High Prevalence of blaNDM Variants Among Carbapenem-Resistant Escherichia coli in Northern Jiangsu Province, China

The continuous emergence of carbapenem-resistant Escherichia coli (CRECO) presents a great challenge to public health. New Delhi metallo-lactamase (NDM) variants are widely disseminated in China, so the research on the prevalence and transmission of diverse blaNDM variants is urgently needed. In the present study, 54 CRECO isolates were collected from 1,185 Escherichia coli isolates in five hospitals in Northern Jiangsu Province, China from September 2015 to August 2016. Antimicrobial susceptibility tests, PCR detection of resistance determinants, multi-locus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) were performed to characterize these strains. Plasmid conjugation experiments were carried out to determine the transferability of resistant genes from selected isolates. PCR-based replicon typing (PBRT), S1 nuclease-PFGE, and Southern blotting were conducted for plasmid profiling. Carbapenemase genes were detectable in all CRECO isolates, among which thirty-one CRECO isolates were found to carry blaNDM−5 (54.7%), while, blaNDM−1, blaNDM−7, blaNDM−4, blaNDM−9, and blaKPC−2 were identified in 14, five, two, one, and one isolates, respectively. MLST results revealed 15 different STs and four new STs were first reported to be linked with NDM-producing isolates. PFGE typing showed that no more than two isolates with the same ST appeared to the same band pattern except three ST410 isolates. Twenty-six selected NDM-producing isolates were successfully transferred to E. coli J53 by conjugation experiments. Notably, 50.0% (13/26) of blaNDM variants were found to be carried by ~55 kb IncX3 plasmid. Our study reported a high prevalence of blaNDM variants, especially blaNDM−5, in Northern Jiangsu province, China. Diverse blaNDM variants were mainly carried by ~55 kb IncX3 plasmids, suggesting that the fast evolution and high transferability of this kind of plasmid promote the high prevalence of blaNDM variants. Therefore, large-scale surveillance and effective infection control measures are also urgently needed to prevent diverse blaNDM variants from becoming epidemic in the future.


INTRODUCTION
Carbapenem, a β-lactam that is highly potent against Gramnegative bacteria, has been recognized as a last resort for treating of infections caused by multidrug-resistant bacteria. However, the increasing number of carbapenem-resistant Enterobacteriaceae (CRE) is unexpected despite infection control efforts, and it poses a great challenge to clinic (Zilberberg and Shorr, 2013). Carbapenem resistance is predominantly attributed to the presence of carbapenemases, among which Class A (bla KPC ), Class B (bla NDM , bla VIM , bla IMP ), and Class D (bla OXA−48 ) types are most common for Enterobacteriaceae (Walsh, 2010;Albiger et al., 2015). An emerging carbapenemase, New Delhi metallo-lactamase (NDM), was first reported in a Swedish patient with a hospitalization history in India, it exhibited resistant to all β-lactams except for monobactams, and has great potential to cause global health crisis (Yong et al., 2009). Initially, bla NDM gene was endemic to India subcontinent and NDM-producing isolates tested worldwide have geographical links with these high prevalence areas. However, an increasing number of regions worldwide have reported that patients with bla NDM− positive isolates have never been abroad, indicating that bla NDM genes are also associated with some special clones (Leverstein- Van et al., 2010).
In China, since the first report of bla NDM gene in four carbapenem-resistant Acinetobacter baumannii isolates (Chen et al., 2011), increasing Enterobacteriaceae have been identified as carriers of the bla NDM gene. Escherichia coli, an important member of Enterobacteriaceae, are often spread globally through some epidemiological lineages. Although the prevalence of NDM-producing CRE strains is low, outbreaks caused by bla NDM -positive isolates have been identified in several regions of China, indicating high transferability of the bla NDM gene and the severity of infections caused by bla NDM−1 -positive organisms (Wang et al., 2014;Jin et al., 2015;Yu et al., 2016). Furthermore, Kaase et al. (2011) first reported a novel bla NDM variant, bla NDM−2 , which differs by one amino acid substitution (Pro28Ala) from bla NDM−1 , and the subsequent discovery of other bla NDM variants highlights the rapid evolution of this multi-drug resistance gene. In 2012, the NDM enzyme reservoir, India, first reported diverse bla NDM variants among Enterobacteriaceae and bla NDM variants exhibited higher minimum inhibitory concentration (MIC) levels of carbapenem compared with bla NDM−1 (Rahman et al., 2014). Although the bla NDM gene is continuously recoverable in China, data on the prevalence and characteristics of bla NDM variants among Enterobacteriaceae are still needed for preventing its transmission. Notably, a study conducted by Hu et al. (2017) have discovered that various species of bacteria harbored several kinds of bla NDM variants in China, which were mainly carried by diverse plasmids with different sizes. In the present study, we reported a high prevalence of bla NDM variants among E. coli from five hospitals in Northern Jiangsu Province, China. Moreover, these diverse bla NDM variants were mainly located on the same plasmid.

