Characterization of an NDM-5 carbapenemase-producing Escherichia coli ST156 isolate from a poultry farm in Zhejiang, China

Background The emergence of carbapenem-resistant Enterobacteriaceae strains has posed a severe threat to public health in recent years. The mobile elements carrying the New Delhi metallo-β-lactqtamase (NDM) gene have been regarded as the major mechanism leading to the rapid increase of carbapenem-resistant Enterobacteriaceae strains isolated from clinics and animals. Results We describe an NDM-5-producing Escherichia coli strain, ECCRA-119 (sequence type 156 [ST156]), isolated from a poultry farm in Zhejiang, China. ECCRA-119 is a multidrug-resistant (MDR) isolate that exhibited resistance to 27 antimicrobial compounds, including imipenem and meropenem, as detected by antimicrobial susceptibility testing (AST). The complete genome sequence of the ECCRA-119 isolate was also obtained using the PacBio RS II platform. Eleven acquired resistance genes were identified in the chromosome; four were detected in plasmid pTB201, while six were detected in plasmid pTB202. Importantly, the carbapenem-resistant gene blaNDM-5 was detected in the IncX3 plasmid pTB203. In addition, seven virulence genes and one metal-resistance gene were also detected. The results of conjugation experiments and the transfer regions identification indicated that the blaNDM-5-harboring plasmid pTB203 could be transferred between E. coli strains. Conclusions The results reflected the severe bacterial resistance in a poultry farm in Zhejiang province and increased our understanding of the presence and transmission of the blaNDM-5 gene. Electronic supplementary material The online version of this article (10.1186/s12866-019-1454-2) contains supplementary material, which is available to authorized users.

isolated from pigs [22,23], dairy cows [24] and vegetables [25]. The complete sequences of bla NDM-5 -harboring plasmids have been helpful for the study of the transmission of the bla NDM-5 gene, although not all of these plasmids have been reported.
In this study, we first describe the NDM-5-producing carbapenem-resistant E. coli strain, ECCRA-119, isolated from a layer hen farm in Zhejiang, China. We obtained the complete genome sequence, predicted the possible mechanism of multidrug resistance and assessed the transmission ability of the plasmid harboring bla NDM-5 from the ECCRA-119 isolate. These results increased our understanding of the diversity and complexity of the strains harboring bla NDM-5 .

Strain features
Two hundred nineteen of the samples studied tested positive for E. coli, and E. coli isolates from all of these samples were obtained and characterized by antimicrobial susceptibility testing (AST) using the VITEK® 2 COMPACT system (BioMérieux, France). The highest overall levels of resistance were observed toward ampicillin, with 74.43% of all isolates resistant to this antimicrobial. High rates of resistance were also observed toward trimethoprim (54.34%), with lower levels of resistance observed toward piperacillin (1.83%), amikacin (2.29%), and amoxicillin (0.91%). No strain was determined to be resistant to tigecycline. One hundred eighty isolates (82.2%) were resistant to at least one antimicrobial agent, and 92 isolates (42.01%) were resistant to three or more antimicrobial agents. Of the 219 E. coli isolates, a carbapenem-resistant strain was identified that showed resistance toward ertapenem and imipenem, which is rare in poultry.
The minimum inhibitory concentrations (MICs) of the ECCRA-119 isolate toward different antibiotics are shown in Table 1. The ECCRA-119 isolate was susceptible to colistin (MIC < 0.125 mg/L), polymyxin B (MIC 1 mg/L) and amikacin (MIC ≤4 mg/L), exhibited intermediate resistance toward gentamicin (MIC 8 mg/L), and was resistant to 27 different compounds from 7 antimicrobial classes that are frequently used in medical treatments, food animal feed and animal medicine (Table 1). In particular, this isolate was resistant to two carbapenems, imipenem (MIC 4 mg/ L) and meropenem (MIC 8 mg/L). Therefore, we classified the ECCRA-119 isolate as a multidrug-resistant strain (MDR) due to its nonsusceptibility to many antimicrobial agents, including imipenem and meropenem.
Characterization of the genome sequence of strain ECCRA-119 The genome of the ECCRA-119 isolate consisted of a single circular chromosome and three circular plasmids ( Table 2, Figs. 1 and 2b). The chromosome sequence of ECCRA-119 was determined to be 4,893,130 bp in length, have a GC content of 50.77% and encodes 5042 proteins that account for 90.96% of the genome. The average depth of coverage was 210.5×, and 22 rRNAs, 87 tRNAs, and 2 CRISPRs were detected. Three plasmids in the ECCRA-119 isolate were identified, pTB201, pTB202 and pTB203. The plasmid pTB201, which is a combination of IncFII-and IncFIB-type plasmid, was determined to be 146,268 bp in length and have an average GC content of 51.35%. The plasmid pTB202, a p0111-IncN-type plasmid, was determined to be 139,629 bp in length and have an average GC content of 49.13%. In addition, the bla NDM-5 -harboring plasmid pTB203, an IncX3-type plasmid, was determined to be 46,161 bp in length and have an average GC content of 46.65%. Moreover, the three plasmids were characterized by S1-PFGE (Fig. 1a), the results of which were consistent with the whole genome sequencing analysis. Multilocus sequence typing (MLST) analysis classified E. coli ECCRA-119 as ST156, suggesting that E. coli ST156 strains have the potential to harbor bla NDM-5 -like genes.
Twenty-two acquired resistance genes were identified in the ECCRA-119 genome that belong to eight antibiotic resistance categories (Table 3). Among these genes, 11 are located on the chromosome, four on plasmid pTB201, six on plasmid pTB202 and one on plasmid pTB203. In addition, several gene mutations were identified in the quinolone and fluoroquinolone resistance-determining region on the chromosome (Additional file 1: Table S1). Double gyrA mutations (giving rise to the amino acid substitutions S83 L and D87Y), parC mutation (giving rise to the amino acid substitution S80I) and parE mutations (giving rise to the amino acid substitution S458A) were also predicted in the ECRRA-119 isolate.
Seven virulence factors were detected in the whole genome sequence (Additional file 1: Table S2), four in the chromosome and three in the pTB201 plasmid, indicating the potential virulence of the ECCRA-119 isolate. These virulence factors are grouped into five classes (iss, gad, lpfA, iroN, and cma), which are related to serum survival, glutamate decarboxylase, long polar fimbriae, enterobactin siderophore receptor protein, and colicin M, respectively. In addition, one mercury resistance-related gene, merA, was identified on plasmid pTB201 (Additional file 1: Table S3).

