antimicrobials, and 20.47% E. coli isolates were resistant to colistin. It has been revealed that MDR isolates that existed in swine industry are associated with the widespread use of antibiotics (38,39). Overuse of colistin is considered to contribute to the emergence and spread of mcr-1 (40). The 36 mcr-1-positive E. coli isolates recovered from pigs with PWD showed MDR (29). In this study, we used WGS to analyze the five MDR (including colistin resistance) of E. coli isolates from pigs with PWD and found that they demonstrate different serotype and sequence type and carry different antimicrobial-resistant genes and virulence genes.
The E. coli CAU16175 carries IS26-enriched MDR region in pCAU16175_1 (IncHI2 type). The IncHI2-type plasmids have been discovered as genetic elements mediating the transmission of MDR genes (16). IS26-flanked Tns play an increasingly critical role in the mobilization and development of antimicrobialresistant genes (43)(44)(45). So far, there are 51 Tns related with IS26 from the Tn registry website. In an individual E. coli strain heterogeneous resistance-encoding plasmid, polymorphic MDR regions driven by IS26-flanked Tns have been detected (44). From the BLAST analysis, we found that there were abundant IS26-enriched MDR regions in Salmonella and E. coli, which were mostly isolated from humans and animals. The results showed that the plasmid carrying IS26-enriched MDR region has been widely distributed in humans and consumption animals (Supplementary Table 1). Combined with the antimicrobial susceptibility testing, we can deduce that the E. coli CAU16175 isolate has MDR and is difficult to control.
The E. coli CAU16175 isolate also carries the mcr-1.1 gene in pCAU16175_3 (IncFII type). The emergence of the mcr-1 gene can be traced back to the E. coli isolated in the 1980s, and the outbreak of chicken-derived mcr-1-containing E. coli started in 2009 (46).The mcr-1 gene has been characterized in various genetic backgrounds and observed on a variety of plasmid type, including Incl2, IncX4, IncHI2, IncP, IncHI1, IncFII, IncFI, IncFIB, F18:A-:B+, IncY, IncK, and phage-like plasmid (29). Most of the recently reported the mcr-1 genes were primarily mobilized by an ISApl1 composite Tns Tn6330 and Tn6390 (47,48). The ISApl1 would be lost over time, leading to the stability of the mcr-1 gene on plasmids (18,49). Thus, only the truncated ISApl1 will lose the ability to transfer the mcr-1 gene.
Furthermore, the mcr-1.1 gene is located within the Tn2 derivative in pCAU16175-3. From the Tn registry website (https://transposon.lstmed.ac.uk/), we noticed the mcr-5 gene and the mcr-3.6 gene could be mobilized by Tn6452 and Tn6518 (belonged to Tn2 family). For the Tn6452, the mcr-5 gene was embedded within a Tn3-family Tn with 38-bp inverted repeats and flanked by 5-bp DRs, which were usually generated during the insertion (50,51). Although DRs appear at the flanking site of the Tn2 derivative, the whole structure did not move by transposition because of truncated tnpA and deleted tnpR, which are essential to occur transposition for Tn2.

CONCLUSIONS
The current work shows the genetic characteristics of five DEC strains that exhibited MDR, including colistin resistance. Our data indicate that the ST4214 ETEC/STEC carried MDR and multivirulence genes and that the E. coli CAU16175 contains IS26-enriched MDR region and the mcr-1.1 gene, which is located within a Tn2 derivative. The coexistence of MDR and multivirulence in DEC may seriously compromise the effectiveness of clinical therapy, and heightened efforts are needed to control their dissemination.

ETHICS STATEMENT
The animal study was reviewed and approved by the Animal Ethics Committee of the China Agricultural University under the protocol CAU20140616-1.

AUTHOR CONTRIBUTIONS
LG and YZ conceived and designed the experiments. LG, JW, SW, JS, and XW performed the experiments. LG analyzed the sequencing data and wrote the manuscript. YZ revised the manuscript. All authors contributed to the article and approved the submitted version.