Characterization of an NDM-5-producing hypervirulent Klebsiella pneumoniae sequence type 65 clone from a lung transplant recipient

ABSTRACT The emergence of New Delhi Metallo-β-lactamase (NDM)-producing Klebsiella pneumoniae has aroused critical concern worldwide. Herein, we reported the first emergence of NDM-5-producing K. pneumoniae isolates in a 68-year-old lung transplant recipient, who died of septic shock 13 days after surgery. The K. pneumoniae strain KP22937 isolated from the bloodstream of the patient was analyzed for phenotypes and genotypes. KP22937 belonged to sequence type (ST) 65 and capsule serotype K2, contained iucABCDiutA and iroBCDN virulence clusters, showed high virulence to mice, and was therefore considered a hypervirulent K. pneumoniae. The blaNDM-5 gene was located on a genomic island region of the IncX3-type plasmid pNDM22937, which was successfully transferred to Escherichia coli EC600 with insignificant fitness costs. The transconjugant demonstrated similar antimicrobial susceptibility and growth kinetics to the recipient E. coli EC600. The plasmid pNDM22937 was almost identical to the blaNDM-5-carrying IncX3 plasmids previously reported in K. pneumoniae strains with different ST types and in other species. Our findings raise concerns about the horizontal spread of blaNDM-5 gene mediated by IncX3 plasmid, where hypervirulent K. pneumoniae strains are also involved. Stricter control measures are needed to prevent the dissemination of the novel clone in hospital settings.

New Delhi Metallo-β-lactamase (NDM) producing Klebsiella pneumoniae strains are capable of hydrolyzing all β-lactams except monobactam. The bla NDM-5 gene was first identified in 2011 from an Escherichia coli strain EC045 from a patient with a hospitalization history in India [1]. Subsequently, this carbapenemase gene has been detected in K. pneumoniae, and has caused sporadic outbreaks worldwide [2]. It has been identified in various ST types of K. pneumoniae, among which ST29 K. pneumoniae strain SCNJ1 and ST35 K. pneumoniae strain RJY9645 have been confirmed to be carbapenem-resistant hypervirulent K. pneumoniae (CR-hvKp) [3,4]. The treatment options are limited for severe infection caused by CR-hvKp [4]. Herein, we reported an NDM-5-producing hypervirulent K. pneumoniae ST65 strain in a post-transplant patient, which is a serious public health concern.
In June 2019, a 68-year-old lung transplantation recipient experienced a severe postoperative infection.
Multiple specimens were sent for pathogen testing on postoperative day 2, 5, 6 and 7, and empirical antibacterial treatment was administered according to the patient's symptoms until the pathogen results were returned ( Figure 1A). Aztreonam was immediately administered after receiving the antimicrobial susceptibility test (AST) result, and the patient's temperature and circulation gradually improved. However, the patient developed chest emphysema on postoperative day 10, and bronchoscopy revealed a large amount of pus moss. His condition dramatically deteriorated, and he died of septic shock.
The analysis showed that strain KP22937 belonged to ST65 and capsule serotype K2, and carried several hypervirulent determinants such as iucABCDiutA and iroBCDN, as well as a hypermucoviscous phenotype regulator gene rmpA. Mouse lethality assay was performed for assessing the in vivo virulence of strain KP22937. The result showed that all eight pathogenfree female BALB/c mice (6-8 weeks and 18-20 g) injected with KP22937 at a concentration of 10 5 CFU/ml died within 12 h, while mice injected with low-virulence clinical K. pneumoniae strain KPZ03 (GenBank accession number SAMN17245928) and PBS survived for over a week ( Figure S1). The above results indicated that the ST65 K. pneumoniae strain KP22937 that caused infection in the lung transplant recipient was hvKp, which was consistent with a previous report [10].
