blaNDM-5 carried by a hypervirulent Klebsiella pneumoniae with sequence type 29

Background A carbapenem-resistant hypermucoviscous Klebsiella pneumoniae isolate was recovered from human sputum. Methods Whole genome sequencing of this isolate was carried out to reveal its clonal background, antimicrobial resistance determinants and virulence factors. Virulence assays were performed using wax moth larvae. The transfer of blaNDM-5 between bacterial strains was tested using conjugation. 59 genome assemblies of ST29 K. pneumoniae and 230 IncX3 plasmids regardless of the carriage of resistance gene were employed for phylogenetic analysis, respectively. Results The strain carried a virulence plasmid pVir-SCNJ1 bearing the virulence gene rmpA and exhibited a high virulence in wax moth. This hypervirulent strain belongs to sequence type 29 and carries blaNDM-5, which is located on a conjugative plasmid, designated pNDM5-SCNJ1, belonging to type IncX3. pNDM5-SCNJ1 was fully sequenced and shows high similarity with pNDM_MGR194, except some deletion inside the ISAba125 region. Phylogenetic analysis of IncX3 plasmids revealed that although blaNDM-5 can be evolved from blaNDM-1 via point mutations within some IncX3 plasmids, most of blaNDM-5-carrying IncX3 plasmids probably have acquired blaNDM-5 in multiple events. Conclusions In this study, we characterized a blaNDM-5-positive hypervirulent K. pneumoniae of sequence type 29 in China. Our results highlight the need for active surveillance on this lineage of carbapenem-resistant K. pneumoniae. Electronic supplementary material The online version of this article (10.1186/s13756-019-0596-1) contains supplementary material, which is available to authorized users.


Introduction
Hypervirulent Klebsiella pneumoniae (hvKP) is a worldwide concern due to its capacity to cause lifethreatening, community-acquired infections in healthy individuals with high morbidity and mortality [1,2]. hvKP strains are usually less resistant to most antimicrobials than classic K. pneumoniae [3], but the increasing emergence of carbapenemase-producing hypervirulent K. pneumoniae (CP-hvKP) compromises options of antimicrobial agents for infection control and drives a global crisis [2,4]. These CP-hvKP strains are thought to be the result of acquiring plasmid-mediated resistance and virulence markers, either by transferring of resistance plasmids into hvKP strains or virulence plasmids into carbapenem-resistant strains [5]. There have now been several reports of infections caused by carbapenemaseproducing hypervirulent K. pneumoniae strains [2,4,[6][7][8][9]. These CP-hvKP isolates mainly produce KPC (a group of serine-lactamases), followed by IMP (a group of metallo-β-lactamases), and they belong to the widely distributed sequence type (ST) 11 [2,8] and several other STs, e.g., ST25, ST65 [6] and ST36 [4]. Here, we identified an ST29 CR-hvKP clinical strain with K54 serotype carrying bla NDM-5 gene and reported on its characterization.

Bacterial identification and PCR analysis
The strain SCNJ1 was recovered from the sputum of a patient with an acute bronchiolitis in a hospital of Sichuan Province in November 2018. The initial species identification was performed using the Vitek-2 compact system (bioMérieux, Marcy-l'Étoile, France). A further species confirmation was performed by PCR amplifying of the 16S rRNA gene using the primer pair 27F/1492R [10]. PCR products were purified and then sequenced by Sanger sequencing. The resulting 16S rRNA gene sequences were compared with sequences in GenBank (NCBI) database using BLAST software. The presence of the acquired carbapenemase genes bla KPC , bla NDM , bla GES , bla IMP , bla OXA-48 , and bla VIM in this isolate was screened via PCR using primers as previously described [11][12][13][14].

String test
String test was performed by stretching a mucoviscous string from the colony using a standard bacteriologic loop as described previously [16]. Strains that formed viscous strings > 5 mm in length were defined as hypermucoviscous.