Study Design
From September 2015 to July 2016, five hospitals (two in Xuzhou, two in Suqian, and one in Lianyungang) in Northern Jiangsu Province of China collected 1,185 E. coli isolates to examine the prevalence and molecular epidemiology of carbapenemresistance isolates. Initial species identification and antimicrobial susceptibility testing was performed by the Vitek 2 system (bioMe'rieux, France) and MALDI-TOF MS (Bruker Microflex LT, Bruker Daltonik GmbH, Bremen, Germany) according to the manufacturer's instructions.

Antimicrobial Susceptibility Testing
Initial susceptibility testing was examined by Vitek 2 system. Further MICtesting was conducted by agar dilution method for cefoxitin, ceftriaxone, ceftazidime, cefepime, aztreonam, amikacin, ciprofloxacin, tigecycline, and piperacillin/tazobactam. The MICs of imipenem, meropenem, and ertapenem were determined by E-tests. For colistin, MIC values were tested by broth microdilution method. The agar dilution method and Etest were performed according to the standard Clinical and Laboratory Standards Institute guideline (M100-S26) (CLSI, 2017). The breakpoints of Food and Drug Administration (FDA) and European Committee on Antimicrobial Susceptibility Testing were used for tigecycline and polymyxin, respectively.

PFGE and MLST
Molecular typing of 54 NDM-producing E. coli isolates was performed by pulsed-field gel electrophoresis (PFGE). The plugs containing genomic DNA were prepared according to the procedure described by Pereira et al. (2015). The DNA fragments digested with restriction endonuclease XbaI (TaKaRa Biotechnology, Dalian, China) were separated by PFGE on 1% SeaKem Gold agarose (Lonza, Rockland, ME, USA) using the CHEF Mapper XA PFGE system (Bio-Rad, USA) for 18 h at 14 • C. The electrophoretic switch times were 6.8-35.4 s. Salmonella H9812 was used as reference marker. Dice coefficients was used to calculate the similarity of PFGE patterns. Dendrograms were constructed by the unweighted pair group method with arithmetic averages (UPGMA) using BioNumerics software version 5.10. Isolates were categorized to be of the same cluster when their dice similarity index was ≥85%. Multi-locus sequence typing of E. coli was conducted by PCR as previously described (Wirth et al., 2006). The allelic profiles and sequence types were identified by amplifying and sequencing the seven housekeeping genes (adk, fumC, gyrB, icd, mdh, purA, recA) according to the reference website (https://enterobase.warwick.ac.uk/species/ index/ecoli). A minimum spanning tree of 54 bla NDM -positive isolates was also constructed by BioNumerics software version 5.10.

Conjugation Assay
The conjugation experiment was implemented by mix broth mating among 26 selected isolates. The donor (clinical strains harboring the bla NDM gene) and recipient (sodium azideresistant E. coli J53) were mixed and cultured in broth at 37 • C overnight. The transconjugants were selected on MH agar with sodium-azide (180 µg/mL) and imipenem (1 µg/mL).. Initial species identification was conducted by Vitek MS system. Transformants were regarded as transconjugants when it exhibited resistance to carbapenem and harbored the bla NDM gene.

