Characterization of IncHI1B Plasmids Encoding Efflux Pump TmexCD2-ToprJ2 in Carbapenem-Resistant Klebsiella variicola, Klebsiella quasipneumoniae, and Klebsiella michiganensis Strains

Abstract

Tigecycline serves as one of the last-resort antibiotics to treat severe infections caused by carbapenem-resistant Enterobacterales. Recently, a novel plasmid-mediated resistance-nodulation-division (RND)-type efflux pump gene cluster, TmexCD1-ToprJ1, and its variants, TmexCD2-ToprJ2 and TmexCD3-ToprJ3, encoding tetracyclines and tigecycline resistance, were revealed. In this study, we reported three TmexCD2-ToprJ2-harboring Klebsiella species strains, collected from two teaching tertiary hospitals in China, including one K. quasipneumoniae, one K. variicola, and one K. michiganensis. The three strains were characterized by antimicrobial susceptibility testing (AST), conjugation assay, WGS, and bioinformatics analysis. AST showed that K. variicola and K. quasipneumoniae strains were resistant to tigecycline with MIC values of 4μg/ml, whereas the K. michiganensis was susceptible to tigecycline with an MIC value of 1μg/ml. The TmexCD2-ToprJ2 clusters were located on three similar IncHI1B plasmids, of which two co-harbored the metallo-β-lactamase gene blaNDM-1. Conjugation experiments showed that all three plasmids were capable of self-transfer via conjugation. Our results showed, for the first time, that this novel plasmid-mediated tigecycline resistance mechanism TmexCD2-ToprJ2 has spread into different Klebsiella species, and clinical susceptibility testing may fail to detect. The co-occurrence of blaNDM-1 and TmexCD2-ToprJ2 in the same plasmid is of particular public health concern as the convergence of “mosaic” plasmids can confer both tigecycline and carbapenem resistance. Its further spread into other clinical high-risk Klebsiella clones will likely exacerbate the antimicrobial resistance crisis. A close monitoring of the dissemination of TmexCD-ToprJ encoding resistance should be considered.

Epidemiology of Klebsiella michiganensis Carrying Multidrug-Resistant IncHI5 Plasmids in the Southeast Coastal Area of China

“…From a geographic view, the distribution of pCP024641 and pKOX_R1 is relatively close; from the perspective of a phylogenetic tree, branches of pCP024641 and pKOX_R1 are also close, which may indicate a transmission event from Taiwan to Fujian. Later, IncHI5 plasmids coexisting with K. michiganensis were reported in Guangzhou, Hangzhou 17 and other places, 16 and all these locations are geographically located in the southeast coastal area of China. This distinctive geographic distribution may imply that the acquisition of IncHI5 plasmids in K. michiganensis is related to some unique geographic-related conditions, although the relevant internal mechanisms are still unclear.…”

“… 9 , 12 , 40 , 41 The K. michiganensis carrying IncHI5 plasmids were identified frequently in the last two years. 16 , 17 The similarities and differences of the genome structure of IncHI5 plasmids and the population and geographic distribution within the K. michiganensis species have urgently needed to be studied.…”

“…Then, starting from 2020, K. michiganensis carrying IncHI5 plasmids have been reported several times. 16 , 17 K. michiganensis carries IncHI5 plasmids at a low frequency, probably because some K. michiganensis was misclassified as K. oxytoca and thus not counted. Perhaps it is also because K. michiganensis have only recently attracted attention, and the rapid development of sequencing has also led to an increase in related sequencing samples in recent years.…”

“…There were five complete sequences of IncHI5 plasmids in K. michiganensis uploaded to NCBI since 2020 (p12084-HI5, pCP024641, pJNQH491-2, 16 p1, and pKOX7525_1 17 ). Among them pCP024641 was the first reported case, which was collected from Fujian Provincial Hospital, Fuzhou Fujian China.…”

“…The sequence of repHI5B (the key identified replicon of IncHI5 plasmids) was used to perform BLAST alignment against the GenBank 15 of the National Center for Biotechnology Information (NCBI) to cover all known IncHI5 plasmids. From the alignment, a total of six IncHI5 plasmids whose host strain were K. michiganensis were obtained (last accessed on December 12, 2021): pKOX_R1 (accession number CP003684), 14 p12084-HI5 (accession number MW810613), pCP024641 (accession number CP024641), pJNQH491-2 (accession number CP075883), 16 p1 (accession number CP067094), and pKOX7525_1 (accession number CP065475). 17 …”