Characterization of a Carbapenem-Resistant Kluyvera Cryocrescens Isolate Carrying Blandm-1 from Hospital Sewage

Carbapenem-resistant Enterobacteriaceae have been a global public health issue in recent years. Here, a carbapenem-resistant Kluyvera cryocrescens strain SCW13 was isolated from hospital sewage, and was then subjected to whole-genome sequencing (WGS). Based on WGS data, antimicrobial resistance genes were identified. Resistance plasmids were completely circularized and further bioinformatics analyses of plasmids were performed. A conjugation assay was performed to identify a self-transmissible plasmid mediating carbapenem resistance. A phylogenetic tree was constructed based on the core genome of publicly available Kluyvera strains. The isolate SCW13 exhibited resistance to cephalosporin and carbapenem. blaNDM-1 was found to be located on a ~53-kb self-transmissible IncX3 plasmid, which exhibited high similarity to the previously reported pNDM-HN380, which is an epidemic blaNDM-1-carrying IncX3 plasmid. Further, we found that SCW13 contained a chromosomal blaKLUC-2 gene, which was the probable origin of the plasmid-born blaKLUC-2 found in Enterobacter cloacae. Phylogenetic analysis showed that K. cryocrescens SCW13 exhibited a close relationship with K. cryocrescens NCTC10483. These findings highlight the further dissemination of blaNDM through clonal IncX3 plasmids related to pNDM-HN380 among uncommon Enterobacteriaceae strains, including Kluyvera in this case.


Introduction
Carbapenem antibiotics are commonly considered as the most effective treatment option for infections caused by extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae. However, carbapenemase-producing Enterobacteriaceae have become a major threat to public health due to its rapid global dissemination [1]. New Delhi metallo-β-lactamase (NDM) is a type of metallo-β-lactamase that is able to hydrolyze a wide range of clinically available β-lactams (including carbapenems) [2]. Since the initial discovery of bla NDM-1 from a Klebsiella pneumoniae isolate in India in 2008 [3], 24 variants of NDM have been identified thus far [2]. Among them, NDM-1 is the most prevalent one with a worldwide distribution. It has been found in various bacterial species, and has disseminated among humans, food animals, as well as the environment [4,5]. In addition, bla NDM-1 was found to be carried on a variety of plasmid types [2,6], such as IncX3, IncFIA, IncFIB, and IncA/C, which facilitated the horizontal transfer of bla NDM-1 among members of Enterobacteriaceae, raising a great challenge for clinical treatment.

Genome Characteristics of SCW13
Draft genome sequence of SCW13 was assembled into 79 contigs (69 were >1000 bp in length) with a 53.4% GC (Guanine and Cytosine) content, which comprised 5,436,727 bp. Average nucleotide identity (ANI) calculation confirmed that SCW13 belonged to K. cryocrescens, as it had an OrthoANIu value of 99.23% against the reference strain K. cryocrescens NBRC 102467, which is obviously above the 95-96% cut-off usually used to define a bacterial species [20].

