Comparative genomic analysis of strong biofilm-forming Klebsiella pneumoniae isolates uncovers novel ISEcp1-mediated chromosomal integration of a full plasmid-like sequence

Background: The goal of the current study was to elucidate the genomic background of biofilm formation in Klebsiella pneumoniae.Methods: Clinical isolates were screened for biofilm formation using the crystal violet assay. Antimicrobial resistance (AMR) profiles were assessed by disk diffusion and broth microdilution tests. Biofilm formation was correlated to virulence and resistance genes screened by PCR. Draft genomes of three isolates that form strong biofilm were generated by Illumina sequencing.Results: Only the siderophore-coding gene iutA was significantly associated with more pronounced biofilm formation. ST1399-KL43-O1/O2v1 and ST11-KL15-O4 were assigned to the multidrug-resistant strain K21 and the extensively drug-resistant strain K237, respectively. ST1999-KL38-O12 was assigned to K57. Correlated with CRISPR/Cas distribution, more plasmid replicons and prophage sequences were identified in K21 and K237 compared to K57. The acquired AMR genes (blaOXA-48, rmtF, aac(6')-Ib and qnrB) and (blaNDM-1, blaCTX-M, aph(3')-VI, qnrS, and aac(6')-Ib-cr) were found in K237 and K21, respectively. The latter showed a novel ISEcp1-mediated chromosomal integration of replicon type IncM1 plasmid-like structure harboring blaCTX-M-14 and aph(3')-VI that uniquely interrupted rcsC. The plasmid-mediated heavy metal resistance genes merACDEPRT and arsABCDR were spotted in K21, which also exclusively carried the acquired virulence genes mrkABCDF and the hypervirulence-associated genes iucABCD-iutA, and rmpA/A2. Pangenome analysis revealed NTUH-K2044 accessory genes most frequently shared with K21.Conclusions: While less virulent to Galleria mellonella than ST1999 (K57), the strong biofilm former, multidrug-resistant, NDM-producer K. pneumoniae K21 (ST1399-KL43-O1/O2v1) carries a novel chromosomally integrated plasmid-like structure and hypervirulence-associated genes and represents a serious threat to countries in the area.