Comprehensive study reveals phenotypic heterogeneity in Klebsiella pneumoniae species complex isolates

Abstract

Here, we conducted a comprehensive analysis of 356 Klebsiella pneumoniae species complex (KpSC) isolates that were classified as classical (cl), presumptive hypervirulent (p-hv) and hypermucoviscous-like (hmv-like). Overall, K. pneumoniae (82.3%), K. variicola (2.5%) and K. quasipneumoniae (2.5%) were identified. These isolates comprised 321 cl-KpSC, 7 p-hv-KpSC and 18 hmv-like-KpSC. A large proportion of cl-KpSC isolates were extended-spectrum-β-lactamases (ESBLs)-producers (64.4%) and 3.4% of isolates were colistin-resistant carrying carbapenemase and ESBL genes. All p-hv-KpSC showed an antibiotic susceptible phenotype and hmv-like isolates were found to be ESBL-producers (8/18). Assays for capsule production and capsule-dependent virulence phenotypes and whole-genome sequencing (WGS) were performed in a subset of isolates. Capsule amount differed in all p-hv strains and hmv-like produced higher capsule amounts than cl strains; these variations had important implications in phagocytosis and virulence. Murine sepsis model showed that most cl strains were nonlethal and the hmv-like caused 100% mortality with 3 × 10⁸ CFUs. Unexpectedly, 3/7 (42.9%) of p-hv strains required 10⁸ CFUs to cause 100% mortality (atypical hypervirulent), and 4/7 (57.1%) strains were considered truly hypervirulent (hv). Genomic analyses confirmed the diverse population, including isolates belonging to hv clonal groups (CG) CG23, CG86, CG380 and CG25 (this corresponded to the ST3999 a novel hv clone) and MDR clones such as CG258 and CG147 (ST392) among others. We noted that the hmv-like and hv-ST3999 isolates showed a close phylogenetic relationship with cl-MDR K. pneumoniae. The information collected here is important to understand the evolution of clinically important phenotypes such as hypervirulent and ESBL-producing-hypermucoviscous-like amongst the KpSC in Mexican healthcare settings. Likewise, this study shows that mgrB inactivation is the main mechanism of colistin resistance in K. pneumoniae isolates from Mexico.

Keywords: Capsule; Colistin-resistance; Hypermucoviscosity; Plasmids; Virulence.

Figure 1

(a) Species distribution and sample types of 356 KpSC isolates. (b) Occurrence of presumptive hypervirulent, hypermucoviscous-like and classical phenotypes amongst 356 isolates of KpSC.

Figure 2

Assays for evaluating capsule production and phagocytosis. (a) Quantification of uronic acid and (b) Phagocytosis killing assays for presumptive hypervirulent, hypermucoviscous-like and classical MDR KpSC isolates. * Indicate p < 0.0002. Bars represent the mean levels ± 95%. (c) Correlation matrix of virulence traits. Numbers indicate Spearman’s correlation and stars identify significant correlations (p < 0.05). A − 1 value represent a strong negative correlation and a + 1 value a strong positive correlation.

Figure 3

Kaplan–Meier plots showing the percent survival of BALB/c mice over twelve days post-infection with hypervirulent and atypical hypervirulent (10², 10³ and 10⁸ CFUs), hypermucoviscous-like (3 × 10⁸ CFUs), and classical (3 × 10⁸ CFUs) strains. The characteristics of each strain such as ST, capsular type and ARGs are included. The panel of classical isolates included two colistin resistant isolates (COL^R) due to mgrB disruption (14669) and mutations in pmrB gene (5166). Mice challenged with classical MDR strain 11271 and the hypervirulent 10271 ST86-KL2 were used as controls. *This isolate correspond to hv K. quasipneumoniae subsp. similipneumoniae.

Figure 4

Molecular epidemiology of KpSC isolates. The phylogenetic tree was constructed with the concatenated sequence of the seven MLST genes. Repertoire of virulence and AMR genes, plasmid types and mobility/transfer components are shown as a presence and absence matrix.

Figure 5

Phylogenomic diversity of KpSC isolates. Whole-genome SNP-based tree inferred with maximum likelihood approach. The classification, AMR profile and source of each isolate is denoted in the tree. An antibiotic susceptible profile is denoted as white boxes. Clades in which hv or hmv-like isolates clustered with ESBL- and/or carbapenemase-producing K. pneumoniae are highlight in red.