Dissemination of Blandm-5 in Escherichia Coli via the Incx3 Plasmid From Different Regions in China

Background: Recently, the spread of NDM-5-producing Escherichia coli has become a severe challenge in clinical therapy, which necessitates reliable detection and surveillance methods. However, limited information is available regarding the prevalence and dissemination of the bla NDM-5 gene in Escherichia coli in China. Therefore, we investigated the dissemination of the bla NDM-5 gene in carbapenem-resistant Escherichia coli isolates from different regions in China. Methods: A total of 1,180 carbapenem-resistant enterobacteriaceae strains were obtained from patients admitted to the 20 sentinel hospitals in eight cities. Strains positive for bla NDM-5 were detected using the Vitek 2 compact system, 16S rRNA gene sequencing, PCR, the S1-pulsed-eld gel electrophoresis assay, and Southern blot hybridization. The horizontal-transfer capability of the bla NDM gene was assessed by lter mating with a standard E. coli J53 azide-resistant strain as the recipient. Genotyping, susceptibility testing, and whole genome sequencing were performed. Results: Seven strains of bla NDM-5 -positive E.coli was detected in 1180 clinical strains from different regions in China. The bla NDM-5 -carrying strains showed resistance to multiple tested antibiotics and belonged to two widespread sequence types, ST167 and ST405. Antimicrobial resistance genes including bla CTX-M , bla OXA , bla CMY , and two novel bla TEM variants (bla TEM-230 and bla TEM-231 ) were also identied. Southern blotting located the bla NDM-5 gene on 46-kb IncX3 plasmids in all isolates, which showed only two single nucleotide differences between EJN003 and the other strains. Conclusions: This study further conrms the increasing occurrence of bla NDM-5 -carrying IncX3 plasmids and the dissemination of carbapenem resistance in E. coli isolates via the plasmid from different parts in China, which warrants stringent surveillance and control measures.


Introduction
Escherichia coli is one of the most common causative agents of infection in humans, and the emergence of resistance to third-generation cephalosporins by [1] extended-spectrum beta-lactamases (ESBLs) has led to an increased use of carbapenem compounds. The growing incidence of resistance to carbapenems among Enterobacteriaceae is of major concern worldwide. Among the newly emerged lactamases, New Delhi metallo-lactamase (NDM) represents the latest threat for public health. Since NDM-1 was rst reported in 2009 in a Swedish patient of Indian origin [2], NDM-producing bacteria have spread globally and have caused various types of clinical infections [3]. To date, 24 bla NDM gene variants have been described worldwide, and some variants confer elevated carbapenem resistance [4,5]. The rapid evolution and dissemination of NDMs represent a crucial challenge for clinical infection treatments, which necessitates reliable detection and surveillance method [6].
NDM-5, a variant with higher carbapenemase activity than NDM-1, was rst identi ed in an E. coli strain isolated in 2011 from a patient in the UK with a recent history of hospitalization in India [4]. It has two amino acid substitutions (Val88Leu and Met154Leu) relative to NDM-1, and confers increased resistance to extended-spectrum cephalosporins and carbapenems. Since then, NDM-5-producing strains have been identi ed in many areas including China. The widespread occurrence of NDM-5 in recent years highlights the need for international attention. Previous studies have reported the prevalence and dissemination of bla NDM-5 among E.coli from different regions in China [7,8]. Herein, we further evaluated the potential transmission of bla NDM-5 -harboring E. coli from different regions in China in the current study. Whole genome analysis and molecular analysis were performed to provide a clear and solid molecular epidemiological description of NDM-5-producing E. coli.