Analysis of the Bla NDM-1 -Harboring Plasmid PNDM1_SCW13
In the strain SCW13, bla NDM-1 was able to be transferred to E. coli J53 at a frequency of~10 −4 (transconjugant/recipient). Compared with the recipient strain J53, transformants containing bla NDM-1 showed significantly increased resistance to carbapenems and cephalosporins ( Table 1), suggesting that bla NDM-1 in SCW13 was functional and pNDM1_SCW13 was a self-transmissible plasmid.
pNDM1_SCW13 was 52,941 bp in size with an average GC content of 49.28% and contained 66 open reading frames (ORFs); none of the other resistance genes listed above except bla SHV-12 was co-harbored with bla NDM-1 on pNDM1_SCW13. A complete nucleotide sequence search against the GenBank database showed that pNDM1_SCW13 was almost identical to previously described IncX3 plasmids pRJA274 (GenBank accession no. KF877335) from Raoultella planticola in Shanghai in 2012 [6], and pNDM-HN380 (GenBank accession no. JX104760) from K. pneumoniae in Hongkong in 2011 [21], with only two and four single nucleotide polymorphisms (SNPs), respectively. It also showed high similarity (>99% identity) to p128379-NDM (Accession no. MF344560) from Enterobacter hormaechei, pZHDC33 (Accession no. KX094555) from E. coli, and pABC80-NDM-1 (Accession no. MK372383) from Citrobacter freundii. In China, IncX3-type plasmids carrying bla NDM variants have been widely found among Enterobacteriaceae isolates [2,[22][23][24][25]. Our present study further supplements those previous studies and expands the host range to Kluyvera spp. Figure 1 showed a linear comparison of pNDM1_SCW13 with three other reference IncX3 plasmids. These plasmids had a highly conserved backbone, with sequence polymorphism in the accessory region between res and hns. Compared to the counterpart on pNDM-HN380 and pRJA274, bla NDM−1 clusters on pNDM1_SCW13 showed a conserved linear organization (IS3000-∆ISAba125-IS5-∆ISAba125-bla NDM-1 -ble MBL -trpF-dsbC-cutA-groES-groL-insE), whereas the length of the remnant of ISAba125 element between IS3000 and IS5 varied, with 917 bp on pNDM-HN380, 28 bp on pRJA274, and 33 bp on pNDM1_SCW13. A deletion of 82 bp in the truncated umuD gene upstream of SHV-12 was observed on pNDM1_SCW13.

Analysis of the BlaNDM-1-Harboring Plasmid PNDM1_SCW13
In the strain SCW13, blaNDM-1 was able to be transferred to E. coli J53 at a frequency of ~10 −4 (transconjugant/recipient). Compared with the recipient strain J53, transformants containing blaNDM-1 showed significantly increased resistance to carbapenems and cephalosporins ( Table 1), suggesting that blaNDM-1 in SCW13 was functional and pNDM1_SCW13 was a self-transmissible plasmid.

Analysis of the Chromosomally-Encoded KLUC-2
In the process of genome sequence analysis, we found a bla CTX-M-1 -like gene on the chromosome of SCW13, which was not identified by ResFinder. BLASTp analysis using the amino acid sequence of the bla CTX-M-1 -like gene as a query matched (100% query coverage and 100% identity) KLUC-2 from E. cloacae 7506 in France in 2008. Analysis of the genetic environment of bla KLUC-2 showed that, in SCW13, bla KLUC-2 was sedentarily located on the chromosome, whereas bla KLUC-2 was located downstream of an ISEcp1 transposase on a plasmid in E. cloacae 7506 (Figure 2). It was proposed that the ISEcp1 element contributed to the mobilization of bla KLUC-2 from the K. cryocrescens chromosome to plasmids [19,[26][27][28]. In this light, bla KLUC-2 in the K. cryocrescens strain SCW13 is most likely to be the chromosomal counterpart of plasmid-born bla KLUC-2 . Besides, the genetic context of chromosomal bla KLUC-2 was identical to that of bla KLUC-1 on the chromosome of K. cryocrescens NBRC 102467 (Figure 2), suggesting that bla KLUC-2 may have evolved from bla KLUC-1 via a point mutation (G352→A)-or the other way around. The lack of more genetic information of bla KLUC-2 on plasmids was a limitation in this comparative analysis.

Analysis of the Chromosomally-Encoded KLUC-2
In the process of genome sequence analysis, we found a blaCTX-M-1-like gene on the chromosome of SCW13, which was not identified by ResFinder. BLASTp analysis using the amino acid sequence of the blaCTX-M-1-like gene as a query matched (100% query coverage and 100% identity) KLUC-2 from E. cloacae 7506 in France in 2008. Analysis of the genetic environment of blaKLUC-2 showed that, in SCW13, blaKLUC-2 was sedentarily located on the chromosome, whereas blaKLUC-2 was located downstream of an ISEcp1 transposase on a plasmid in E. cloacae 7506 (Figure 2). It was proposed that the ISEcp1 element contributed to the mobilization of blaKLUC-2 from the K. cryocrescens chromosome to plasmids [19,[26][27][28]. In this light, blaKLUC-2 in the K. cryocrescens strain SCW13 is most likely to be the chromosomal counterpart of plasmid-born blaKLUC-2. Besides, the genetic context of chromosomal blaKLUC-2 was identical to that of blaKLUC-1 on the chromosome of K. cryocrescens NBRC 102467 ( Figure  2), suggesting that blaKLUC-2 may have evolved from blaKLUC-1 via a point mutation (G352→A)-or the other way around. The lack of more genetic information of blaKLUC-2 on plasmids was a limitation in this comparative analysis.

