Genomic characterization of carbapenem resistant Escherichia coli from multiple hospitals in Nanjing, China: focusing on frequent co-occurrence of blaNDM and blaKPC-2

Background: The increasing emergence of carbapenem resistant Escherichia coli (CREC) poses a potential threat to public health, hence genomic characterization of isolates is needed for a better understanding of its transmission and implementation of infection control measures. Materials and methods (cid:0) Eleven CREC isolates were collected in 2015 from 6 hospitals in Nanjing, China, and analyzed using whole genome sequencing. Resistance determinants, virulence elements, multi-locus sequence type (MLST), serotypes, phylogeny and mH types were determined. Results: All of the CREC carried at least one carbapenemase. NDM-5 (n=9) was the most frequent carbapenemase, followed by KPC-2 (n=3) and NDM-1 (n=2); three isolates produced NDM-5 and KPC-2. Ten out of the 11 isolates co-carried blaCTX-M variants. MLST analysis found 7 distinct STs, including ST410 (n=2), ST3489 (n=1), ST156 (n=1), ST683 (n=1), ST297 (n=1), ST167 (n=1), and ST361 (n=1). Six distinct serotypes and 8 Fim types were identied. A great diversity of plasmid proles was observed with plasmid replicon IncX3 being the most frequent (n=11). Phylogenetic analysis showed great diversity between the 11 CREC isolates and also between 6 additional isolates co-carrying blaNDM and blaKPC which were selected from the strains collection of Nanjing Drum Tower Hospital for comparison. Conjugation assays demonstrated that blaNDM was transferable. Conclusion: NDM is the major carbapenemase among CREC, with NDM-5 being the main variant which can be horizontally disseminated by IncX3 plasmids. These isolates displayed genetic diversity by MLST, Fim typing and serotyping. We herein provided the rst report on emergence of NDM-5 producing E. coli ST297, ST683, ST3489, and NDM-1 producing E. coli ST361.


Introduction
Escherichia coli mainly inhabits the lower intestinal tract of warm-blooded animals. It is a major pathogen for numerous types of infections, such as intestinal, urinary, and respiratory tract infections in humans and other animals (1) . In recent years, carbapenems have been increasingly used as the most effective antibiotic in clinical therapy for infections caused by multidrug-resistant (MDR) strains, due to production of extended spectrum β-lactamases (ESBLs) or AmpC-type β-lactamases (2). Therefore, the frequent occurrence of carbapenem-resistant Escherichia coli (CREC) worldwide has been posing a threat to public health (3,4), Production of carbapenemases has been so far the main mechanism for carbapenem resistance in Enterobacterales (5), and New Delhi metallo-β-lactamase (NDM) is the major carbapenemase in E. coli all over the world (6). It is worthy to note that the co-occurrence of multiple ß-lactamases among single bacteria species (7), especially carbapenemase, such as co-production of NDM-1 and Klebsiella pneumoniae carbapenemase 2 (KPC-2)(8), co-occurrence of KPC-2 and OXA-48, have so far been frequently described in multiple clinical Enterobacterales, such as K. pneumoniae, Enterobacter cloacae, and Citrobacter freundi (8)(9)(10). Thus, such a frequent co-occurrence of carbapenemase in one isolate are causing rapidly rising carbapenem resistance, leading to increasing CREassociated morbidity and mortality (11) Report from the China CRE Network showed that the overall CREC infection incidence differed signi cantly by region, with the highest in Shandong (9.1%) and the lowest in Qinghai (0 %) during January to December 2015 (12); another study showed a 4.6% prevalent rate of CREC in in Northern Jiangsu Province, China during September 2015 to August 2016 (13). However, strains analyzed were predominantly collected from tertiary hospitals. Specialized hospitals, Children's hospital and level II hospitals were less involved.
Moreover, multiple studies have shown that that blaNDM is often located on IncX3 plasmids, (14,15), but little information on virulence genes, serotyping and m typing is available for such strains.
In this study, we tried to characterize the genomic epidemiology of CREC strains including resistance determinants, virulence factors, serotyping, m typing and plasmid replicons. Furthermore, the molecular characterization of strains co-producing NDM-5 and KPC-2 screened from clinical CREC in our hospital during 2013-2017 were investigated.
Secondly, considering the high prevalence of E. coli isolates co-producing KPC-2 and NDM among the 11 CREC strains from 6 hospitals, we tried to investigate the distribution of these strains among clinical CREC isolates. Therefore, a total of 43 consecutive non-duplicate isolates collected in Nanjing Drum Tower hospital during 2013-2017 were further analyzed for isolates co-producing KPC-2 and NDM by PCR and DNA sequencing. Among them, 4 strains were isolated in 2013, 10 in 2014, 2 in 2015, and 11 from 2016 and 16 in 2017. The source of the samples was as follows: urine (n=18), blood (n=9), sputum (n=6), secretion (n=3), bile (n=3), abdominal dropsy (n=2), and pus (n=1).
Isolates resistant to at least one carbapenem (imipenem, meropenem, ertapenem) were included in the study.

