Abstract
Carbapenem-resistant Enterobacteriaceae (CRE) infections, high in morbidity and mortality, pose serious clinical challenges due to limited treatment options. A previous CRE surveillance study on 1,507 patients from 43 hospitals in Osaka, Japan, revealed that 12% of patients carried CRE and that 95% of the CRE isolates were IMP-type carbapenemase producers. Here, the mechanisms for this regional dissemination of a single carbapenemase gene were investigated. Since the dissemination of CRE is primarily due to the transmission of carbapenemase genes located on plasmids, we analyzed the plasmidome of 230 CRE isolates carrying bla IMP by whole-genome sequencing and Southern blotting. bla IMP-6 was found to be predominantly disseminated among chromosomally distinct isolates through the pKPI-6 plasmid. Underlying the vast clonal dissemination of pKPI-6, various subpopulations deriving from pKPI-6 were identified, which had acquired advantages for the dissemination of CRE isolates. A cluster exhibiting heteroresistance against meropenem by the transcriptional regulation of bla IMP-6 caused an outbreak likely through covert transmission of bla IMP-6 For stable carriage of bla IMP-6, they occasionally integrated bla IMP-6 on their chromosomes. In addition, we detected one isolate that broadened the range of antimicrobial resistance through a single point mutation in bla IMP-6 on pKPI-6. Multifaceted analysis of the plasmidome granted us more accurate perspectives on the horizontal spread of CRE isolates, which is difficult to trace only by comparing the whole genomes. This study revealed the predominant spread of a specific carbapenemase-encoding plasmid accompanying the emergence of phenotypically diverse derivatives, which may facilitate further dissemination of CRE in various environments.IMPORTANCE Global dissemination of carbapenem-resistant Enterobacteriaceae (CRE) threatens human health by limiting the efficacy of antibiotics even against common bacterial infections. Carbapenem resistance, mainly due to carbapenemase, is generally encoded on plasmids and is spread across bacterial species by conjugation. Most CRE epidemiological studies have analyzed whole genomes or only contigs of CRE isolates. Here, plasmidome analysis on 230 CRE isolates carrying bla IMP was performed to shed light into the dissemination of a single carbapenemase gene in Osaka, Japan. The predominant dissemination of bla IMP-6 by the pKPI-6 plasmid among genetically distinct isolates was revealed, as well as the emergences of pKPI-6 derivatives that acquired advantages for further disseminations. Underlying vast clonal dissemination of a carbapenemase-encoding plasmid, heteroresistance was found in CRE offspring, which was generated by the transcriptional regulation of bla IMP-6, stabilization of bla IMP-6 through chromosomal integration, or broadened antimicrobial resistance due to a single point mutation in bla IMP-6.
Highlights
Carbapenem-resistant Enterobacteriaceae (CRE) infections, high in morbidity and mortality, pose serious clinical challenges due to limited treatment options
All blaIMP-positive CRE isolates of Escherichia coli (n ϭ 135) and Klebsiella pneumoniae (n ϭ 95) were classified into seven groups based on the results of S1-PFGE followed by Southern blotting hybridization with probes for the blaIMP and repA genes encoded on the IncN-type plasmid pKPI-6, sporadically reported as a plasmid carrying blaIMP-6 [11] (Fig. 1)
We compared the similarity between pKPI-6 and 39 representative plasmids categorized as group pKPI-6 based on whole-genome sequencing (WGS) data using Illumina HiSeq 3000 or Illumina MiSeq
Summary
Carbapenem-resistant Enterobacteriaceae (CRE) infections, high in morbidity and mortality, pose serious clinical challenges due to limited treatment options. We previously conducted a surveillance study of CRE in 1,507 patients from 43 hospitals in northern Osaka (population, 1,170,000; area, 307 km2), Japan [10], and we reported that 12% of the patients carried CRE and 95% of CRE isolates harbored blaIMP-6, the predominant carbapenemase in Japan. The predominance of this particular carbapenemase gene might have resulted from vigorous horizontal spreading of a specific plasmid carrying blaIMP-6 in this region. The aim of the present study was to analyze the plasmidome transmitting carbapenemase genes in order to unveil the mechanisms for their regional dissemination
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