Case Report of bla NDM-7-Harboring IncX3 Plasmid in ST196 Klebsiella quasipneumoniae in China.

Klebsiella quasipneumoniae isolate SBH035 was recovered from a patient in Jiangsu Province, China. The isolate showed resistance to ampicillin, cefazolin, cefotaxime, meropenem, ceftazidime-avibactam, and fosfomycin. The carbapenemase-encoding gene bla NDM-7 was identified, and whole genome sequencing analysis indicated that bla NDM-7 was located in an IncX3 plasmid with a conserved structure of IS26-ΔcutA-tat-trpF-ble MBL -bla NDM-7-ISAba125-IS3000-ΔTn2. To date, this is the first identification of a bla NDM-7-harboring IncX3 plasmid in ST196 K. quasipneumoniae from a patient in China. Greater attention to controlling the dissemination of IncX3 plasmids is needed owing to potential horizontal transfer via mobile genetic elements.

Plain Language Summary

We observed carbapenem resistance in ST196 Klebsiella quasipneumoniae.

The blaNDM-7 gene was located in a conjugative IncX3 plasmid associated with a commonly observed core structure.

The blaNDM-7 gene can confer ceftazidime–avibactam resistance.

Carbapenemase-producing Enterobacterales, especially New Delhi metallo-β-lactamase (NDM) enzyme-producing strains, has become an increasing threat to public health. Since blaNDM-1 was first reported in 2009, 40 NDM allelic variants have been identified so far (#; last accessed March 2, 2022). As a member of the Klebsiella pneumoniae complex, K. quasipneumoniae has also become a cause for concern. To date, blaNDM-1,1,2 blaNDM-5,3,4 blaKPC-2,5,6 and blaKPC-36 have been identified in K. quasipneumoniae and IncX3, IncX5, IncX6, and IncF are the main plasmids accounting for carbapenem resistance. ST196 K. quasipneumoniae has rarely been reported but can be present in health care centers worldwide, possibly leading to hospital-acquired infections.7

In 2013, blaNDM-7 was identified for the first time in Escherichia coli from a patient who had traveled to Burma and was hospitalized in France.8 Owing to Asp-130-Asn and Met-154-Leu substitutions, NDM-7 has greater carbapenem-hydrolyzing activity than NDM-1.9 Subsequently, plasmid-mediated blaNDM-7 has been found in different species such as K. pneumoniae,10 Enterobacter cloacae,11 E. aerogenes,12 and Citrobacter freundii.13 Here, we report the first detection of K. quasipneumoniae harboring blaNDM-7 isolated from a patient in Jiangsu Province, China.

K. quasipneumoniae isolate SBH035 was recovered from a urine sample collected from a 67-year-old male patient in a tertiary hospital of Jiangsu Province in July 2018. Species identification was performed using the VITEK® MS system (bioMérieux, Marcy-l’Étoile, France). Antibiotic susceptibility testing against 17 antibiotics was performed using the agar dilution method or broth microdilution method (limited to colistin and tigecycline). The results were interpreted according to guidelines of the Clinical and Laboratory Standards Institute or European Committee on Antimicrobial Susceptibility Testing. The isolate was resistant to ampicillin, cefazolin, cefotaxime, meropenem, ceftazidime–avibactam, and fosfomycin but was susceptible to streptomycin, gentamicin, amikacin, tetracycline, tigecycline, chloramphenicol, florfenicol, nalidixic acid, ciprofloxacin, trimethoprim/sulfamethoxazole, and colistin (). Carbapenemase-encoding genes were further detected, as previously described,14 and the blaNDM-7 gene was identified.

Whole genome sequencing of SBH035 was performed using the Illumina NovaSeq platform (Illumina Inc., San Diego, CA, USA). The reads were assembled using SPAdes v. 3.10.0 and 85 contigs (>200 base pairs; bp) were obtained (GenBank accession no. PRJNA823891). The Center for Genomic Epidemiology pipeline () was used to identify sequence type (ST), antimicrobial resistance genes, mutations, and plasmid replicon type. K. quasipneumoniae strain SHB035 was identified as ST196 and contained several resistance genes conferring resistance to fosfomycin (fosA), quinolone (oqxAB), and β-lactam (blaNDM-7, blaOKP-A-5).