Transferability of plasmids
Conjugation assays confirmed that bla NDM-5 could be transferred between E. coli strains, with an observed transfer frequency of (1.39 ± 0.12) × 10 − 5 . The antibiotic susceptibility testing results showed that the transconjugants, confirmed by PCR and sequencing, were resistant to meropenem (4 mg/L). The transfer regions of the three plasmids of strain ECCRA-119 were successfully identified (Figs. 1 and 2b) by oriTfinder, including the origin of transfer region (oriT), relaxase gene, bacterial type IV secretion system (T4SS) apparatus gene clusters and the type IV coupling protein (T4CP) gene. Plasmid pTB201 was observed to possess an oriT (52,884-52,969 bp in the plasmid), relaxase gene, T4CP and T4SSs, indicating a high potential for self-transferability [26]. Plasmid pTB202 was observed to harbor a relaxase but lacked an oriT, T4CP and/or T4SS, indicating that it is not a mobilizable plasmid [26]. Plasmid pTB203 possess a relaxase gene, T4CP and T4SSs, but lacked a typical oriT sequence, demonstrating its potential to be transferred to other bacteria [26], with its transfer ability having been confirmed experimentally.
Phylogenetic analysis of strain ECCRA-119 with other E. coli ST156 isolates MLST analysis classified E. coli strain ECCRA-119 as ST156. Thus, we built a phylogenetic tree to determine its relationship among ST156 E. coli strains based on a SNP analysis (Additional file 1: Figure S1). We identified 52,076 SNPs from the 37 genome sequences available in Gen-Bank. Of these, 17,953 and 34,123 were identified as core and noncore SNPs, respectively. We excluded the noncore SNPs for the further analysis and constructed a phylogenetic tree based on the genome-wide core SNPs. The core genome analysis identified 5 groups (Additional file 1: Figure S1). E. coli strain ECCRA-119 is grouped with the strains 174,900, SCEC020022 and VREC0575, which were isolated from Bangladesh, China and the United Kingdom, respectively. There were 7 group-specific core SNPs in this group. The number of strain-specific SNPs identified in strains ECCRA-119, 174,900, SCEC020022 and VREC0575 was 59, 71, 134 and 160, respectively. Interestingly, most isolates identified from the same region or source are not in the same lineage. Isolates from different countries were observed to be clustered together (strains 157-1949 and SE11). Similarly, strains isolated from different hosts (wild animals, livestock and poultry, and dog) clustered into the same branch (strains MOD1-EC5693, CVM N33633PS and MOD1-EC6498).
Phylogenetic and comparative analysis of pTB203 and other bla NDM-5 -harboring IncX3 plasmids An SNP-based phylogenetic analysis was conducted using the 52 complete sequences of bla NDM-5 -harboring IncX3 plasmids available in GenBank (Fig. 2a). Among these sequences, 41 originated from bacterial strains from humans, 1 from a pig, 5 from geese, 1 from a vegetable, 1 from a layer hen, 1 from sewage, and 2 from unknown sources. Our results showed that the IncX3 plasmids have an extensive host range. Among these 52 plasmids, 43 were isolated in China and 33 were from E. coli. Five plasmids from geese became available in January 2019 but were not published. Among these plasmids, 9 published plasmids were selected and constructed by BRIG (Fig. 2b Figure  S2). The results of our analysis showed that bla NDM-5 -harboring IncX3 plasmids with an~46 kb backbone have extensive host adaptability in Enterobacteriaceae.

