Klebsiella pneumoniae Prevents The Release of Neutrophil Extracellular Traps by Human Neutrophils

CASTILLO L; LANDONI V.I; RODRIGUEZ-RODRIGUES N; FERNÁNDEZ G C; BIRNBERG-WEISS F; MARTIRE-GRECO D

Cancun

Congreso; ALAI 2018; 2018

ALAI

Bacterial infections remain an important cause of morbidity and mortality worldwide, especially in immune-compromised patients. Although much progress has been made in its treatment, the appearance of new strains with acquired multiple antibiotic resistances and the growing number of immunosuppressed patients represents great therapeutic challenges. Klebsiella pneumoniae carbapenemase (KPC)-producing bacteria are a group of emerging highly drug-resistant Gram-negative bacilli causing infections associated with significant mortality, mainly associated to carbapenem resistance, one of last option of antimicrobial treatment. Neutrophil (PMN)-mediated response is essential for host to first combat bacterial infection. However, previous results showed that Klebsiella pneumoniae (Kpn) may be resistant to PMN bactericidal mechanism such as phagocytosis. The release of neutrophil extracellular traps (NET?s) is a major immune mechanism intended to capture and destroy pathogens. These histone- and protease-coated DNA structures are released by PMN in response to a variety of stimuli and have been identified in the airways of patients with respiratory infection, cystic fibrosis, peritonitis, acute lung injury, chronic obstructive pulmonary disease and septicemia. Some bacteria such as Staphylococcus aureus, Yersinia enterocolitica, Yersinia pseudotuberculosis and E. coli have been reported to induce NETosis. Since the discovery of NET?s over a decade ago, evidence that "NET evasion" might act as an immune protection strategy for bacterial pathogens, including group "A" Streptococcus, Bordetella pertussis, and Haemophilus influenzae, has been growing, with the majority of these studies being published in the past 2 years. Hence, since the evasion of NET?s appears to be a widespread strategy to allow pathogen proliferation and dissemination, and is currently a topic of intense research interest, the aim of this study was to investigate whether Kpn was able to modulate the bactericidal response of PMN, focusing on NET?s formation. For this purpose, we determine NETosis on human purified PMN in response to three different strains, two Kpn, the KPC producer strain (Kpn-KPC) and the non carabapenem resistant Kpn ATCC 700603 strain (Kpn-ATCC), and other opportunistic enterobacteria such as Escherichia coli (E. coli) ATCC 25922. PMN-bacteria were incubated for 3 hours, NET?s released was observed by confocal microscopy and the area was measured by the Image J® software, after DNA and Elastase staining. In addition, PMN-released Myeloperoxidase (MPO) and DNA were measured in supernatants of PMN-bacteria co-cultures. We first found that PMN failed to produce NETosis when were challenged with Kpn-KPC or Kpn-ATCC strains, in spite to different PMN:Kpn ratios (1:10 and 1:1) as determined by both confocal microscopy and measure of NET´s released, while E. coli was a potent NET?s inducer, compared to unstimulated PMN (Ctrl) (NET?s area, µM2= Ctrl: 66.5±12, Kpn-KPC: 95.7±16, Kpn-ATCC: 174±43, E. coli: 21754.4±2289; Released DNA, ng/mL= Ctrl: 234.8±29, Kpn-KPC: 195.4±37, Kpn-ATCC: 160±26, E. coli: 930±52; Released MPO, O.D= Ctrl: 0.05±0.01, Kpn-KPC: 0.08±0.004, Kpn-ATCC: 0.08±0.005, E. coli: 0.37±0.03, p< 0.05 E. coli vs all others). This data was in accordance with PMN-bacterial killing determined by colony forming units (CFU) quantification after 3 hours of PMN-bacteria co-cultures. In this sense, the percentage of bacterial survival was significantly higher for Kpn-KPC and Kpn-ATCC compared to E. coli (p< 0.05). Previous results from our laboratory have shown an inability of Kpn to induce a respiratory burst in PMN. Considering this result and the fact that intracellularly produced reactive oxygen species are necessary for NET?s formation, we decided to investigate if the failure in NET?s formation was related to the absence of one of this reactive oxygen species. Constant supply of peroxide (H2O2) can be guarantied by glucose oxidase (GO) addition in a culture medium with glucose as GO substrate. Therefore, PMN were incubated for 3 hours in the presence of Kpn-KPC and GO and NET?s were evaluated after the incubation period. In spite of constant reactive oxygen species supply by GO, Kpn-KPC still failed to induce NETosis; moreover, the presence of Kpn-KPC inhibited the intrinsic capacity of GO to induce NETosis through the production of peroxide (Released DNA, ng/mL= GO: 496±29, Kpn-KPC: 126±17, Kpn-KPC+GO: 180±21, p