Virulence Determinants of Colistin-Resistant K. pneumoniae High-Risk Clones

Abstract

We proposed the hypothesis that high-risk clones of colistin-resistant K. pneumoniae (ColR-Kp) possesses a high number of virulence factors and has enhanced survival capacity against the neutrophil activity. We studied virulence genes of ColR-Kp isolates and neutrophil response in 142 patients with invasive ColR-Kp infections. The ST101 and ST395 ColR-Kp infections had higher 30-day mortality (58%, p = 0.005 and 75%, p = 0.003). The presence of yersiniabactin biosynthesis gene (ybtS) and ferric uptake operon associated gene (kfu) were significantly higher in ST101 (99%, p ≤ 0.001) and ST395 (94%, p < 0.012). Being in ICU (OR: 7.9; CI: 1.43-55.98; p = 0.024), kfu (OR:27.0; CI: 5.67-179.65; p < 0.001) and ST101 (OR: 17.2; CI: 2.45-350.40; p = 0.01) were found to be predictors of 30-day mortality. Even the neutrophil uptake of kfu+-ybtS+ ColR-Kp was significantly higher than kfu--ybtS- ColR-Kp (phagocytosis rate: 78% vs. 65%, p < 0.001), and the kfu+-ybtS+ ColR-Kp survived more than kfu--ybtS- ColR-Kp (median survival index: 7.90 vs. 4.22; p = 0.001). The kfu+-ybtS+ ColR-Kp stimulated excessive NET formation. Iron uptake systems in high-risk clones of colistin-resistant K. pneumoniae enhance the success of survival against the neutrophil phagocytic defense and stimulate excessive NET formation. The drugs targeted to iron uptake systems would be a promising approach for the treatment of colistin-resistant high-risk clones of K. pneumoniae infections.

Keywords: K. pneumoniae; colistin resistance; iron uptake; net formation; phagocytosis.

Figure 1

The phagocytosis of ColR-Kp by neutrophils. Phagocytosis rate of kfu+-ybtS+ and kfu--ybtS- isolates (A) and survival of kfu+-ybtS+ and kfu--ybtS- isolates after being phagocytosed by neutrophils (B).

Figure 2

Confocal microscopic images of NETs. The samples were stained consecutively with myeloperoxidase (MPO, red) and histone 3 (H3, green). The nuclei were counterstained with DAPI (blue). Neutrophils were seen intact with K. pneumonia ATCC 700,831 control (A–D). The kfu- -ybtS- isolates depicted rare and weak NET formation (E–L). The rectangular area in image H was magnified in images I–L. The kfu+ -ybtS+ isolates showed abundant NET formation with excessive histone and MPO release in extracellular matrix (M–U). The rectangular area in image p was magnified in images R–U. Bars: A–H, M-p = 25 μm; I–L, R–U = 10 μm.