Enterococcus faecalis Produces Abundant Extracellular Structures Containing DNA in the Absence of Cell Lysis during Early Biofilm Formation

Abstract

Enterococcus faecalis is a common Gram-positive commensal bacterium of the metazoan gastrointestinal tract capable of biofilm formation and an opportunistic pathogen of increasing clinical concern. Dogma has held that biofilms are slow-growing structures, often taking days to form mature microcolonies. Here we report that extracellular DNA (eDNA) is an integral structural component of early E. faecalis biofilms (≤4 h postinoculation). Combining cationic dye-based biofilm matrix stabilization techniques with correlative immuno-scanning electron microscopy (SEM) and fluorescent techniques, we demonstrate that—in early E. faecalis biofilms—eDNA localizes to previously undescribed intercellular filamentous structures, as well as to thick mats of extruded extracellular matrix material. Both of these results are consistent with previous reports that early biofilms are exquisitely sensitive to exogenous DNase treatment. High-resolution SEM demonstrates a punctate labeling pattern in both structures, suggesting the presence of an additional, non-DNA constituent. Notably, the previously described fratricidal or lytic mechanism reported as the source of eDNA in older (≥24 h) E. faecalis biofilms does not appear to be at work under these conditions; extensive visual examination by SEM revealed a striking lack of lysed cells, and bulk biochemical assays also support an absence of significant lysis at these early time points. In addition, some cells demonstrated eDNA labeling localized at the septum, suggesting the possibility of DNA secretion from metabolically active cells. Overall, these data are consistent with a model in which a subpopulation of viable E. faecalis cells secrete or extrude DNA into the extracellular matrix.

Streptococcus mutans Extracellular DNA Is Upregulated during Growth in Biofilms, Actively Released via Membrane Vesicles, and Influenced by Components of the Protein Secretion Machinery

“…While programmed cell death and lysis is widely considered the major source of eDNA (25)(26)(27)(28)(29), there is evidence that eDNA can also be actively released/secreted independently of cell lysis (18,30,31). In Enterococcus faecalis, growth in biofilms triggered eDNA release, resulting in increases of eDNA by more than 3 logs compared to the planktonic counterparts (30).…”

Combination of Cold Atmospheric Plasma and Vitamin C Effectively Disrupts Bacterial Biofilms

“…Extracellular polymeric substances (EPS) produced by bacterial cell constitute the matrix of the bacterial biofilm, which holds bacterial communities together. The EPS matrix, which is composed of polysaccharides, proteins and extracellular DNA, is known to protect the bacterial communities from various antimicrobial agents, making them several fold more resistant [10,11]. Generally, the dense polymeric matrix is protecting the bacterial cells from antimicrobial exposure by preventing the penetration/diffusion of antimicrobial molecules.…”

The Environment of “Mycobacterium avium subsp. hominissuis” Microaggregates Induces Synthesis of Small Proteins Associated with Efficient Infection of Respiratory Epithelial Cells

“…Microaggregate and biofilm formation has been described in various other pathogens and is a key component in establishing infection as well as persistence in the host (17)(18)(19)(20)(21)(22). M. avium subsp.…”

“…(17)(18)(19)(20)(21)(22). Microaggregate formation is often mediated by cell surface proteins, such as fimbriae and pili, and can have a protective effect in the presence of antibiotics, enhance bacterial adherence to host cells, and serve as a prelude to early biofilm formation (18,20,23).…”