Phylogenetic Analysis of Different Kluyvera Species
A phylogenetic tree (Figure 3) was inferred based on 36,734 qualified SNPs. We found that SCW13 was closely related to K. cryocrescens NCTC10483, which was isolated in the United Kingdom in 2018. Five K. cryocrescens strains were included in the phylogenetic analysis, and they were tightly clustered with each other with the exception of a distinct member, K. cryocrescens L2. We suggested that K. cryocrescens L2, in fact, may not be a member of K. cryocrescens. Similarly, the K. intermedia strain FOSA7093 showed a greater evolutionary relatedness with K. intestini strain GT-16, but was less related to other K. intermedia members. We suggested that K. intermedia FOSA7093 should be recategorized as a K. intestini isolate. Thus, a taxonomic re-evaluation of the Kluyvera genus is required. Figure 2. Comparative analysis of the genetic context of bla KLUC-2 and bla KLUC-1 . Genes and insertion sequences are indicated by arrows. Light gray shades denote shared regions with a high degree of homology. The construction of sequence comparison was performed using BLAST [29] and Easyfig version 2.2.3.

Phylogenetic Analysis of Different Kluyvera Species
A phylogenetic tree (Figure 3) was inferred based on 36,734 qualified SNPs. We found that SCW13 was closely related to K. cryocrescens NCTC10483, which was isolated in the United Kingdom in 2018. Five K. cryocrescens strains were included in the phylogenetic analysis, and they were tightly clustered with each other with the exception of a distinct member, K. cryocrescens L2. We suggested that K. cryocrescens L2, in fact, may not be a member of K. cryocrescens. Similarly, the K. intermedia strain FOSA7093 showed a greater evolutionary relatedness with K. intestini strain GT-16, but was less related to other K. intermedia members. We suggested that K. intermedia FOSA7093 should be recategorized as a K. intestini isolate. Thus, a taxonomic re-evaluation of the Kluyvera genus is required.

Strain Identification
K. cryocrescens strain SCW13 was recovered from the influx mainstream of hospital sewage at the affiliated hospital of Southwest Medical University, Luzhou in Western China, in April 2019. A volume of 10 mL of collected water was concentrated by centrifugation. The sediment was resuspended in 100 µL of Luria-Bertani broth culture and spread onto McConkey agar plates containing 1 µg/mL meropenem. Species identification was performed by sequencing of the 16S rRNA gene amplified with the universal primers 27F and 1492R [30].

Antimicrobial Susceptibility Testing
To examine the phenotypic resistance profile of SCW13, minimum inhibitory concentrations (MICs) of meropenem, imipenem, colistin, ceftriaxone, cefotaxime, cefoxitin, aztreonam, ciprofloxacin, gentamycin, amikacin, and tigecycline against the strain were determined using the microdilution broth method following the recommendations of the Clinical Laboratory Standards Institute (CLSI) [31]. The breakpoints of colistin and tigecycline were interpreted according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (http://www.eucast.org/). Otherwise, we applied those defined by the CLSI.

Conjugation Assay
Conjugation experiments were carried out in broth using azide-resistant E. coli J53 as the recipient strain at 37 °C. Bacteria were spread on Luria-Bertani agar plates containing 150 µg/mL sodium azide plus 1 µg/mL meropenem, for selecting the transconjugants carrying blaNDM-1. The presence of blaNDM-1 in transconjugants was further confirmed by PCR and sequencing.