Conjugation
Conjugation experiments were carried out using broth-based methods with the azide-resistant Escherichia coli strain J53 as the recipient and transconjugants were selected using 2 μg/ml meropenem plus 150 μg/ml sodium azide. The presence of bla NDM-5 in transconjugants was confirmed by PCR and sequencing.

Virulence assay
The virulence potential of the SCNJ1 strain was assessed using wax moth (Galleria mellonella) larvae weighing 250 to 350 mg (Tianjin Huiyude Biotech Company, Tianjin, China) with method described previously [17]. Overnight cultures of K. pneumoniae strains were adjusted with phosphate-buffered saline (PBS) to concentrations of 1 × 10 4 CFU/ml, 1 × 10 5 CFU/ml, 1 × 10 6 CFU/ml, 1 × 10 7 CFU/ml after being washed with PBS. 10 μl of inoculum was injected into the hemocoel of sixteen larvae using a 25-μl Hamilton syringe via the last left proleg. The larvae were then incubated at 37°C and the number of live larvae was counted at 12 h intervals for 3 days. Two bla KPC-2 -carrying carbapenemresistant K. pneumoniae clinical isolates of ST11:K47, KPNJ2 and KPLZ1050, without rmpA and rmpA2, were used as the control.

Genome sequencing and analysis
The strain was subjected to whole genomic sequencing using an Illumina HiSeq 2000 system with the 150-bp paired-end approach and 150 × coverage. Reads were trimmed using Trimmomatic [18]. Draft genome was then assembled using the SPAdes program [19]. Annotation was carried out using Prokka [20]. Sequence type and capsular type of this strain were determined using the assembled contigs to query the Multi-Locus Sequence Typing (MLST) v 2.0 (https://cge.cbs.dtu.dk/ services/MLST/) and wzc genotyping system as previously described [21], respectively. In addition, the wzi genotyping system [22] and KLeborate (https://github. com/katholt/Kleborate/) were employed to confirm the sequence type and capsular type. Clonal complexes (CCs) were determined by using eBURST v3 based on K. pneumoniae MLST data (https://eburst.mlst.net). Virulence genes were identified using the Virulence Factors Database (VFDB) available at http://www.mgc. ac.cn/VFs/main.htm. Antimicrobial resistance genes were identified using the ResFinder v3.1 software of the Center for Genomic Epidemiology (CGE, http://genomicepidemiology.org/).
Plasmids pVir-SCNJ1 and pNDM5-SCNJ1 were completely circularized with gaps between the contigs closed by PCR and respective amplicons sequenced using Sanger sequencing, respectively. Identification of plasmid incompatibility types were performed on complete sequences of plasmids via the online service PlasmidFinder v2.0 at CGE (https://cge.cbs.dtu.dk/services/PlasmidFinder/). The annotations of the plasmid sequences were conducted using the RAST tools and edited manually [23]. Sequence alignment of bla NDM-5carrying plasmids was performed using BLAST and visualized with Easyfig v 2.2.3 [24]. Alignments with highly homologous complete plasmid sequences of pVir-SCNJ1 available in NCBI were performed by using the BRIG tool [25]. The circular map of pNDM5-SCNJ1 was also generated using BRIG [25].

