Interactions of Klebsiella pneumoniae with the innate immune system vary in relation to clone and resistance phenotype

Abstract

Apart from inadequate antimicrobial treatment, specific virulence factors contribute to the high attributable mortality of infections caused by multidrug-resistant (MDR) Klebsiella pneumoniae. We explored the roles of MDR and clones as virulence determinants of K. pneumoniae and their interaction with innate immunity. Twenty isolates were studied and characterized by resistance phenotype and multilocus sequence type (MLST). Human peripheral blood mononuclear cells (PBMCs) were stimulated for the production of proinflammatory cytokines by live and heat-killed isolates and plasmid DNA; modulation by cellular pathway inhibitors was explored. Survival of 30 mice was recorded after intraperitoneal challenge with susceptible and K. pneumoniae carbapenemase (KPC)-producing isolates. Splenocytes of mice were stimulated for the production of pro- and anti-inflammatory cytokines. Isolates were divided into different patterns of production of tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β) poststimulation in relation to both the MLST clone and resistance phenotype. The sequence type 383 (ST383) clone producing Verona integron-encoded metallo-β-lactamase (VIM) stimulated high production of both TNF-α and IL-1β. Clone ST17 producing KPC elicited low TNF-α production; this was reversed by Toll-like receptor 9 (TLR9) antagonists, indicating an effect of plasmid DNA. This isolate was linked with early death of mice compared to high-TNF-α-producing isolates. We conclude that KPC-producing isolates seem to be highly virulent in a low-TNF-α-release environment, suggesting an immunoparalysis induction mechanism. KPC plasmids may directly contribute to the immune system stimulation.

FIG 1

Pattern of overtime cytokine production by Klebsiella pneumoniae in relation to multilocus sequence typing (MLST) cloning and resistance phenotype. (A) High, high-tumor necrosis factor alpha (TNF-α)-producing isolates (ST383 VIM, ST258 ESBL, ST528 susceptible); Moderate, moderate-TNF-α-producing isolates (ST258 KPC, ST147 susceptible, ST147 ESBL, ST17 VIM, ST643 susceptible, ST1237 VIM, ST347 KPC); Low, low-TNF-α-producing isolates (ST147 VIM, ST340 KPC, ST383 ESBL, ST17 KPC); (C) High, high-interleukin-1β (IL-1β)-producing isolates (ST383 VIM, ST347 KPC); Low, low-IL-1β-producing isolates (all other except nontypeable). a, high versus low; b, high versus moderate. (B and D) IL-6 kinetics in relation to TNF-α and IL-1β production. Comparisons were performed by the Mann-Whitney U test with Bonferroni's correction. Six experiments were run using cells from two healthy volunteers. Abbreviations: ST, sequence type; VIM, Verona integron-encoded metallo-β-lactamase; ESBL, extended-spectrum β-lactamases; KPC, Klebsiella pneumoniae carbapenemase.

FIG 2

Tumor necrosis factor alpha (TNF-α) (A) and interleukin-1β (IL-1β) (B) stimulation at 4 h by sequence type 383 (ST383) Verona integron-encoded metallo-β-lactamase (VIM) versus stimulation by all other isolates. Comparisons were performed by the Mann-Whitney U test. Six experiments were run using cells from two healthy volunteers.

FIG 3

Growth at 1.5 h of incubation of Klebsiella pneumoniae isolates in relation to the pattern of tumor necrosis factor alpha (TNF-α) (A) and interleukin-1β (IL-1β) (B) production. (A) High, high-TNF-α-producing isolates; Moderate, moderate TNF-α-producing isolates; Low, low-TNF-α-producing isolates; (B) High, high-IL-1β-producing isolates; Low, low-IL-1β-producing isolates. pNS, difference nonsignificant. Comparisons were performed by the Mann-Whitney U test. Five experiments were run using cells from two healthy volunteers.

FIG 4

Mechanism of tumor necrosis factor alpha (TNF-α) modulation by Klebsiella pneumoniae in relation to multilocus sequence typing (MLST) cloning and resistance phenotype. (A) TNF-α production after stimulation of mononuclear cells with plasmid DNA in the presence of Toll-like receptor 9 (TLR9) inhibitor; (B and C) TNF-α production after stimulation of mononuclear cells by heat-killed isolates in the presence of the respective inhibitors. High, high-TNF-α-producing isolates; Moderate, moderate-TNF-α-producing isolates; Low, low-TNF-α-producing isolates. a, high versus low; b, high versus moderate. Comparisons were performed by the Mann-Whitney U test with Bonferroni's corrections. Five experiments were run using cells from two healthy volunteers.

FIG 5

(A) Kaplan-Meier survival curves of mice after intraperitoneal challenge by Klebsiella pneumoniae in relation to patterns of tumor necrosis factor alpha (TNF-α) production. High, high-TNF-α-producing isolates; Moderate, moderate-TNF-α-producing isolates; Low, low-TNF-α-producing isolates. Comparisons were performed by the log rank test. Six experiments were run. (B) Bacterial growth of K. pneumoniae in liver and kidney of mice sacrificed 24 h after intraperitoneal challenge by high/moderate- and low-TNF-α-producing isolates. pNS, difference nonsignificant. Comparisons were performed by the Mann-Whitney U test. Six experiments were run.

FIG 6

Release of interleukin-10 (IL-10), IL-17, tumor necrosis factor alpha (TNF-α), and interferon gamma (IFN-γ) after stimulation of murine splenocytes. P values represent comparisons of the percentage of change of TNF-α production after lipopolysaccharide (LPS) stimulation for high/moderate- versus low-TNF-α-producing isolates (A), IFN-γ production after Candida albicans stimulation for high/moderate- versus low-TNF-α-producing isolates (B), IL-10 production after C. albicans stimulation for high/moderate- versus low-TNF-α-producing isolates (C), and IL-17 production after C. albicans stimulation for high/moderate- versus low-TNF-α-producing isolates (D). pNS, difference nonsignificant. Comparisons were performed by the Mann-Whitney U test. Six experiments were run.

FIG 7

Correlation between bacterial growth of Klebsiella pneumoniae in liver of mice after intraperitoneal challenge and survival time. Comparisons were performed by Spearman's rank correlation. Six experiments were run.