Plasmid Analysis
Twenty-six selected isolates, including bla NDM -5 and bla NDM -1 gene with different STs from the aforementioned five different hospitals and all bla NDM -4 (one isolates was lost in transit), bla NDM -7 , and bla NDM -9 -positive isolates, were subjected to further plasmid analysis. Incompatibility groups of plasmids extracted from transconjugants were determined by PCR-based replicon typing as described previously (Carattoli et al., 2005;Johnson et al., 2012). S1-PFGE and Southern blotting were conducted to isolate and locate resistance plasmids. Briefly, the gel plugs embedded with bla NDM -positive isolates were digested with S1 nuclease (TaKaRa Biotechnology, Dalian, China) and linear plasmids were separated by CHEF-Mapper XA PFGE system (Bio-Rad) as described above. The universal primers (F: GAAGCTGAGCACCGCATTAG; R: GGGCCGTATGAGTGATTGC) were used for probe synthesis. The plasmid DNA were transferred to positive-charged nylon membranes (Millipore, USA), and DIG-labeled bla NDM− specific probe served to hybridize plasmids according to the instructions of the DIG High Prime DNA Labeling and Detection Starter Kit (Roche, USA).

DISCUSSION
Metallo-lactamase, NDM, is an emerging carbapenem-resistant β-lactamase that is of major public concern due to its high medical and economic burden (Otter et al., 2017), especially for developing countries such as India, Pakistan, and the Balkan countries. As the most populous country in the world, there are major difficulties in preventing the dissemination of multidrug resistant genes in China. Therefore, comprehensive, extensive studies on diverse bla NDM variant-positive E. coli are needed to provide clear information to optimize antibiotic policy in endemic areas. Generally, the prevalence of the bla NDM gene has continuously increased worldwide. As of now, the NDM enzyme has been identified in almost all of the world, including many countries in Asia, Africa, Europe, the Americas, and Australia (Berrazeg et al., 2014). A study from India also analyzed the occurrence of the bla NDM gene among carbapenem resistant isolates, and it accounted for 45.4% of them (Rahman et al., 2018). Recently,   a survey from the French National Reference Center revealed that among 140 carbapenem-resistant isolates 21% were NDM producer (Gauthier et al., 2018). In 2017, a nationwide study of clinical CRE strains in China demonstrated that 49% were NDM producer among carbapenem-resistant E. coli . Furthermore, a multicenter study of the China CRE network revealed that among 39 carbapenem-resistant E. coli isolates, 74.4% were NDM producer, suggesting that there is a serious challenge in combating infections caused by this "superbug" in China (Zhang et al., 2018). In the present study, we identified 53 bla NDM -carrying isolates among 54 CRECO, which is much higher than in any other region of China (Wang S. et al., 2016;Hu et al., 2017;Liang et al., 2017). To the best of our knowledge, this is also the first report on the bla NDM gene in Northern Jiangsu Province. Moreover, the emergence of such a high prevalence of bla NDM variants indicates that the bla NDM gene is increasing in this area. Since its first identification in 2009, the bla NDM gene has evolved at a fast pace during the past 10 years. Twenty-one bla NDM variants have been identified in different countries, all of which are archived at http://www.lahey.org/Studies/other.asp. Khan et al. (2017) reported that the Asian continent, especially China and India, was a reservoir of NDM producers, in which about a 58.2% abundance of the bla NDM−1 variants was found. Among these bla NDM variants, the bla NDM−1 gene has been reported as the most prevalent type worldwide (Nordmann and Poirel, 2014). In our study, five bla NDM variants were identified as responsible for MBL production, with the bla NDM−5 FIGURE 3 | Dendrogram of PFGE profiles of 54 CRECO isolates. The UPGMA algorithm was used to construct dendrogram based on the dice similarity coefficient. Isolates were categorized to be of the same cluster when their dice similarity index was ≥ 85%. gene being the most prevalent. Compared with NDM-1, NDM-5 producers exhibited higher hydrolytic activity and toxicity toward carbapenem and cephalosporin (Mei et al., 2017). The bla NDM−5 gene is an emerging bla NDM variants and has tended to surpass bla NDM−1 recently, and it differs from the NDM-1 enzyme at two amino acid substitutions, exhibited increased carbapenem resistance . The bla NDM−4 and bla NDM−7 genes have been discovered among E. coli in China with relatively low prevalence, involving in 4 and 6 patients, respectively. Moreover, clinical bla NDM−9 -positive E. coli has only been found in Taiwan (Lai et al., 2017), with this being the first report on the mainland China. Moreover, not merely NDM-5, amino acid substitutions in NDM-4 (M154L), NDM-7 (D130A), and NDM-9 (E152A) could also result in high levels of carbapenem resistance (Düzgün, 2018). Stewart et al. (2017) reported that the substitution in M154L, which is found in most bla NDM variants, could enhance resistance to ampicillin at low zinc(II) concentrations relevant to infection sites.
