AUTHOR=Khamwong Muthita , Phanthanawiboon Supranee , Salao Kanin , Chareonsudjai Sorujsiri 

TITLE=Burkholderia pseudomallei biofilm phenotypes confined but surviving in neutrophil extracellular traps of varying appearance

JOURNAL=Frontiers in Immunology

VOLUME=13

YEAR=2022

URL=https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2022.926788

DOI=10.3389/fimmu.2022.926788

ISSN=1664-3224

ABSTRACT=<p>Melioidosis is a fatal infectious disease caused by <italic>Burkholderia pseudomallei</italic>. Complications following treatment are usually due to antibiotic resistance and relapse is mainly caused by <italic>B. pseudomallei</italic> biofilm. Although the release of neutrophil extracellular traps (NETs) is crucial to capture and eliminate bacterial pathogens, to date response of NETs to <italic>B. pseudomallei</italic> biofilm is poorly understood. Here we compare the NETs produced by neutrophils in response to <italic>B. pseudomallei</italic> H777 (a biofilm-producing strain containing the <italic>bpsl0618</italic> gene), a biofilm-defect strain lacking this gene (<italic>B. pseudomallei</italic> M10) and a <italic>bpsl0618</italic> biofilm-complemented strain, <italic>B. pseudomallei</italic> C17, in which function of <italic>bpsl0618</italic> was restored. Co-cultivation of these strains with healthy human neutrophils at MOI 10 with or without cytochalasin D demonstrated that H777 significantly resisted neutrophil-mediated killing and non-phagocytotic mechanisms compared to M10 (<italic>p</italic> &lt; 0.0001). Three distinct morphotypes of NETs were seen: “aggregated”, “spiky” and “cloudy”. These were induced in different proportions by the different bacterial strains. All types of NETs were shown to confine all <italic>B. pseudomallei</italic> strains. Strains H777 and C17 could stimulate production of twice as much extracellular DNA (234.62 ng/mL and 205.43 ng/mL, respectively) as did M10 (111.87 ng/mL). Cells of H777 and C17 were better able to survive in the presence of neutrophil killing mechanisms relative to M10 (<italic>p</italic> &lt; 0.0001) and NET formation (<italic>p</italic> &lt; 0.0001 and 0.05). These findings suggest that NET stimulation was insufficient to eradicate <italic>B. pseudomallei</italic> H777 and C17 despite their possession of <italic>bpsl0618</italic>, a sugar-transferase gene associated with biofilm formation ability. Our findings demonstrate that <italic>B. pseudomallei</italic> biofilm phenotype may be a key factor in assisting pathogens to escape killing by neutrophils. This work provides a better understanding of how <italic>B. pseudomallei</italic> biofilm-associated infections induce and survive NET formation, resulting in bacterial persistence and increased severity of disease.</p>