Molecular Characterization of blaIMP–4-Carrying Enterobacterales in Henan Province of China

Carbapenem-resistant Enterobacterales (CRE) pose a serious threat to clinical management and public health. We investigated the molecular characteristics of 12 IMP-4 metallo-β-lactamase-producing strains, namely, 5 Enterobacter cloacae, 3 Escherichia coli, 2 Klebsiella pneumoniae, and 2 Citrobacter freundii. These strains were collected from a tertiary teaching hospital in Zhengzhou from 2013 to 2015. The minimum inhibitory concentration (MIC) results showed that each blaIMP–4-positive isolate was multidrug-resistant (MDR) but susceptible to colistin. All of the E. coli belonged to ST167, two C. freundii isolates belonged to ST396, and diverse ST types were identified in E. cloacae and K. pneumoniae. S1-PFGE, Southern blotting, and PCR-based replicon typing assays showed that the blaIMP–4-carrying plasmids ranged from ∼52 to ∼360 kb and belonged to FII, FIB, HI2/HI2A, and N types. N plasmids were the predominant type (8/12, 66.7%). Plasmid stability testing indicated that the blaIMP–4-carrying N-type plasmid is more stable than the other types of plasmids. Conjugative assays revealed that three of the blaIMP–4-carrying N plasmids were transferrable. Complete sequence analysis of a representative N type (pIMP-ECL14–57) revealed that it was nearly identical to pIMP-FJ1503 (KU051710) (99% nucleotide identity and query coverage), an N-type blaIMP–4-carrying epidemic plasmid in a C. freundii strain. PCR mapping indicated that a transposon-like structure [IS6100-mobC-intron (K1.pn.I3)-blaIMP–4-IntI1-IS26] was highly conserved in all of the N plasmids. IS26 involved recombination events that resulted in variable structures of this transposon-like module in FII and FIB plasmids. The blaIMP–4 gene was captured by a sul1-type integron In1589 on HI2/HI2A plasmid pIMP-ECL-13–46.


INTRODUCTION
The Zn(II)-containing metallo-β-lactamases (MBLs) comprise Imipenemase (IMP), New Delhi metallo-β-lactamase (NDM), and Verona Integron-encoded Metallo-βlactamase (VIM) types that belong to class B β-lactamase according to the Ambler classification. MBLs can hydrolyze nearly all β-lactams, including carbapenems, which are important antibiotics in clinical practice and the "last line" drugs for treating infections caused by multiple drug-resistant (MDR) Gram-negative bacteria (Boyd et al., 2020). The rapid spread of MBLs among Enterobacterales has led to the increased prevalence of carbapenem-resistant Enterobacterales (CRE), and this presents a challenge for infection treatment worldwide (Nordmann and Poirel, 2019). Unlike NDMs, IMP-type β-lactamases are not often detected in CRE from China (Zhang et al., 2017;Wang et al., 2018). The most commonly encountered bla IMP−4 gene has been found captured by class 1 integrons and carried by plasmids belonging to multiple replicon types including HI2, L/M, A/C, and N for dissemination Matsumura et al., 2017). An epidemic N plasmid in Enterobacterales isolates was recently recovered from Shanghai, Guangdong, and Fujian provinces of China and was responsible for the dissemination of bla IMP−4 gene (Wang et al., 2017). It is not known if this type of plasmid is prevalent in other regions of China and if it is involved in the spread of bla IMP genes. We conducted a retrospective study to investigate the prevalence and molecular characterization of IMP-positive Enterobacterales isolates in Henan Province within the north central region of China.

Bacterial Isolates and Antimicrobial Susceptibility Testing
From January 2013 to December 2015, a retrospective survey for MBLs in CRE isolated from a tertiary teaching hospital of Zhengzhou University identified 12 bla IMP−4 positive isolates, which were recovered from different types of clinical specimens ( Table 1). The study and consent procedure was approved by the Ethical Committee of Zhengzhou University. PCR and sequencing were used to identify MBL encoding genes, including bla IMP , bla NDM , and bla VIM , as described previously (Doyle et al., 2012). Antimicrobial susceptibility of the 12 bla IMP−4positive isolates and their transconjugants was determined using microbroth and agar dilution methods according to the Clinical and Laboratory Standards Institute (CLSI) guidelines (CLSI, 2019). Escherichia coli ATCC25922 was used as the quality control.
Conjugation Assay, S1-PFGE, and Southern Blotting Conjugation experiments were conducted using methods described previously at 25, 30, and 37 • C. Briefly, the bla IMP−4 -positive isolates served as the donor, while E. coli EC600 (rifampin resistant) was used as the recipient strain. Transconjugants were selected on Mueller-Hinton (MH) agar supplemented with sodium rifampin (200 µg/ml) and meropenem (2 µg/ml). The presence of the bla IMP−4 gene and other resistance genes in transconjugants was confirmed by PCR, DNA sequencing, and antimicrobial susceptibility. S1-PFGE and Southern blotting were conducted, according to published methods, to estimate sizes of bla IMP−4 plasmids (Qin et al., 2014).