Strain Identification
K. cryocrescens strain SCW13 was recovered from the influx mainstream of hospital sewage at the affiliated hospital of Southwest Medical University, Luzhou in Western China, in April 2019. A volume of 10 mL of collected water was concentrated by centrifugation. The sediment was resuspended in 100 µL of Luria-Bertani broth culture and spread onto McConkey agar plates containing 1 µg/mL meropenem. Species identification was performed by sequencing of the 16S rRNA gene amplified with the universal primers 27F and 1492R [30].

Antimicrobial Susceptibility Testing
To examine the phenotypic resistance profile of SCW13, minimum inhibitory concentrations (MICs) of meropenem, imipenem, colistin, ceftriaxone, cefotaxime, cefoxitin, aztreonam, ciprofloxacin, gentamycin, amikacin, and tigecycline against the strain were determined using the microdilution broth method following the recommendations of the Clinical Laboratory Standards Institute (CLSI) [31]. The breakpoints of colistin and tigecycline were interpreted according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (http://www.eucast.org/). Otherwise, we applied those defined by the CLSI.

Conjugation Assay
Conjugation experiments were carried out in broth using azide-resistant E. coli J53 as the recipient strain at 37 • C. Bacteria were spread on Luria-Bertani agar plates containing 150 µg/mL sodium azide plus 1 µg/mL meropenem, for selecting the transconjugants carrying bla NDM-1 . The presence of bla NDM-1 in transconjugants was further confirmed by PCR and sequencing.

Genome Sequencing and Bioinformatic Analysis
Total genomic DNA of SCW13 was extracted using a Rapid Bacterial Genomic DNA Isolation Kit (Sangon Biotech, Shanghai, China). Purified DNA was subjected to whole genomic sequencing on the Illumina HiSeq 2000 system with the 150-bp paired-end approach and 100 × coverage. Reads were trimmed using Trimmomatic [32] and assembled using the Spades program [33]. Annotation was carried out using Prokka [34]. Genome-based species identification was performed by ANI analysis using the web program ANI Calculator [35].
Plasmids were completely circularized using PCR and Sanger sequencing to fill in the gaps between contigs. The plasmid replicon type was determined using the PlasmidFinder [36]. Annotations of the plasmid sequences were conducted using Prokka combined with BLASTp/BLASTn searches against the NCBI database. Antimicrobial resistance genes carried by plasmids were identified using the online database ResFinder [37]. Multiple and pairwise sequence comparisons were performed using BLAST and visualized with Easyfig v 2.2.3 [38].
Core genomes for Kluyvera strains (n = 16, accessed on 12 July, 2019) available in the GenBank database were aligned with that of the K. cryocrescens reference strain NBRC 102467 (Accession no. BCTM01000000) using CSI Phylogeny 1.4 [39]. Gubbins (version 2.3.4) was used to remove single nucleotide polymorphisms (SNPs) on recombination sites [40]. The filtered SNPs were used as input for inferring a phylogenetic tree using RAxML with the GTRGAMMA model and 1000 bootstraps [26]. Antimicrobial resistance genes in these genomes were identified using ABRicate (https://github.com/tseemann/abricate).

Nucleotide Sequence Accession Numbers
A draft genome sequence of SCW13 has been deposited into GenBank under the accession no. VKGH00000000. The complete sequences of pNDM1_SCW13, pSCW13-1 and pSCW13-2 have been deposited into GenBank under accession no. MN178638, MN178639 and MN178640, respectively.

Conclusions
In summary, we reported a bla NDM−1 -carrying K. cryocrescens isolate for the first time. bla NDM−1 was carried by a pNDM-HN380-like IncX3 plasmid in this K. cryocrescens strain, which is a worrying development, as it highlights that bla NDM−1 has further spread to uncommon Enterobacteriaceae strains by epidemic IncX3 plasmids. Therefore, extensive surveillance and effective actions are urgently needed to control the spread of bla NDM -encoding IncX3 plasmids. In addition, our work also identified a bla KLUC-2 gene on the chromosome of K. cryocrescens SCW13, which represents the origin of the plasmid-mediated resistance gene bla KLUC-2 .