Emergence of IncHI2 Plasmid-Harboring blaNDM-5 from Porcine Escherichia coli Isolates in Guangdong, China

Carbapenem resistance has posed potential harmful risks to human and animals. The objectives of this study were to understand the prevalence of blaNDM-5 in pigs and investigate the molecular characteristics of NDM-5-producing Escherichia coli isolates in Guangdong province in China. Carbapenem-resistant E. coli isolates were isolated from pigs and obtained using MacConkey plates containing 0.5 mg/L meropenem. Conjugation assay and antimicrobial susceptibility testing were conducted for the isolates and their transconjugants. Whole-genome sequence (WGS) was used to analyze the plasmid genetic feature. A total of five blaNDM-5-carrying E. coli isolates were obtained in the present investigations. They belonged to five ST types. The blaNDM-5 genes were found to be in IncX3 and IncHI2 plasmid. The IncX3 plasmid was 46,161 bp in size and identical to other reports. IncHI2 plasmid was 246,593 bp in size and similar to other IncHI2-ST3 plasmids. It consisted of a typical IncHI2 plasmid backbone region and a multiresistance region (MRR). The blaNDM-5 was closely associated with the IS3000-ISAba125-blaNDM-5-bleMBL-trpF-tat-IS26 unit. We first reported the blaNDM-5-carrying IncHI2 in E. coli isolates recovered from pigs and revealed the molecular characterization. Continued surveillance for the dissemination of blaNDM-5 among food-producing animals is required.


Introduction
Carbapenems are considered to be the most active and potent agents in the treatment of severe infections caused by multidrug-resistant (MDR) Gram-negative pathogens [1,2]. In the past few years, the emergence and spread of carbapenem-resistant Enterobacteriaceae (CRE) has led to a variety of infectious diseases with high mortality rates, which has posed a critical threat to clinical treatment and a public health concern [3,4]. New Delhi metallo-β-lactamase (NDM) is a type of metallo-β-lactamase (MBL) that hydrolyzes most carbapenems except for monobactams [5]. Since the bla NDM-5 gene was first reported, it has been the most prevalent carbapenem resistance gene in Enterobacteriaceae from humans and animals as demonstrated by several epidemiological investigations published in recent years [6,7].
Plasmids considered to be an important vehicle, play a vital role in the dissemination of the resistance genes. The bla NDM-5 gene is reported to be located on a variety of plasmid replicon types, such as IncFII-IncFIB [8], with IncX3 being the major carrier [9][10][11]. Recently, bla NDM-5 gene was found from the surrounding via IncX3 plasmid. These findings warn the wide-dissemination possibility of carbapenem resistance gene and emphasize the importance of monitoring the prevalence. Herein, the aim of our study was to investigate the prevalence of bla NDM-5 -positive Escherichia coli strains from pigs in Guangdong province, China, and revealed the bla NDM-5 -carrying plasmid in E. coli isolates recovered from pigs.

Results
In this study, five isolates positive for bla NDM-5 gene from one swine farm were obtained. The susceptibility test indicated that five isolates were phenotypically resistant to β-lactams and displayed patterns of MDR, such as ampicillin, ceftazidime, meropenem, apramycin, florfenicol, sulfamethoxazole/trimethoprim, but sensitive to gentamycin, amikacin, colistin, and tigecycline, as shown in Table 1. According to PFGE analysis, five isolates exhibited five different PFGE patterns, designated A to E (Figure 1). Considering the source of isolates, we presumed that the dissemination of the bla NDM-5 gene may be due to horizontally transfer.
Based on the whole-genome sequence analysis, the MLST of five isolates were divided into five distinct sequence types, namely ST10, ST48, ST155, ST2937, and ST4063. The isolates harbored numerous antimicrobial resistance genes such as bla TEM-1B , bla OXA-10 , bla CTX-M-14 , aadA2, cmlA1 and floR, as seen in Figure 1. These resistance genes were associated with antibiotic-resistant phenotype. The five bla NDM-5 -positive isolates were successfully transferred the bla NDM-5 -5 gene to E. coli J53 at frequencies of 0.15 × 10 −6 to 5.98 × 10 −6 transconjugant/recipient ( Table 1). The transconjugants were resistant to ampicillin, cefoxitin, ceftazidime, meropenem, florfenicol, and sulfamethoxazole/trimethoprim (Table 1). warn the wide-dissemination possibility of carbapenem resistance gene and emphasize the importance of monitoring the prevalence. Herein, the aim of our study was to investigate the prevalence of blaNDM-5-positive Escherichia coli strains from pigs in Guangdong province, China, and revealed the blaNDM-5-carrying plasmid in E. coli isolates recovered from pigs.