Bacterial Strains, Detection of the NDM Gene, and Antimicrobial Susceptibility Testing
A total of 1,180 carbapenem-resistant enterobacteriaceae strains obtained from patients admitted to the 20 sentinel hospitals in eight cities from January 2014 to December 2015 in China were identi ed using the VITEK 2 system (BioMérieux, France). In a retrospective study, the presence of bla NDM was screened by PCR amplification and sequencing using primers described in our previous study [9]. Seven bla NDM−5positive E.coli strains were identified. The antimicrobial susceptibility testing was performed using the VITEK 2 system (BioMérieux). The minimal inhibitory concentration (MIC) results were interpreted according to the Clinical and Laboratory Standards Institute (CLSI) guidelines [10]. E. coli ATCC25922 was used for quality control. Collection of the clinical isolates was part of the routine surveillance of carbapenem-resistant Enterobacteriaceae. This study was approved by the institutional ethics committees of Academy of Military Medical Sciences.
2.2 S1-Pulsed-eld gel electrophoresis (PFGE), Southern Blotting and Plasmid Conjugation Genomic DNA from the seven strains was prepared in agarose plugs and digested with S1 nuclease (Takara, Dalian, China). Linearized plasmids and partially digested genomic DNA were separated using a CHEF-DR III system (Bio-Rad, Hercules, CA, USA). Southern blot analysis to locate the bla NDM genes was performed using specific digoxigenin-labeled probes (Roche) [11]. The NDM-1-producing Acinetobacter isolate 1750 reported by our laboratory [12] was used as a positive control, S. enterica H9812 was used as a reference size standard and a negative control.
The horizontal-transfer capability of the bla NDM-5 gene was assessed by broth and filter mating using the seven strains as donors and a standard E. coli J53 azide-resistant strain as the recipient. The donor and recipient cultures were mixed in LB broth and incubated at 37°C for 24 hours. MacConkey agar containing 100 mg/liter sodium azide and 0.5 mg/liter meropenem was used to select E. coli J53 transconjugants [13]. Antimicrobial susceptibility testing and PCR amplification of the transconjugants were subsequently performed to confirm the transfer of the plasmid to the recipient.

Whole Genome Sequencing and Determination of Genetic Relatedness
Genomic DNA was extracted from seven E. coli isolates using the QIAamp DNA Mini Kit following the manufacturer's protocol (Qiagen, Inc., Valencia, CA, USA). Whole genome sequencing was performed on the Illumina Hiseq 2500 platform with a 350-bp insert at the Beijing Novogene Technology Co., Ltd. Paired-end reads of 150 bp were assembled using SOAPdenovo (v2.04) with coverage ranging from 113to 157-fold. Scaffolding and gap lling were performed using SSPACE [14] and GapFiller. The plasmid pNDM-QD28 [15] was selected as a reference. Gaps were closed using reference-guided assembly and manually checked by re-mapping raw reads against the reference. Plasmid sequences were annotated using RAST. Plasmid replicon type was assigned using PlasmidFinder (Enterobacteriaceae) [16].
The STs of seven E. coli isolates were identi ed using the MLST web server [17]. PFGE was performed after XbaI digestion with reference to the standard PulseNet conditions. Restriction patterns were compared visually using BioNumerics software with the Salmonella enterica serovar Braenderup H9812 as a size marker. E. coli strain K-12_DH10B (GenBank accession number CP000948) was used as the reference for alignment. Single nucleotide polymorphisms (SNPs) were identi ed using BWA (v0.7.12) [18] and SAMtools (v1.3). The concatenated SNPs were aligned to construct a Maximum-Likelihood phylogenetic tree using RAxML (v8.2.4) with the GTR model and the gamma distribution [19]. Genome sequences of ten pNDM5-ESY001-like plasmids were also downloaded for phylogenetic analysis as described above.

Bacterial Strains and Antimicrobial Susceptibility Testing
Seven bla NDM-5 -positive isolates were recovered from seven hospitalized patients from three sources in three different cities in China. EBJ001 and EBJ003 were isolated from Beijing, ESY001, ESY002, and ESY003 from Shenyang, EJN001 and EJN003 from Jinan. ( Table 1). None of the patients had ever traveled abroad. All E. coli strains were resistant to carbapenems, cephalosporins, quinolones, aztreonam, ampicillin, and sulfamethoxazole/trimethoprim, and were mostly susceptible to amikacin ( Table 2).

Genetic Relatedness of all Strains
MLST analysis revealed that strain EBJ003 belonged to ST405, which fell into phylogenetic group D and was distinguishable from other strains by PFGE. The other six strains belonged to ST167 and phylogenetic group A (Fig. 1). ESY001 and ESY002 were classi ed into the same pulsotype, whereas EJN001, EJN003, ESY003, and EBJ001 were classi ed into different pulsotypes (Fig. 2), suggesting both clonal and non-clonal dissemination.