Phylogenetic analysis
All assembled K. pneumoniae genomes (n = 6823; accessed by March 1, 2019) were retrieved from Gen-Bank. MLST typing was performed using the script (https://github.com/tseemann/mlst). A total of 59 assemblies of ST29 K. pneumoniae were included and aligned with that of strain SCNJ1 using CSI Phylogeny 1.4 (https://cge.cbs.dtu.dk/services/CSIPhylogeny/) (Additional file 1 : Table S2). Gubbins (version 2.3.4) was used to remove single nucleotide polymorphisms (SNPs) on recombination sites [26]. The filtered SNPs were then used as input for inferring a phylogenetic tree using RAxML with the GTRG AMMA model and 1000 bootstraps [27]. ABRicate (https://github.com/tseemann/abricate) was used to identify antimicrobial resistance genes in these genomes and the capsular typing of K. pneumoniae was performed with the wzc genotyping system.
The sequence of all available IncX3 plasmids regardless of the carriage of resistance gene (n = 230; accessed by March 24, 2019) were retrieved from the GenBank (Additional file 1 : Table S4). Orthogroups were identified using OrthoFinder [28] and used for multiple sequence alignments (MSA) with MAFFT [29]. The species tree was inferred from the concatenated MSA using FastTree [30]. The STRIDE algorithm (Specie Tree Root Inference from Duplication Events) was used to root the species tree in OrthoFinder.
PCR and sequencing showed that bla NDM-5 was the only carbapenemase-encoding gene carried by the strain SCNJ1. NDM-5, a variant of NDM (New Delhi Metallo-β-lactamase), was first identified in an E. coli ST648 isolate (EC045) in the UK in 2011 from a patient with a recent hospitalization history in India [31]. Although bla NDM-5 has been widely found in K. pneumoniae strains since its first discovery (Additional file 1 : Table S1), bla NDM-5 -carrying hypermucoviscous K. pneumoniae remains uncommon. We found one publication that described a bla NDM-5 -positive K. pneumoniae isolate (K2/ST14) in China in 2015 [32], which was speculated to be hypermucoviscous on the basis of genome analysis. However, no experimental data was included.
The gene bla NDM-5 is described for the first time in a strain of K. pneumoniae ST29 in our work, as demonstrated by the phylogenetic tree based on filtered SNPs of all available ST29 K. pneumoniae strains (Fig. 1). The phylogenetic analysis also showed that strain SCNJ1 was clustered with four isolates recovered in China and was closest to strain SCLZ15-011 (GCA_ 001630805, carrying no carbapenemase gene, recovered in 2016 in China) with 198 SNPs difference (Fig. 1). It should be noted that sporadic cases due to ST29 K. pneumoniae were frequently detected, mainly from liver abscess patients [37,43,45,46], and multidrug-resistant ST29 hvKP strains have been reported in different locations of China [5,33,47]. This highlights the need to monitor the epidemiology of the ST29 clones of K. pneumoniae isolates in China.
Conjugation assays showed that strain SCNJ1 transferred a plasmid carrying bla NDM-5 to E. coli J53 at a frequency of 10 − 6 (transconjugant/recipient) by mating, suggesting that bla NDM-5 was carried on a selftransmissible plasmid, which was assigned pNDM5-SCNJ1. In addition to the bla NDM-5 , strain SCNJ1 had a few chromosomal resistance genes, including the ESBL gene bla SHV-187 , fluoroquinolone-resistance genes oqxA and oqxB, and fosfomycin-resistance gene fosA.
pNDM5-SCNJ1 was a 45,255-bp IncX3 plasmid, with an average GC content of 46.83% and had no other known antimicrobial resistance genes except bla NDM-5 . pNDM5-SCNJ1 consists of a 30-kb backbone comprising several sets of genes (pir and bis encoding replication initiation protein, parA for plasmid partitioning, hns and topB for maintenance and a gene cluster responsible for conjugation) and a genetic load region with high GC content between the Fig. 1 Phylogenetic tree and resistance gene profile of K. pneumoniae strain SCNJ1 with other 59 ST29 K. pneumoniae genomes available from GenBank. SCNJ1 is indicated in red. Isolates from China are marked with an asterisk and resistance gene bla NDM-5 is marked with a red rectangle resolvase and the hns gene, which are typical of IncX3 plasmids (Fig.4a). BLASTn revealed that the sequence of pNDM5-SCNJ1 was highly similar (100% coverage and 99.99% identity) to the plasmid pNDM_MGR194 (GenBank accession no. KF220657) recovered from a K.