Complete sequences of plasmids harboring bla NDM variants from China
At present, 24 variant bla NDM sequences are available in GenBank, all of which were aligned by ClustalX (Additional file 1: Figure S3 and S4). These sequences are 813 bp in length, with the exception of bla NDM-18 , and only 1-6 SNPs are observed among these sequences. In particular, the bla NDM-5 gene has the closest homology with bla NDM-17 , bla NDM-20 and bla NDM-21 (Additional file 1: Figure S4). Relative to bla NDM-5 , bla NDM-17 , bla NDM-20 and bla NDM-21 contained point mutations at positions 508

Comparative analysis of plasmids pTB201 and pTB202
We compared the plasmids pTB201 and pTB202 with the corresponding homologous plasmids from GenBank via BLAST analyses. The results showed that plasmid pTB201 shares homology with plasmid pSMS35_130 (CP000971), plasmid pJIE186_2 (JX077110) and the p300 iro gene cluster (AY205565) (Fig. 4a); plasmid pTB202 showed homology with plasmid p1079-#IncFIB-N (MG825383) and part of plasmid pD90-3 (CP022453) (Fig. 4b). These comparisons revealed that these plasmids do not have full-length matching plasmids in the GenBank database, although they shared backbones with many other plasmids.

Discussion
The extensive use of antibacterials has led to the emergence of drug resistance as an increasingly serious issue, which poses a great threat to public health. There have been widespread reports of the isolation of multidrug resistant E. coli from hospitals, poultry, livestock, food, and the environment [33]. In this study, we identified a bla NDM-5 -harboring E. coli isolate from a layer hen farm in Zhejiang, China, and we obtained detailed data through bioinformatics and experimental analyses. The AST results showed that the ECCRA-119 isolate is resistant to 27 different compounds used as therapeutics and food animal feeding, indicating its strong environmental adaptability under antibiotic selection pressure.
There is no doubt that the multidrug resistance of this strain may present a serious risk to clinical and veterinary medicine. Apart from a few cases, acquired antimicrobial resistance genes and genomic mutations can largely explain drug resistance phenotypes. The identification of the antimicrobial resistance genes acquired by this strain show that this isolate may have a broad spectrum of drug resistance. For example, the presence of the fosA3 gene in the chromosome could result in fosfomycin resistance [34], but further experiments are needed to confirm this possibility.
The iss gene was detected on both the chromosome and on plasmid pTB201, indicating the potential virulence of  the ECCRA-119 isolate. The protein encoded by the iss gene is part of an outer membrane protein and is involved in the anti-complement effect of bacteria, possibly enhancing the serum resistance of E. coli and enabling the strain to rapidly proliferate in the host. It is widely believed that the iss gene is closely associated with the virulence of avian E. coli [35]. The merA gene was detected in plasmid pTB201, which can confer resistance to mercury and increase the viability of the ECCRA-119 isolate. Furthermore, the results suggested that plasmid pTB201 had a high potential for self-transferability. Therefore, it is likely that the mercury resistance of the ECCRA-119 isolate may be transferred to other bacteria [36]. Thus, the ECCRA-119 isolate has strong environmental resilience and a high potential to survival in a complicated breeding environment for a long time.
To the best of our knowledge, this is the first time a bla NDM-5 -harboring plasmid has been reported in layer chickens. E. coli ST156 has not been a predominant multidrug-resistant clone observed worldwide in the past, but it is associated with the distribution of bla NDM-1 and bla CTX-M-15 in humans and poultry [37,38]. The genes mcr-1 and bla NDM-5 have been reported to be detected in E. coli ST156 from Muscovy duck in China [39]. E. coli ST156 has spread to many countries and can be isolated from many types of hosts, suggesting that E. coli ST156 has the potential to play an important role in the transmission of the bla NDM-5 gene. In this study, the bla NDM-5 -harboring plasmid was first detected from E. coli ST156 in the feces of a layer hen in China, which may increase our understanding of the transmission of bla NDM-5 .
IncX3 plasmids are narrow host range plasmids of Enterobacteriaceae and are believed to have a low prevalence [40]. Since the first discovery of bla NDM-5 in China, this gene has been identified in a variety of Enterobacteriaceae [21,31], with IncX3 being the primary type of Inc. to harbor bla NDM-5 [41]. From our results, the IncX3 plasmids harboring bla NDM-5 were highly similar to each other in different countries and host sources, suggesting its ability to be an efficient vehicle for bla NDM-5 dissemination among humans, animals, food and the environment, potentially indicating its role in the rapid spread of bla NDM-5 -harboring isolates [21,28]. The BRIG analysis results showed that bla NDM-5 -harboring IncX3 plasmids have a conserved backbone of~46 kb, indicating that these plasmids had a common ancestor, and the conjugation/type IV secretion components in the backbone may be a factor promoting its transmission.
The bla NDM-5 -harboring plasmids were initially detected from isolates from human [4,27,28]. However, they have also been detected in food, the environment and livestock and poultry sources in recent years. For example, plasmid pNDM5_025943 (unpublished) was detected in sewage, and plasmid pVH1 was detected from a cucumber [25]. Carbapenem resistance is well known to be a universal phenomenon because of its frequent usage in clinics. Thus, it is interesting that the bla NDM-5 -harboring plasmid has an increasing host range, which reflects the development of serious carbapenem resistance. In particular, the bla NDM-5 gene has been detected from livestock animals in recent years, such as swine [23] and dairy cows [24]. In this study, the complete sequence of a bla NDM-5 -harboring plasmid isolated from layer hen feces was first published, which is important evidence of bla NDM-5 transmission in poultry in China.