Virulence and Resistance Genes in All Strains
The seven strains harbored type II and III secretion systems. Certain virulence factors were exclusively observed in EBJ003, such as m (encoding type I mbriae regulatory proteins), iuc (encoding aerobactin siderophore biosynthesis proteins), and irp and ybt (encoding yersiniabactin biosynthetic proteins) genes.
In addition to bla NDM-5 , other antimicrobial resistance genes were also frequently identi ed in the seven E. Also, bla CMY-42 was detected in EJN001 and EBJ001, and bla OXA-1 was identi ed in EBJ003. Strain

Discussion
NDM-5-producing Enterobacteriaceae show increased resistance to extended-spectrum cephalosporins and carbapenems, posing a signi cant threat to public health throughout the world. The coexistence of bla NDM-5 and other resistance genes confers multidrug resistance to pathogens, which is a serious problem for clinical treatment. This study identi ed seven NDM-5-producing E. coli isolates from three different cities in China.
Six of the seven strains belong to the ST167 type, which is an internationally disseminated clone of the ST10 complex. ST167 E. coli strains carry bla NDM-1 , bla NDM-5 [26], bla NDM-7 [30], and a variety of bla CTX-M genes [26,31] in various countries. ST167 is the most prevalent bla NDM -bearing ST in China, accounting for 42% of NDM-producing E. coli isolates according to the nationwide surveillance [32]. Recent study reported several cases of clinical infection related to bla NDM-5 -positive E. coli ST167 in Shanghai or Zhejiang China [33,34]. Strain EBJ003 belongs to ST405 and a different phylogenetic group D, suggesting distinct clonal dissemination from others. ST405 is a high-risk clone found in diverse hosts and it carries the bla NDM and bla CTX-M genes [35]. The bla NDM-5 -positive strains in this study were associated with ESBL/AmpC genes or CTX-M-types, demonstrating their ability to acquire different resistance genes. Considering that ST167 and ST405 are both widely disseminated in China, further spread of multidrugresistant E. coli could become a crucial issue in the clinical setting. In addition, an increasing number of virulence factors were identi ed in ST405 strain EBJ003 compared with ST167 strains, underscoring the potential danger of ST405 as an epidemic pathogen.
The nucleotide sequences of the bla NDM-5 -carrying plasmids pNDM5-ESY001 and pNDM5-EJN003 were nearly identical to those of other IncX3 plasmids from China, suggesting that the pNDM5-ESY001-like plasmid is an important reservoir and plays a critical role in the dissemination of bla NDM-5 in China. The pNDM5-ESY001-like plasmid (pNDM_MGR194) was rst reported in Klebsiella pneumoniae in India and later in Australia [36] and Denmark. The bla NDM-5 carriers from Australia and Denmark were isolated from patients who had travelled to India, suggesting the linkage to Indian bla NDM-5 dissemination. The bla  carriers isolated from China showed little association with the Indian subcontinent. However, considering that international travel is common and transmission routes between people are often unrecognized, the origin of the prevalence of pNDM5-ESY001-like plasmids in China remains unclear. IncX-type plasmids were earlier believed to have a narrow host range but were recently found to be prevalent in various members of Enterobacteriaceae. We previously reported the presence of an IncX3 plasmid similar to pNDM5-ESY001 in S. enterica [20]. The identi cation of pNDM5-ESY001-like plasmids in K. pneumoniae, S. enterica, and E. coli of different STs demonstrates their ability to mediate intra-and inter-species transfer of bla NDM-5 , which would facilitate the wide distribution of NDM-5 across diverse enterobacterial species. Further surveillance should be devoted to monitoring the transfer of such bla NDM-5 -carrying IncX3 plasmids among Enterobacteriaceae.
Phylogenetic analysis of plasmids revealed that the bla NDM-4 -carrying plasmid pJEG027 [24] in Australia and the bla NDM-7 -carrying plasmid pKpN01-NDM7 [25] in Canada are indistinguishable from the pNDM5-ESY001-like plasmids. Because bla NDM-5 and bla NDM-7 have a single nucleotide substitution relative to bla NDM-4 (G388A and G262T, respectively), pNDM5-ESY001 and pKpN01-NDM7 are likely to have arisen from a pJEG027-like plasmid through acquisition of different variations.
In conclusion, we report seven drug-resistant E. coli isolates harboring bla NDM-5 and multiple ESBL/AmpC genes from patients in different geographic locations in China. The coexistence of resistance to antimicrobials of different groups in clinical NDM-5-producing E. coli isolates is a serious concern. These isolates belonged to ST167 and ST405 and contained a transferable plasmid, pNDM5-ESY001 (pNDM5-EJN003), which serves as an important reservoir for the dissemination of bla NDM-5 . The present ndings underscore the threat of NDM-5 carbapenemase circulation via IncX3 plasmids and the urgency of implementing stringent surveillance and control measures.

Nucleotide sequence accession number
The shotgun whole genome sequences of strains ESY001, ESY002, ESY003, EJN001, EJN003, EBJ001 and EBJ003 have been deposited in NCBI GenBank under accession numbers PUJM000000 to PUJS00000000.

Declarations
Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable.
Availability of data and material: The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.