bla NDM variant-positive isolates exhibited multi-drug resistance. Variable resistances to aztreonam, amikacin, and ciprofloxacin were identified in NDM-1 and NDM-5-producing E. coli. The NDM-7, NDM-4, and NDM-9-producing isolates exhibited pan-resistant phenotypes, showing resistance to almost all commonly used clinical antibiotics except for colistin and tigecycline. In view of this, the expert recommended polymyxins was regarded as the last resort for NDM-producing isolates (Yamamoto and Pop-Vicas, 2014).
ST167 was confirmed as significant carrier in the present study, and is reported to be associated with the production of the NDM enzyme, especially NDM-5 . Notably, four bla NDM -positive ST617 strains were identified in the world, but we identified 6 isolates in the present study. Moreover, a novel new variant of bla NDM, bla NDM−21, belonged to ST617, which   should deserve more attention (Liu et al., 2018). Furthermore, four new STs ST361, ST46, ST2376, and ST2083 were firstly reported to be linked with NDM-producing isolates. The high efficiency of transfer renders the NDM producer ubiquitous throughout the world. Torres-González et al. (2015) reported an outbreak caused by bla NDM−1 -carrying plasmid, which is easily transferred between E. coli and Klebsiella pneumoniae. Similarly, in the present study, the plasmids of 26 selected CRECO isolates were also tested for transfer. All selected bla NDM−5 genes were identified to be located on 55 kb plasmid, among which IncX3 was the most common replicon type. Notably, bla NDM−5 gene was frequently reported to be carried by the IncX3 plasmid of 55 kb in size, which was widely reported in China , Inidia (Krishnaraju et al., 2015), Australia (Wailan et al., 2015), and Damark (Hammerum et al., 2015). Moreover, Li et al. (2018) reported that IncX3 type plasmids play an important role in the transmission of the bla NDM−5 gene in Enterobacteriaceae and this kind of plasmid occurred in different species. Further illustrated the challenge of preventing the dissemination of the bla NDM−5 gene. The widespread bla NDM−1 -carrying plasmid has been found to be associated with multiple replicon types, including IncX3, IncF, and IncA/C etc. In agreement with previous studies (Göttig et al., 2013;Wang L. H. et al., 2016), four bla NDM−7 -positive plasmids were identified as ∼55 kb IncX3 type, while the bla NDM−7 gene was also detected in the IncFI plasmid, which have been identified in India (Rahman et al., 2014). As for bla NDM−9 , to the best of our knowledge, this is the first report on ∼105 kb IncI1 type plasmid harboring bla NDM−9 gene. Overall, most plasmids harboring bla NDM variants were identified as 55 kb IncX3 types, hinting that amino acid mutations might occur in the process of plasmid transfer, resulting in the emergence of bla NDM variants. The high prevalence of the bla NDM genes due in part to plasmid transfer, meanwhile, the fast evolution of this multidrug resistance gene also favors the persistence of such bacteria harboring it.
In summary, the present study reported high prevalence of bla NDM variants, especially bla NDM−5 , among carbapenemresistant E. coli in Northern Jiangsu Province. The presence of five different variants further increases the threat to public health because of the limited treatment options. Notably, diverse bla NDM variants were mainly located on ∼55 kb IncX3 plasmids, indicating that the fast evolution and high transferability of this kind of plasmid has led to the high prevalence of bla NDM variants. Timely detection of NDM enzyme and antimicrobial susceptibility testing are necessary so that infections caused by NDM producers receive appropriate and effective therapy. Similarly, large-scale surveillance and effective infection control measures are also urgently needed to prevent diverse bla NDM variants from becoming epidemics in the future.

AUTHOR CONTRIBUTIONS
The laboratory measurements were performed by RB, ZK, and HQ. BG and PM participated in experimental design and manuscript revision. Data analysis were implemented by RB, ZK, HQ, FJ, and HK.