Abstract
Carbapenem-resistant Klebsiella pneumoniae ST258 (CRKP-ST258) are a global concern due to their rapid dissemination, high lethality, antibiotic resistance and resistance to components of the immune response, such as neutrophils. Neutrophils are major host mediators, able to kill well-studied and antibiotic-sensitive laboratory reference strains of K. pneumoniae. However, CRKP-ST258 are able to evade neutrophil phagocytic killing, persisting longer in the host despite robust neutrophil recruitment. Here, we show that neutrophils are unable to clear a CRKP-ST258 isolate (KP35). Compared to the response elicited by a prototypic K. pneumoniae ATCC 43816 (KPPR1), the neutrophil intracellular response against KP35 is characterized by equivalent production of reactive oxygen species (ROS) and myeloperoxidase content, but impaired phagosomal acidification. Our results ruled out that this phenomenon is due to a phagocytosis defect, as we observed similar efficiency of phagocytosis by neutrophils infected with KP35 or KPPR1. Genomic analysis of the cps loci of KPPR1 and KP35 suggest that the capsule composition of KP35 explain the high phagocytosis efficiency by neutrophils. Consistent with other reports, we show that KP35 did not induce DNA release by neutrophils and KPPR1 only induced it at 3 h, when most of the bacteria have already been cleared. l-arginine metabolism has been identified as an important modulator of the host immune response and positively regulate T cells, macrophages and neutrophils in response to microbes. Our data show that l-arginine supplementation improved phagosome acidification, increased ROS production and enhanced nitric oxide consumption by neutrophils in response to KP35. The enhanced intracellular response observed after l-arginine supplementation ultimately improved KP35 clearance in vitro. KP35 was able to dysregulate the intracellular microbicidal machinery of neutrophils to survive in the intracellular environment. This process, however, can be reversed after l-arginine supplementation.
Highlights
Klebsiella pneumoniae (KP) is an extremely versatile bacterium able to colonize different environments including different kinds of soil, water courses, medical devices and mucosal surfaces (Tzouvelekis et al, 2012; Paczosa and Mecsas, 2016)
We evaluated neutrophil phagocytosis of two different KP strains, KPPR1 (K. pneumoniae ATCC 43816), a classical KP strain that is killed by neutrophils (Figure 1A) and KP35, a clinical CRKP-ST258 isolate that is resistant to neutrophil killing (Figure 1A)
After 10 min, the percentage of FITC+ neutrophils were equivalent when neutrophils were infected with KPPR1 and KP35; this was MOI-dependent, with greater uptake of bacteria when the neutrophils were infected with a MOI of 50 (Figure 1E)
Summary
Klebsiella pneumoniae (KP) is an extremely versatile bacterium able to colonize different environments including different kinds of soil, water courses, medical devices and mucosal surfaces (Tzouvelekis et al, 2012; Paczosa and Mecsas, 2016). A recent study reported considerable heterogeneity in capsule composition among CRKP-ST258 isolates due to mutations in capsule-related genes located in the capsular polysaccharide synthesis (cps) locus (Ernst et al, 2020); and a different study described the ability of phagocytosed KP to survive within the macrophage-phagosome by preventing the maturation of the phagolysosome (Cano et al, 2015). These two studies suggest that the ability of CRKP-ST258 to evade phagocytosis by neutrophils is not restricted to an uptake defect, and these bacteria potentially survive intracellularly by disrupting phagosomal machinery in these immune cells
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