Plasmid Stability
Stability tests for plasmids were conducted as described previously (Wang et al., 2017). Briefly, the bla IMP−4 -harboring transformants from ECL14-57, CF-15-127, and ECL-13-46, which were representative of bla IMP−4 -carrying N, F, and HI2 plasmids characterized in this study, respectively, were used as the test strains. The overnight growths of the bacteria in LB broth were inoculated into 2 ml of a fresh LB broth and incubated for 12 h at 37 • C (time zero). The above process was repeated every 12 h (equivalent to 10 generations each). At time zero and after passage without antibiotic for 50, 100, 150, and 200 generations, a sample of the culture was diluted and spread onto a LB plate. One hundred colonies were picked and replica plated onto a pair of plain and antibiotic-containing (0.5 µg/ml meropenem) LB plates. Plasmid stability was determined by the percentage of colonies growing on the antibiotic-containing plates.

Overview of the bla IMP−4 -Positive Isolates
A total of 12 (12/317, 3.79%) bla IMP−4 positive isolates, namely, 5 E. cloacae, 3 E. coli, 2 K. pneumoniae, and 2 C. freundii strains, were obtained from 317 CRE. These strains were recovered from different sample types including urine, blood, wound, abdominal drainage, sputum, and cerebrospinal fluid ( Table 1). Over half of the bla IMP−4 -carrying isolates (7/12, 58.33%) were collected from the ICU department, and the mortality among the patients infected with a bla IMP−4 -positive isolate was 25% (3/12) ( Table 1). These patients were diagnosed with different clinical diseases and none of them had a history of foreign travel.
For the antimicrobial susceptibility profiles, all the bla IMP−4positive isolates were susceptible to colistin [minimum inhibitory concentrations (MICs) of ≤2 µg/ml]; tigecycline also had high activity against these isolates (MIC 50 = 0.5 µg/ml) ( Table 2). Our observation is consistent with previous data from both China and other countries which showed that colistin and tigecycline are effective for the treatment of infections caused by CRE .

Bacterial Genotyping, Conjugation, and Plasmid Analysis
MLST was performed for all the IMP-4-positive E. cloacae, E. coli, C. freundii, and K. pneumoniae isolates. Based on the MLST results, five E. cloacae isolates were distributed to four ST types, namely, ST133 (n = 2), ST231 (n = 1), ST754 (n = 1), and ST97 (n = 1). All of the three E. coli isolates belonged to ST167, which is regarded as the most common clone of E. coli in China (Zhang et al., 2017;Wang et al., 2018). Two C. freundii isolates belonging to ST396. ST14-and ST17-type K. pneumoniae carried bla IMP−4 in this study (Table 1). Overall, the observation of diversity in the isolates of E. cloacae for carrying bla IMP−4 indicated that the mobile genetic elements, such as conjugative plasmids and transposons, might be responsible for the horizontal transfer of bla IMP−4 among different clones. The bla IMP−4 gene was always carried by a plasmid, so S1-PFGE and Southern blotting were performed to identify bla IMP−4 harboring plasmids. The bla IMP−4 genes in all 12 CRE isolates were located on plasmids with sizes ranging from ∼52 to ∼360 kb. The ∼52 kb plasmids were predominant among those carrying bla IMP−4 (8/12, 66.7%). Conjugative assays revealed that only three ∼52 kb bla IMP−4 -carrying plasmids were successfully transferred to E. coli EC600 from the donors by conjugation at frequencies of 3.2 × 10 −4 -4 × 10 −5 per donor cell. The other nine IMP-4-encoding plasmids which failed to transfer to the recipient strain by conjugation were electrotransformed into E. coli DH5α. PCR-based replicon typing analysis for both transconjugants and transformants showed that all the ∼52 kb bla IMP−4 -carrying plasmids were distributed in four E. cloacae, three E. coli, and one K. pneumoniae isolates belonging to plasmid replicon type N (Table 1 and Figure 1A). The details concerning plasmid name, size, and replicon type are summarized in Table 1.

Sequence Analysis of bla IMP−4 -Carrying Plasmids and Genetic Environments of bla IMP−4
A representative N-type bla IMP−4 -carrying plasmid named pIMP-ECL14-57, which came from E. cloacae strain ECL14-57, had 51,795 bp, with an average GC content of 50.52%, encoding 54 predicted open reading frames (ORFs). It shared extensive similarity with pIMP-FJ1503 (99% nucleotide identity and query coverage) (KU051710), an N-type bla IMP−4carrying plasmid in a carbapenem-resistant C. freundii strain CRE1503 isolated from Hong Kong (Figure 1A). Comparative genomic analysis between these two plasmids revealed only two differences: (1) the intact ISkpn19 element downstream of qnrS1 that was carried by pIMP-FJ1503 was inserted by an IS26 element in pIMP-ECL14-57 and (2) the Int1 gene immediately upstream of bla IMP−4 was complete in pIMP-ECL14-57 but was truncated in pIMP-FJ1503 ( Figure 1A). Only two resistance genes, namely bla IMP−4 and qnrS1, conferring resistance to carbapenems and quinolones, respectively, were identified in each plasmid. The bla IMP−4 gene-associated class 1 integron In823 was carried by a transposon-like structure [IS6100-mobC-intron (K1.pn.I3)-bla IMP−4 -IntI1-IS26] bracketed by two 5 bp direct repeats (DR: AACAG) inserted between the EcorII and uvp1 genes. In addition, this bla IMP−4 -carrying transposon-like structure was also identified in the other seven N plasmids by using PCR mapping and sequencing ( Figure 1D).
Overall, the bla IMP−4 -associated In823 flanked by IS6100 and IS26 in N plasmids was conserved, and IS26 involved recombination events that resulted in variable structures of this transposon-like module in the FII and FIB plasmids. Analysis of a ∼46 kb bla IMP−4 -carrying segment from the HI2/HI2A plasmid pIMP-ECL-13-46 (failure to obtain complete sequence by WGS) revealed that the bla IMP−4 gene was present in the sul1-type integron In1589, which was first identified in an HI2 plasmid pIMP-4-EC62 obtained from E. cloacae EC62 of swine origin (Zhu et al., 2019).