Results
In this study, five isolates positive for blaNDM-5 gene from one swine farm were obtained. The susceptibility test indicated that five isolates were phenotypically resistant to β-lactams and displayed patterns of MDR, such as ampicillin, ceftazidime, meropenem, apramycin, florfenicol, sulfamethoxazole/trimethoprim, but sensitive to gentamycin, amikacin, colistin, and tigecycline, as shown in Table 1. According to PFGE analysis, five isolates exhibited five different PFGE patterns, designated A to E (Figure 1). Considering the source of isolates, we presumed that the dissemination of the blaNDM-5 gene may be due to horizontally transfer.

Discussion
Plasmid-mediated horizontal transmission of drug-resistant genes has been recognized as an important pathway for the rapid transmission of drug-resistant genes in En-

Discussion
Plasmid-mediated horizontal transmission of drug-resistant genes has been recognized as an important pathway for the rapid transmission of drug-resistant genes in En-

Discussion
Plasmid-mediated horizontal transmission of drug-resistant genes has been recognized as an important pathway for the rapid transmission of drug-resistant genes in Enterobacteriaceae, of which IncX3, IncFII and IncHI2 plasmids play a key role in the transmission of carbapenem-resistant bla NDM-5 genes in Gram-negative bacteria [12]. Until now, bla NDM-5 gene has been reported to be prevalent in various members of Enterobacteriaceae. Recently, plasmid-encoded bla NDM-5 gene was found in Salmonella typhimurium of pork origin [13,14] and Klebsiella pneumonia [4,15] via IncX3 plasmids. IncX3 is a group of narrowhost-range plasmid, with the size of 46,161 bp, and an important transmission vector for bla NDM-5 gene, with a variety of bla NDM-5 subtypes, such as bla NDM-1 , bla NDM-4 , bla NDM-5 , bla NDM-7 and bla NDM-21 [16,17]. IncX3 plasmid has been reported in many regions of the world, and has been widely reported in clinical human medicine, food animals and the environment [9]. In this study, three strains of bla NDM-5 -positive E. coli were detected in IncX3 plasmid. Genomic sequence analysis showed that the IncX3 plasmid was highly similar to that reported in the Enterobacteriaceae such as E. coli and Klebsiella pneumoniae from human clinic, environment and food animals published in the database [18,19], suggesting that IncX3 plasmid has been widely prevalent.
In this study, for the first time, we detected and revealed the bla NDM-5 -carrying IncHI2 in E. coli isolates recovered from pigs. Although the sample number is small and only two strains carry the IncHI2 plasmid, the presence of this plasmid should not be ignored. IncHI2 is a group of low-copy number, wide-host plasmid, which was widely distributed in Enterobacteriaceae. IncHI2 plasmid were described to carry a variety of drug-resistant genes, such as oqxAB, bla CTX-M , bla NDM , bla KPC , fosA3 and mcr-1 gene [13,[20][21][22]. Reports on the bla NDM-5 -carrying IncHI2 plasmid are scarce. Previous reports have also suggested that the IncHI2 plasmid was considered to be an important vehicle in the spread of carbapenem resistance genes such as bla NDM-1 , bla NDM-4 , bla NDM-9 and bla IMP-4 among Enterobacteriaceae from human and animals [13,23,24]. Since the discovery of mcr-1 gene in 2015 that threatens the effectiveness of polymyxins, the IncHI2 plasmid that carried mcr-1 has been found in Enterobacteria from a variety of sources, including humans [25], animals (cattle, pigs, chickens, ducks and turkeys) [26,27], food (chicken, beef, pork, milk and vegetables) [28,29] and the environment (farm soil and wastewater) [30] in several countries around the world. In this study, IncHI2 plasmid was co-transmitted with many drug resistance genes such as floR, cmlA, sul1, acc(3)-IV, aph(4) -Ia and arr-2, which undoubtedly exacerbated the resistance crisis of animal borne bacteria. In addition, after carefully looking up relevant information in the NCBI database, IncHI2 plasmids carrying bla NDM-5 gene were found in E. coli from chicken, ducks, and pigs in Guangdong province, but there were few reports in other regions. The present findings suggest that IncHI2 plasmid has become an important transmission vector for bla NDM-5 gene in food animals in Guangdong province. This type of plasmid may have spread in different areas of Guangdong province, and adaptive evolution occurred in the variable region of the plasmid during transmission. In this study, the sequence of porcine E. coli plasmid pNGD64-NDM showed high homotypes with other IncHI2 plasmid such as pNDM33-1 obtained in the duck farm that uploaded by our teaching and research department. We speculate that this phenomenon may be related to the large number of copies of insertion sequence IS26 in the variable region, which lead to the variation of the variable region and then the inversion, deletion, and insertion of some sequences. It is noteworthy that the 9525-bp bla NDM-5 module was found in various replicon plasmids such as IncHI2 plasmids p8C59-NDM and pNDM33-1, IncI-γ plasmid pNDM-TD33 and IncX3 plasmid pHNGD75-NDM, which suggests that the dissemination of bla NDM-5 was closely associated with the IS3000-ISAba125-bla NDM-5ble MBL -trpF-tat-IS26 unit.

