The origin of extracellular DNA in bacterial biofilm infections in vivo

Abstract

Extracellular 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 of Pseudomonas aeruginosa and 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 eDNA in vivo was not a result of NETosis. Together these observations show that, in these in vivo biofilm infections with P. aeruginosa, the majority of eDNA is found external to the biofilm and derives from the host.\n\nAuthor summaryThe role of extracellular DNA (eDNA) has been described in vitro to play a major role in biofilm formation and antibiotic tolerance, but never for biofilm infections in vivo.\n\nTwo 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. Our in vivo findings show eDNA is located outside biofilms as opposed to incorporated within the biofilm as commonly observed in vitro.\n\nWe 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.