Stability of bla IMP−4 -Carrying Plasmids
Plasmid stability analysis revealed that the N-type plasmid pIMP-ECL14-57 in transformants from ECL14-57 could be maintained at 100% over 200 generations of multiplication in the absence of antibiotics. However, drastic loss of the F-type plasmid pIMP-CF-15-127 and HI2-type plasmid pIMP-ECL-13-46 in transformants from CF-15-127 and ECL-13-46 was observed after 50 generations of multiplication, with 35 and 3%, respectively, retaining the bla IMP−4 -harboring plasmid after 150 generations. These results revealed that, among the plasmids carrying bla IMP−4 , the N type is more stable than the F type and HI2 type.

DISCUSSION
The IMP-4-type MBL, first identified in clinical Acinetobacter spp. from Hong Kong (Chu et al., 2001), has spread to Australia but has not been frequently detected as KPC-2 and NDM among CRE in mainland China (Xiong et al., 2016). The incidence (3.79%) of IMP-4-producing Enterobacterales observed in the CRE of this study was comparable to that found in a recent nationwide survey of CRE (3.6%) . The bla IMP−4 gene was found in four species, namely, E. cloacae, E. coli, K. pneumoniae, and C. freundii, which are the most common species carrying bla IMP genes . A report from Australia indicated that IMP-4 was the predominant MBL type among CRE, particularly in carbapenemresistant E. cloacae (CRECL) (Sidjabat et al., 2015). Our previous study together with recent findings from China revealed the dominance of NDM-type MBL among CRECL; whether IMP-4 is the second most common MBL in CRECL needs further study (Liu et al., 2015;Jin et al., 2018).
All of the bla IMP−4 genes in this study were carried by plasmids with diverse replicons. These included HI2, N, F, and especially the predominant N plasmids. The N type is a broad host range plasmid that carries a variety of resistance determinants and shows resistance to extendedspectrum-β-lactams, sulfonamides, quinolones, aminoglycosides, tetracyclines, and streptomycin (Eikmeyer et al., 2012). N plasmids are also associated with the spread of carbapenemresistant determinants, such as bla NDM and bla KPC (Poirel et al., 2011;Partridge et al., 2012;Eilertson et al., 2017;Jiang et al., 2017;Partridge et al., 2018;Schweizer et al., 2019). This type of plasmid was recently identified as an epidemic plasmid for carrying bla IMP−4 among Enterobacterial species in China Wang et al., 2017), and it was responsible for horizontal transmission of bla IMP−6 among Enterobacterales from Japan (Yamagishi et al., 2020). Our findings are consistent with these studies and indicate the prevalence of N bla IMP−4 -carrying epidemic plasmids among CRE in other regions of China. Additionally, FII plasmids, which are carriers of the bla KPC gene in K. pneumoniae (Partridge et al., 2018;Yang et al., 2020), were found to carry the bla IMP gene in this study. Association with these widespread types of plasmids may accelerate dissemination of bla IMP genes among K. pneumonia.

CONCLUSION
In conclusion, we determined the prevalence and molecular characterization of bla IMP−4 -positive Enterobacterales in clinical specimens collected at a teaching hospital in Henan Province. Previously reported epidemic N-type plasmids exhibited superior stability compared with F-and HI2-type plasmids. N-type plasmids were the predominant plasmids carrying bla IMP−4 among the collected Enterobacterales. Associated with selftransmissible N plasmids, widespread FII plasmids and a successful epidemic E. coli ST167 clone might facilitate further dissemination of bla IMP−4 among the Enterobacterales. Surveillance is needed to monitor the spread of bla IMP−4harboring Enterobacterales.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://www.ncbi. nlm.nih.gov/, MH727565, CP068028, CP068026, CP068027, and CP068240.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by the Ethical Committee of Zhengzhou University. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin. Written informed consent was obtained from the individual(s), and minor(s)' legal guardian/next of kin, for the publication of any potentially identifiable images or data included in this article.

AUTHOR CONTRIBUTIONS
SQ and ZW designed the study. WL, TY, CL, and SZ performed the experiments. HD, JC, LL, and XF analyzed the bioinformatics data. SQ and JC wrote the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.