Two plasmids were identified in K. pneumoniae strain KP22937. The plasmid pNDM22937, 45048 bp in size, belongs to the IncX3 group and did not harbour any resistance genes other than bla NDM-5 . BLASTN search for GenBank showed that pNDM22937 was nearly identical to bla NDM-5 -carrying plasmids previously reported in K. pneumonia of different ST types, and other species. Specifically, pNDM22937 showed 100% identity and coverage with plasmid pNDM5_020026 (CP034957) from E. coli SCEC020026, and 100% identity and 97% coverage with the plasmid pNDM_MGR194 (KF220657), which was a typical bla NDM-5 -carrying plasmid recovered from a K. pneumoniae isolate in India ( Figure 1B) [11]. The genetic environment of bla NDM-5 gene in pNDM22937 (ΔTn2-ΔIS3000-IS5-ΔISAba125bla NDM-5 -ble-trpF-dsbC-IS26-ΔumuD) was almost identical to pNDM_MGR194 and pNDM5_020026, except that part of the downstream region of IS3000 and upstream region of ISAba125 were deleted from pKP22937 ( Figure 1C). Moreover, we found a genomic island region with 15 open reading frames (ORFs) in the plasmid pNDM22937 ( Figure 1B). The first ORF shared 44.6% identity with an integrase from Serratia sp, and the last ORF in the genomic island was a twin-arginine translocation (TAT) pathway signal sequence domain protein. The bla NDM-5 gene and its flanking contents were also present in this region.
Several conjugal transfer genes were also identified in pNDM22937, such as virB4, virB8, virB9 and virD4 ( Figure 1B). Conjugation experiment showed that the strain KP22937 transferred the plasmid carrying bla NDM-5 to the recipient E. coli EC600 at a frequency of 10 −5 (transconjugant/recipient), PCR amplification and sequencing of the transconjugants and S1-PFGE experiment ( Figure S2) confirmed successful transfer to the recipient, suggesting that bla NDM-5 was located on a self-transmissible plasmid, which could mediate dissemination of antibiotic resistance. The recipient E. coli EC600 was susceptible to all antibiotics, whereas the transconjugant displayed a similar antibiotic resistance phenotype to the strain KP22937 after obtaining the bla NDM-5 -harbouring plasmid ( Table  S1). The fitness cost of the IncX3 plasmid harbouring bla NDM-5 was evaluated through growth kinetics assays. The results showed that the growth of the transconjugant was almost indistinguishable from that of E. coli EC600, revealing that the acquisition of IncX3 plasmid did not confer a fitness cost to the host. Accordingly, we hypothesized that the strain that caused postoperative infection in lung transplant patients in this study was produced by obtaining a selftransmissible IncX3 plasmid carrying bla NDM-5 gene by a hypervirulent ST65 K. pneumoniae strain.
In the absence of antibiotic pressure, the presence of resistance genes in plasmids will impose fitness costs on their host [12]. However, a recent in vitro study has shown that up to 75.9% (22/29) Enterobacteriaceae strains did not produce fitness costs after obtaining the IncX3 plasmid through conjugation test [13], which may facilitate the dissemination of the plasmid. Genomic islands are clusters of genes of probable horizontal origin in bacterial or archaeal genomes. A previous study revealed that genomic islands are a major driver of genome evolution, and they can enhance the fitness of bacteria within a niche [14]. In this study, the bla NDM-5 gene and its flanking content were located in the genomic island region of plasmid pNDM22937, which may contribute to the lower fitness cost of K. pneumoniae strain KP22937. On all accounts, the lower fitness cost may partly explain the rapid dissemination of bla NDM-5 among Enterobacteriaceae strains.
In the present study, we reported an NDM-5-producing hypervirulent K. pneumoniae ST65 strain. To investigate whether this clone has been previous reported, we retrieved 9157 genomic sequences of K. pneumoniae with different assembly levels from the NCBI genome database on July 1, 2020. MLST analysis of these isolates was performed to include only ST65 clone. Eventually, 30 genomic sequences of ST65 K. pneumoniae were involved. Analysis of resistance and virulence determinants showed that the bla KPC-2 gene was present in many ST65 K. pneumoniae strains, but the occurrence of bla NDM-5 gene in ST65 K. pneumoniae strain was comparatively rare. This study was the first report that found the bla NDM-5 gene in a clinical ST65 K. pneumoniae strain ( Figure S3).
In summary, this study reported a fatal infection in a post-transplant patient caused by an NDM-5producing hypervirulent K. pneumoniae ST65 clone. The bla NDM-5 gene was located on a self-transmissible IncX3 plasmid, which could be transferred to E. coli without significant fitness costs. The current finding raises concerns about horizontal spread of bla NDM-5 gene mediated by IncX3 plasmid, where hypervirulent K. pneumonia strains are also involved.