Ten published genomes of ST196 K. quasipneumoniae were downloaded, and the phylogenetic tree was generated using Parsnp (core genome SNP tree).15 The results showed that the SBH035 isolate in our study was clustered in one separate clade, and isolates from the United States (US) and Qatar were clustered into respective clades. All Qatar strains contained blaNDM-1, and one US strain (CAV1947, from hospital wastewater) co-harbored blaKPC-2 and blaKPC-3; only SBH035 contained blaNDM-7 (Figure 1).

Figure 1

Phylogenetic tree based on core genome and drug resistance genes of ST196 Klebsiella quasipneumoniae. Antimicrobial resistance genes are shown in purple solid circles.

The complete plasmid sequence harboring blaNDM-7 was assembled using PCR and Sanger sequencing () and designated pYUSBH035 (GenBank accession no. LC716358). pYUSBH035 was 46,461 bp in size, with an average GC content of 46.65% and was identified as IncX3 plasmid. BLASTn analysis showed that pYUSBH035 was identical or highly similar to other NDM-7-producing IncX3 plasmids (99.9–100% identity and 100% coverage) as well as IncX3 plasmids harboring other blaNDM alleles, for example, pHN6DS3 (blaNDM-5, MN276078), pHD6415-NDM (blaNDM-33, MZ004933), pNDM-20 (blaNDM-20, MF458176), and pM216_X3 (blaNDM-4, AP018146) (). In pYUSBH035, blaNDM-7 was located in a region with various insertion sequence (IS) elements (IS26, IS5, ISAba125 and IS3000). The blaNDM-7 gene was embedded in an 8906-bp structure, IS26-ΔcutA-tat-trpF-bleMBL-blaNDM-7-ISAba125-IS3000-ΔTn2, in which ISAba125 was interrupted by IS5, and 4-bp direct repeats (5’-CTAA-3’; DRs) were generated. This blaNDM-7 segment was inserted into umuD flanked by 3-bp DRs (5’-TGT-3’) (Figure 2). This genetic context was also found in other blaNDM-carrying IncX3 plasmids, except that 925 bp of the 5’-end of ISAba15 was absent in a blaNDM-7-bearing plasmid pHZW25-P4 (CP025215) from K. pneumoniae in China (Figure 2).

Figure 2

Genetic environment of multidrug resistance region in pYUSBH035 and homologous plasmids.

The plasmid pYUSBH035 could be successfully transferred to the recipient E. coli C600 and the blaNDM-carrying transconjugant displayed resistance to ampicillin, cefazolin, cefotaxime, meropenem, and ceftazidime–avibactam (). The blaNDM-7-carrying plasmid pYUSBH035 was a self-transmissible plasmid, which could transfer carbapenem resistance to E. coli. IncX3 plasmids are efficient vectors for blaNDM transmission between different species.

K. quasipneumoniae was initially considered a commensal intestinal colonizer, but recent studies have proven that it is an etiologic agent in potentially fatal infections. Plasmid-mediated carbapenem resistance has also been observed among K. quasipneumoniae clinical isolate strains, which may complicate treatment regimens. Until now, blaKPC-2, blaKPC-3, blaKPC-9, blaOXA-181, blaGES-5, blaNMD-1, and blaNMD-5 have been reported in K. quasipneumoniae, and the plasmids involved mainly include IncX3, IncX5, IncX6, and IncF ().

To the best of our knowledge, this is the first identification of a blaNDM-7-harboring IncX3 plasmid in ST196 K. quasipneumoniae from a patient in China. Greater attention to controlling the dissemination of IncX3 plasmids is needed owing to potential horizontal transfer via mobile genetic elements.