pneumoniae isolate in India, as well as a number of previously described IncX3 plasmids carrying bla NDM-5 in China.
In the genetic load region of pNDM5-SCNJ1 (Fig. 4b), the umuD gene was split into two fragments at the Fig. 2 Alignment of pVir-SCNJ1 with 3 hypervirulence-encoding plasmids. The alignment was performed using BRIG and pLVPK was used as a reference. Accession numbers for the plasmids are NZ_CP031258 (pL22-1), NC_006625 (pK2044), NC_005249 (pLVPK). The locations of virulence genes rmpA2, iutA, iucDCBA, rmpA and iroNDCB are indicated  Supplementary Table S3. KPLZ1050 and KPNJ2, two bla KPC − 2 -carrying K pneumoniae clinical isolates of ST11 that did not harbour a virulence plasmid, were used as the control nucleotide position 336 bp by the bla NDM-5 -containing structure (IS26-ΔctuA1-tat-trpF-ble MBL -bla NDM-5 -ΔIS-Aba125-IS5-ΔISAba125-IS3000-ΔTn2), resulting in a pair of 3-bp direct repeats (TGT). In such a genetic context, an IncX3 plasmid pEC14_35 (GenBank accession no. JN935899) without any antibiotic-resistance gene, which was isolated from a patient in the USA in 1989, was likely to be the ancestral vector. It is also likely that pNDM5-SCNJ1 has diverged recently from bla NDM-1 -positive plasmids pBJ01 (GenBank accession no. JX296013) by sequential mutations (Fig. 4b). In the subsequent genetic variant, the ISAba125 was truncated by the insertion of IS5 element (at 166 bp upstream bla NDM-5 start codon) and a 4-bp flanking direct repeats (CTAA) was identified. Comparisons of the genetic contexts of bla NDM-5 in pNDM_MGR194, pP744T, pTK1044 and pNDM5-SCNJ1 showed that the remnant of ISAba125 (73 bp of 1087 bp) upstream of bla NDM-5 was conserved, but the length of the remnant of ISAba125 between IS3000 and IS5 differed (pNDM_MGR19: 1002 bp, pP744T: 404 bp, pNDM-SCNJ1: 112 bp, pTK1044: 0 bp), suggesting that IS5 has inserted into ISAba125 at the same position in these plasmids and that gene deletions caused by homologous recombination plays a possible role in the formation of diversified ΔIS-Aba125 region.
Phylogenetic analysis based on concatenated MSA of IncX3 plasmids revealed that pNDM5-SCNJ1 was closely related (100% coverage, 99.99% identity) to plasmid pQDE2-NDM (GenBank accession no. MH917280), which also carried bla NDM-5 that was recovered from a K. pneumoniae isolate in Shandong, China, in 2015 (Fig. 5). The phylogenetic tree also showed that most of bla NDM-5 -carrying IncX3 plasmids are tightly clustered with each other and formed a relatively distinct clade, with only sporadic ones clustered into clades with bla NDM-1 -carrying plasmids, suggesting that although bla NDM-5 is likely to evolve from bla NDM-1 via point mutations on some IncX3 plasmids, most of IncX3 plasmids probably have acquired bla NDM-5 in multiple events.

Conclusion
In conclusion, our work identified an ST29 CP-hvKP carrying the carbapenemase gene bla NDM-5 and provided additional evidence of the rapid dissemination of bla NDM-5 by pNDM-MGR194-like plasmid among Enterobacteriaceae in China. The association of the epidemic IncX3 plasmid carrying bla NDM-5 with a hypervirulent K. pneumoniae lineage, ST29/K54 in this case, is quite worrisome and may pose a great threat to humans. More extensive surveillance and effective action to control its further dissemination are urgently required.

Additional file
Additional file 1: Table S1. Background information on the bla NDM-5positive K. pneumoniae isolates. Table S2. ST29 K. pneumoniae strains with genome sequences available in the GenBank. Table S3. Survival (number of larvae) of G. mellonella after infection by K. pneumoniae strain SCNJ1.   Table 4. Those carrying bla NDM-5 are indicated in red, while pNDM5-SCNJ1 is shown in purple. The species tree was inferred from the concatenated multiple sequence alignments using FastTree