Sample collection and antimicrobial susceptibility testing
Using the sampling method proposed by Leon and Hassan [42,43], 251 samples of chicken feces were collected from 12 large-scale chicken farms in Zhejiang province in 2017.

Whole genome sequencing, assembly and annotation
After genomic DNA extraction and quality checks, a 20-kb fragment library was constructed for the sample when the concentration and purity met the sequencing requirements. Whole-genome sequencing was performed using a PacBio RS II instrument [44]. The assembly of the reads was performed following the Hierarchical Genome Assembly Process (HGAP) workflow [45]. In this process, the Celera Assembler, following the OLC algorithm, was used to assemble the sequences [46], and Quiver was used to optimize the assembly results [45]. The gene prediction and annotation of the genomes was performed using the NCBI Prokaryotic Genome Annotation Pipeline [47]. The complete genome of the ECCRA-119 isolate was deposited in GenBank under the accession numbers CP029242 (chromosome), CP029243 (plasmid pTB201), CP029244 (plasmid pTB202) and CP029245 (plasmid pTB203).

Conjugation assay
Plasmid conjugation experiments were performed on the ECCRA-119 isolate as described previously by Lin et al. [23,61]. A rifamycin-resistant E. coli EC600 strain was used as the recipient in the plasmid conjugation assay to test the transferability of the carbapenem resistance gene and other resistance genes harbored by the ECCRA-119 isolate. Briefly, transconjugants were selected on LB agar plates (Landbridge., Beijing, China) supplemented with rifamycin (400 mg/L) (Sangon Biotech., Shanghai, China) and meropenem (4 mg/L) (J&K Chemical Ltd., Shanghai, China). The transfer frequencies were calculated by dividing the number of colony-forming units (CFUs) of transconjugants by the number of CFUs of the recipients. Genome DNA was extracted from the E. coli transconjugant using a bacterial DNA extract kit (Generay, Shanghai, China). The bla NDM-5 primers (F: 5′-GTCT GGCAGCACACTTCCTA-3′; R: 5′-TAGTGCTCA GTGTCGGCATC-3′) were used to confirm that the transconjugant harbored the plasmid. S1-PFGE S1-PFGE was performed according to a standard protocol using the contour-clamped homogeneous electric field (CHEF) technique with 0.5 × TBE buffer [62]. Salmonella enterica serotype Braenderup H9812 was used as a size marker [63]. The gels were run at 6 V/cm and 14°C with an angle of 120°, and initial and final pulses were set at 2.16 and 63.8 s, respectively. The running time was 16 h using the CHEF apparatus (CHEF MAP-PER XA; Bio-Rad, USA).

Conclusions
In this study, we reported the isolation and characterization of a carbapenem-resistant E. coli strain ST156 harboring the bla NDM-5 gene from a layer hen farm in Zhejiang province, China. Three plasmids in ECCRA-119 were identified based on whole genome sequencing and S1-PFGE. Twenty-two acquired resistance genes were identified, and this finding is consistent with the MDR phenotype of strain ECCRA-119. In particular, the bla NDM-5 gene has a high risk of spreading widely due to the potential transfer ability of the IncX3 plasmid pTB203 in this strain. The results of our study may reflect the level of antimicrobial resistance in poultry breeding in Zhejiang province and increase our knowledge of the presence and transmission of the bla NDM-5 gene.