DNA Extraction
The Ultraclean Microbial DNA Isolation Kit (MOBIO Laboratories, Carlsbad, CA, US) was used to extract genomic DNA. The NanoDrop 2000c spectrophotometer (Thermo Scienti c, Waltham, MA, USA) was used for measuring the DNA concentration and purity for whole genome sequencing.
Whole Genome Sequencing, denovo Assembly, Scaffolding, and Annotation The prepared pair-end DNA library was sequenced on the MiSeq (Illumina, SanDiego, CA, USA). Denovo assembly of the paired-end reads was performed by CLC Genomics Workbenchv7.0.4 (QIAGEN, Hilden, Germany) after quality trimming (Qs ≥ 20). Scaffolding was nished using SSPACE standard version 3.0 and the gaps within scaffolds were further closed by GapFiller (17,18). Then genomes were then submitted to NCBI for annnotation.

The screening of Escherichia coli co-producing NDM and KPC-2 carbapenemases
In order to investigate the prevalence of E. coli isolates co-producing NDM-5 and KPC-2 in our hospital during 2013-2017, genes encoding carbapenemases (KPC and NDM) were detected by PCR and DNA sequencing (22). The positive products were sent to the Qingke Biotechnology Co., Ltd (Nanjing, China) for puri cation and sequencing. Sequences were further analyzed by using the Chromas-Pro application and BLAST (www.ncbi.nlm.nih.gov/BLAST).

Pulsed-eld gel electrophoresis
Six E. coli isolates co-producing NDM-5 and KPC-2 including 3 ones from 11 CREC and 3 ones selected from the 43 CREC were further analyzed for genetic relatedness by PFGE, which was performed according to the protocol as previously described (23). Brie y, fresh colonies were mixed with proteinase K (Merck Sharp & Dohme Ltd, Germany) into plugs. After the plugs were digested by restriction endonuclease XbaI (Fermentas, ABI, Germany), the resultant DNA fragments were separated in a PFGE CHEF-DR III system (Bio-Rad Laboratories, Hercules, CA) in 0.5×Tris-borate-EDTA buffer at 120 V for 19 h. The pulse times ranged from 2.2 s to 54.2 s. Finally, the BioNumerics software (Applied Math, Sint-Maten-Latem, Belgium) was used to analyze the banding patterns.

Conjugation assay
For the 6 isolates co-carrying blaKPC and blaNDM, broth mating was performed in order to analyze the transferability of these genes according to the protocol prescribed previously (24). Azide resistant E. coli J53 was used as the recipient. Brie y, fresh colonies were inoculated into 5 ml LB broth and incubated at 37℃, 200 rpm. After 5 hours, 500 μl recipient cells and 100 ul donor were suspended in 5 ml LB broth for overnight culture at 37℃, 200 rpm, then 100 μl were plated onto the LB plates containing 30 mg/L cefoxitin and 100 mg/L sodium azide for E. coli J53. PCR (ampli cation for blaNDM and blaKPC) and Eric-PCR were used to verify conjugants.

Results
The susceptibilities of the 11 CREC.

Phylogenetic characterization of 11 CREC strains
The phylogenetic tree showed that 11 CREC evolve into 2 main clades albeit a great diversity was observed ( Figure 1). Two isolates from Nanjing Children's hospital displayed close evolutionary relationship.

Prevalence of isolates co-producing NDM-5 and KPC-2
Among the 43 CREC collected from our hospital during 2013-2017, 6 KPC-2 and 23 NDM were identi ed, two strains co-producing NDM-5 and KPC-2 and one strain co-carrying NDM-1 and KPC-2 were found. One was isolated from the urine of an inpatient in ICU in 2014, the other two strains were isolated from abdominal dropsy and sputum of the different patients in 2017.
Genetic relatedness of strains co-carrying blaKPC and blaNDM PFGE displayed a high diversity of the 6 strains co-producing blaKPC and blaNDM (Figure 2), indicating that these strains were not from the same clone.

Transferability of blaKPC and blaNDM
Conjugation assay revealed that the blaNDM of all the 6 isolates was transferable to E. coli J53. However, we could not isolate any conjugants with blaKPC, suggesting that the blaKPC and blaNDM were not on the same plasmid.

