The origin of extracellular DNA in bacterial biofilm infectionsin vivo

Abstract

AbstractExtracellular DNA (eDNA) plays an important role in both the aggregation of bacteria and in the interaction of the resulting biofilms with polymorphonuclear leukocytes (PMNs) during an inflammatory response. Here, transmission electron and confocal scanning laser microscopy were used to examine the interaction between biofilms ofPseudomonas aeruginosaand PMNs in a murine implant model and in lung tissue from chronically infected cystic fibrosis patients. PNA FISH, DNA staining, labeling of PMN DNA with a thymidine analogue, and immunohistochemistry were applied to localize bacteria, eDNA, PMN-derived eDNA, PMN-derived histone H3 (H3), neutrophil elastase (NE), and citrullinated H3 (citH3). Host-derived eDNA was observed surrounding bacterial aggregates but not within the biofilms. H3 localized to the lining of biofilms while NE was found throughout biofilms. CitH3, a marker for neutrophil extracellular traps (NETs) was detected only sporadically indicating that most host-derived eDNAin vivowas not a result of NETosis. Together these observations show that, in thesein vivobiofilm infections withP. aeruginosa, the majority of eDNA is found external to the biofilm and derives from the host.Author summaryThe role of extracellular DNA (eDNA) has been describedin vitroto play a major role in biofilm formation and antibiotic tolerance, but never for biofilm infectionsin vivo.Two important characteristics of human chronic bacterial infections are aggregated bacteria and white blood cells (WBC). Bacteria use eDNA to stabilize biofilms and WBC use eDNA to trap bacteria. Given the importance of eDNA for both bacteria and WBC we show here for the first time that bacterial biofilms do not co-localize with either bacterial or WBC-derived eDNA during chronic infections. Ourin vivofindings show eDNA is located outside biofilms as opposed to incorporated within the biofilm as commonly observedin vitro.We believe that understanding the interplay between biofilms and WBC during chronic infection is essential if we are to elucidate the mechanisms underlying persistent infection and ascertain why WBC fail to eradicate bacteria; this will enable us to develop new treatment strategies.