A Widely Used In Vitro Biofilm Assay Has Questionable Clinical Significance for Enterococcal Endocarditis

Abstract

Biofilm formation may play an important role in the pathogenesis of infections caused by Enterococcus faecalis, including endocarditis. Most biofilm studies use a polystyrene dish assay to quantify biofilm biomass. However, recent studies of E. faecalis strains in tissue and animal models suggest that polystyrene dish results need to be interpreted with caution. We evaluated 158 clinical E. faecalis isolates using a polystyrene dish assay and found variation in biofilm formation, with many isolates forming little biofilm even when different types of media were used. However, all tested clinical isolates were able to form biofilms on porcine heart valve explants. Dextrose-enhanced biofilm formation in the polystyrene dish assay was found in 6/12 (50%) of clinical isolates tested and may explain some, but not all of the differences between the polystyrene dish assay and the heart valve assay. These findings suggest that in studies assessing the clinical relevance of enterococcal biofilm-forming ability, ex vivo biofilm formation on a relevant tissue surface may be warranted to validate results of in vitro assays.

Expression of Adhesive Pili and the Collagen-Binding Adhesin Ace Is Activated by ArgR Family Transcription Factors in Enterococcus faecalis

“…Both AhrC and ArgR2 are required for expression of ebpR, the direct regulator of pilus structural genes. The deletion of ahrC resulted in decreased expression of the ebp pilus locus, and the failure to produce pili could result in the defects we observed in biofilm formation on plastic surfaces (12,16,17) or heart valve explants (16), as well as the attenuation of virulence in endocarditis and urinary tract infection models (17). Therefore, we hypothesized that the AhrC protein was a direct positive regulator of transcription of either the ebpA-C operon or of epbR, which was previously identified as an activator of ebpA-C (11).…”

“…We also identified insertions in diverse regulatory genes, including several argR family transcription factors, which resulted in biofilm defects. These included the ahrC gene (OG1RF_RS03710, corresponding to EF0983 in the V583 genome), whose disruption had dramatic effects on in vitro biofilm formation on plastic, biofilm formation on porcine cardiac valve explants (16), virulence in experimental rabbit endocarditis (17), and virulence in experimental urinary tract infections (17) and in experimental osteomyelitis (18) in mouse models. Overall, ahrC mutants of OG1RF are more attenuated for virulence trait expression than strains with any other single mutation we have identified.…”

Enzymatic modifications of exopolysaccharides enhance bacterial persistence

“…While some systems can closely approximate the conditions encountered in vivo , such as flow cell models for catheter associated infections, biofilms from infections like CF pneumonia or those encountered in epidermal wounds are thought to involve attachment to host cells or host-derived molecules ( Bjarnsholt et al, 2009 ). In these instances it is challenging to extrapolate results obtained in vitro to chronic biofilm infections, as modifications that appear important for attachment to abiotic surfaces may be disposable for attachment to host tissues, or vice versa ( Leuck et al, 2014 ). Furthermore, many biofilms encountered in the clinic are not comprised of a single species of bacteria, but rather contain mixtures that can include pathogenic or non-pathogenic bacteria of host or environmental origin, as well as fungi ( Wargo and Hogan, 2006 ; Elias and Banin, 2012 ).…”

Association of Novel Streptococcus sanguinis Virulence Factors With Pathogenesis in a Native Valve Infective Endocarditis Model

“…Cardiac vegetations, which develop over the course of IE, are considered biofilm-like structures composed of both bacterial and host factors (Elgharably et al, 2016). While in vitro biofilm formation on a plastic surface does not strongly correlate with in vivo virulence (Ge et al, 2008;Leuck et al, 2014), production of an extracellular matrix has been described to occur within streptococcal vegetations (Mills et al, 1984) and production of extracellular polysaccharide (EPS) correlates with streptococcal IE (Mills et al, 1984;Dall and Herndon, 1990). Consistent with a previous report (Ge et al, 2008), supplementation with the same concentration of glucose, a more physiologically relevant sugar, did not produce strong biofilms in this assay (data not shown).…”