Discussion
In this study, we provided data on genomic epidemiology of 11 CREC strains from 6 hospitals in Nanjing city, Jiangsu province. Based on the high cooccurrence of blaNDM-5 and blaKPC-2, 43 CREC strains collected from a tertiary hospital during 2013-2017 were further screened to investigate the prevalence of such strains in our hospital. This is the rst study that provided the genomic epidemiology of the CREC from multiple hospitals in Nanjing.
The high resistance toward the commonly used antibiotics in clinical therapy displayed by the CREC from 6 hospitals were consistent with the previous report (25), leading to a quite limited choice of antimicrobial agents for infections caused by such strains. Fortunately, tigecycline, colistin and aztreonam/avibactam showed the best sensitivity. Of note, the co-occurrence of KPC and NDM among single isolate seems not confer higher resistance to β-lactams when the MICs of β-lactams were compared between the strains carrying blaNDM and the ones co-carrying blaKPC and blaNDM. In addition, it was reported that MICs of ertapenem against strains producing NDM-5 are 4-or 8-fold higher than those against strains producing NDM-1(26), however, we did not observe such a phenomenon, we therefore speculate that the existence of other resistant mechanism such as the production of ESBLs and AmpCs, overexpressed e ux pumps, as well as decreased outer membrane permeability may contribute to the resistance towards β-lactams.
The high prevalence of NDM among 11 CREC in our study is in accordance with the previous report (6), indicating that NDM is the major carbapenemase for carbapenem resistance in E. coli, which may result from the low tness burden of the plasmid harboring blaNDM in E. coli (27). We found that NDM-5 is the most common variant, this is also in agreement with the present epidemiological data (28), demonstrating that NDM-5 is the predominant determinant conferring carbapenem resistance in CREC. Noteworthily, NDM-5 has been predominantly found in high-risk clone ST167, ST410 and ST101 in the hospital (29).This may result from successful expansion of E. coli clonal groups and frequent horizontal gene transfer of NDM-5 expressing plasmids. Notetaceblily, ST131 as a multidrug clone has spread extensively throughout the world (30). However, ST131 was not detected in our study. The multiple distinct STs identi ed in our study indicated the diversity of these CREC, which was also con rmed by the phylogenetic relationship. As known, NDM-5 has been reported in E. coli ST410, ST156, and ST167 (24). However, to the best of our knowledge, NDM-5 producing E. coli ST297, ST683 and ST3489, as well as NDM-1 producing E. coli ST361 has not been reported previously. Moreover, we found a high co-occurrence of KPC-2 and NDM-5, 5 out of the 6 strains that were isolated from our hospital which is a comprehensive tertiary hospital with 3000 beds. The more worse is that multiple resistance determinants including blaOXA-1, blaCMY, blaCTX-M, and fosA3, rmtB, qnr and aac(6')-Ibcr were also identi ed in these NDM-5 and/or KPC-2 producing strains, representing a signi cant challenge for clinical management and public health.
Despite multiple plasmid replicons were found among our CREC, most blaNDM -carrying plasmids belong to limited replicon types (IncX3, IncFII, or IncC) (4) Considering that plasmid replicon InX3 was found among all the NDM-producing E. coli, we speculate that IncX3 is the main host for blaNDM (31). Note worthily, the conjugation assay in our study showed that the spread of blaNDM was not accompanied by transfer of blaKPC-2, indicating that blaNDM and blaKPC-2 were not harbored by the same plasmid.
Virulence gene analysis revealed several major VFs in CREC, among them, gad encodes glutamate decarboxylase, which is the structural component of the major acid resistance system that protects E. coli from strong acid stress (pH < 3), typically encountered in the mammalian gastrointestinal tract (32), lpfA (Long polar mbriae ) is a putative adhesion gene, encoding one of the few mbrial adhesions of enterhemorrhagic E. coli O157:H7 associated with colonization on host intestine, which play essential roles during the bacterial infection process (32). iss (increased serum survival) is the most common avian pathogenic E. coli encoding gene. It has been identi ed as a virulence trait associated with the virulence of E. coli, causing colibacillosis in poultry (33). The high prevalence of these VFs among CREC may suggest that CREC mainly colonize the host intestine, and they might have a lower potential to cause human disease. Noteworthy, a strain isolated from urine belong to a new ST, which not only carried gad and iss, lpfA, but also sat (secreted autotransporter toxin), senB (Plasmid-encoded enterotoxin), sepA (Shigella extracellular protein A), iha (Adherence protein) and cnf1 (Cytotoxic necrotizing factor). It is known that sat can promote cytotoxic effects in several lines of undifferentiated epithelial cells and is highly prevalent in certain E. coli pathogenic groups responsible for urinary and intestinal infections (34). Cnf1 is frequently expressed in clinical UPEC isolates, CNF1-producing and β-hemolytic E. coli strains most notably cause urinary tract and meningeal infections in humans (35). Altogether, the wide presence of these VFs among the urinary CREC may indicate a higher pathogenicity of this strain.
O8:H9 as the most common serotype in our study was consistent with previous report, indicating that O8:H9 is a clinically-relevant serotype correlated with multidrug resistance (36) Altogether, the multiple mH types, the diverse STs and serotypes in our study, indicates a high diversity of these CREC strains. Although further PFGE from the strains co-carrying KPC and NDM excludes an epidemic dissemination of the frequent occurrence of these strains, the emergence of these strains poses a potential public health threat.