Human Neutrophil Response Against the Hyper-Epidemic Clone of Carbapenem Resistant Klebsiella pneumoniae ST258

RODRIGUEZ-RODRIGUES, NAHUEL; MARTIRE-GRECO, DAIANA; FERNÁNDEZ, GABRIELA C.; BIRNBERG-WEISS F; GOMEZ S.A.; CASTILLO, LUIS A.; SCHIERLOH P; LANDONI V.I

Cancun

Congreso; ALAI 2018; 2018

ALAI

Klebsiella pneumoniae (Kpn) has become a relevant nosocomial pathogen due to the acquisition of multiple antibiotic resistance genes, emerging as a serious public health problem. Kpn isolates classified as Sequence Type 258 (ST258) by multi-locus sequence typing are largely responsible for global spread of the blaKPC gene, which codifies for Kpn-Carbapanemase (KPC), an enzyme that mediates resistance to carbapenems, the last option in antibiotic treatment. In order to determine whether the rapid dissemination and persistence of Kpn-KPC ST258 could be related to mechanisms of immune evasion, in this study we evaluated if Kpn-KPC induced a differential response in neutrophils (PMN), compared to another opportunistic pathogen. For this purpose, we evaluated different parameters of activation in human isolated PMN challenged with a local clinical isolate of Kpn-KPC ST258 or Eschericia coli ATCC 25922 (Eco), using a multiplicity of infection (MOI) of 20. CD11b expression was measured using an anti-CD11b antibody, and reactive oxygen species (ROS) using dihydrorhodamine 123 (DHR) by flow cytometry, after 30 min. of stimulation with the different bacteria. Neutrophil extracellular traps (NETs) formation was evaluated after 3 hours of challenge by confocal microscopy using a DNA and elastase staining and by quantification of the NETs-released myeloperoxidase (MPO) (using TMB substrate) and DNA (using Pico-Green) after DNase digestion. Although up-regulation of CD11b expression was similar for both bacteria, NETosis and ROS generation were lower in the presence of Kpn-KPC compared to Eco (NETs area, mm2x10-4: Kpn-KPC=0.22±0.06; Eco=2.17±0.22, p