Genetic, virulence, and antimicrobial resistance characteristics associated with distinct morphotypes in ST11 carbapenem-resistant Klebsiella pneumoniae

Abstract

ST11 is the most common lineage among carbapenem-resistant Klebsiella pneumoniae (CRKP) infections in Asia. Diverse morphotypes resulting from genetic mutations are associated with significant differences in microbial characteristics among K. pneumoniae isolates. Here, we investigated the genetic determinants and critical characteristics associated with distinct morphotypes of ST11 CRKP. An ST11-KL47 CRKP isolate carrying a pLVPK-like virulence plasmid was isolated from a patient with a bloodstream infection; the isolate had the "mcsw" morphotype. Two distinct morphotypes ("ntrd" and "msdw") were derived from this strain during in vitro passage. Whole genome sequencing was used to identify mutations that cause the distinct morphotypes of ST11 CRKP. Transmission electron microscopy, antimicrobial susceptibility tests, growth assays, biofilm formation, virulence assays, membrane permeability assays, and RNA-seq analysis were used to investigate the specific characteristics associated with different morphotypes of ST11 CRKP. Compared with the parental mcsw morphotype, the ntrd morphotype resulted from mutation of genes involved in capsular polysaccharide biosynthesis (wza, wzc, and wbaP), a result validated by gene knockout experiments. This morphotype showed capsule deficiency and lower virulence potential, but higher biofilm production. By contrast, the msdw morphotype displayed competition deficiency and increased susceptibility to chlorhexidine and polymyxin B. Further analyses indicated that these characteristics were caused by interruption of the sigma factor gene rpoN by insertion mutations and deletion of the rpoN gene, which attenuated membrane integrity presumably by downregulating the phage shock protein operon. These data expand current understanding of genetic, virulence, and antimicrobial resistance characteristics associated with distinct morphotypes in ST11 CRKP.

Keywords: ST11; chlorhexidine; morphotype; rpoN; virulence.

Figure 1.

Four variants with distinct morphologies derived from ST11-KL47 CRKP isolate KP10042. (a) A schematic showing the appearance of variants with distinct morphologies during in vitro passage. (b) Colony morphology of variants on LB agar plates. (c) Transmission electron microscopy images of the variants with different morphotypes. Arrows point to the capsule.

Figure 2.

Phenotypic differences between variants with distinct morphologies. (a) Growth curves of the variants in LB broth. (b) Biofilm formation. (c) Mucoviscosity of the different morphotypes. (d) Serum resistance of the different morphotypes. Differences in serum resistance were determined by comparing growth curves in LB broth with those in LB broth supplemented with 10% pooled normal human serum. (e) Survival curves generated by the G. mellonella infection model and the murine intraperitoneal infection model. Ten larvae per group were injected with 20 μL (10⁶ CFU/mL) of each morphotype. Ten mice of each group were injected intraperitoneally with 10⁷ CFU of each morphotype. Survival was monitored for 7 days.

Figure 3.

Increased susceptibility of the msdw morphotype to chlorhexidine and PMB is associated with attenuated membrane integrity. (a) Comparison of membrane permeability of the wild-type and ΔrpoN strains in an assay using 1% sodium dodecyl sulfate (SDS) and 100 μg/mL polymyxin B nonapeptide (PMBN) (both of which compromise the integrity of bacterial membrane). (b) Volcano plot of differentially expressed genes. The red and green dots denote significantly upregulated and downregulated genes, respectively, and the black dots represent non-differentially expressed genes. Representative genes are depicted. (c) Downregulated transcription of the psp operon in the ΔrpoN mutant, validated by RT-qPCR. (d) A diagram of the psp operon in different species.