Bacterial Collection, Species Identification, and Molecular Detection of Carbapenemase Genes
A total of 357 fecal samples were collected from three swine farms in two cities (Maoming and Qingyuan) in Guangdong province from May to November in 2018, with the sample of 180, 81, and 96 in each farm, respectively. Carbapenem-resistant E. coli isolates were screened using MacConkey plates containing 0.5 mg/L meropenem and only one red colony was selected from each fecal sample. Meropenem-resistant isolates from these swine farms were identified as E. coli by MALDI-TOF-MS. PCR and sanger sequencing were further done to confirm whether the resistant strains harbored the bla NDM-5 gene. The primer for the bla NDM-5 was conducted to confirm the presence of bla NDM-5 gene as previous report [31].

Conjugation and Antimicrobial Susceptibility Testing
To determine the transferability of bla NDM-5 -bearing isolates, the transfer frequency for each strain was determined using the broth mating method, with sodium-azide-resistant E. coli J53 used as the recipient strain. The donor strains and the recipient strain were mixed at the ratio of 1:4 in Luria-Bertani (LB) broth, and then incubated at 37 • C for 4 h, the transconjugants were selected on MacConkey agar plates supplemented with 0.5 mg/L meropenem and 200 mg/L sodium azide and then confirmed by PCR method.

Molecular Analysis of bla NDM-5 -Positive Isolates
The genetic typing of bla NDM-5 -positive E. coli isolates was digested with restriction endonuclease Xbal and conducted by pulsed-field gel electrophoresis (PFGE) according to our previous study [32]. The band patterns were analyzed with BioNumerics software version 5.10 (Applied Math s, Austin, TX, USA). Cluster analysis of fingerprinting similarity was conducted on software MasterScripts v4.0. The bacterial DNA of bla NDM-5 -positive E. coli isolates were extracted by the DNA Extraction Kit, and then were sent to laboratory by next-generation sequencing. The multi-locus sequence type (MLST) of E. coli was performed by Escherichia typing database (https://pubmlst.org/bigsdb?db=pubmlst_ escherichia_seqdef, accessed on 10 September 2020).

Conclusions
To the best of our knowledge, this study was the first to identify the bla NDM-5 -carrying IncHI2 recovered from porcine E. coli isolates. Although carbapenems have not been approved for use in food-producing animals, multiple resistance genes were observed in the IncHI2 plasmid pHNGD64-NDM in the present research, which suggests that the spread and evolution of the bla NDM-5 gene may be selected and accelerated under the selective pressure of other antibacterial drugs. Thus, the dissemination of bla NDM-5 among food-producing animals requires continued surveillance.

Conflicts of Interest:
The authors declare no conflict of interest.