Influence of origin of isolates, especially endocarditis isolates, and various genes on biofilm formation by Enterococcus faecalis

Abstract

Endocarditis isolates of Enterococcus faecalis produced biofilm significantly more often than nonendocarditis isolates, and 39% of 79 versus 6% of 84 isolates produced strong biofilm (P < 0.0001). esp was not required, but its presence was associated with higher amounts of biofilm (P < 0.001). Mutants disrupted in dltA, efaA, ace, lsa, and six two-component regulatory systems were largely unaltered, while disruptions in epa (encoding enterococcal polysaccharide antigen), atn (encoding an autolysin), gelE (encoding gelatinase), and fsr (encoding the E. faecalis regulator) [corrected] resulted in fewer attached bacteria, as determined using phase-contrast microscopy, and less biofilm (P < 0.0001).

FIG. 1.

Biofilm formation by E. faecalis isolates derived from different sources. Biofilm formation on a polystyrene surface was assessed after CV staining. Each dot indicates the median OD₅₇₀ value from 12 determinations (three independent experiments, each performed in quadruplicate). The medians (and IQRs) for endocarditis isolates and those from other sources were 1.74 (IQR, 1.32 to 2.35) and 1.31 (IQR, 0.82 to 1.53), respectively (P < 0.0001).

FIG. 2.

Biofilm formation (BF) and primary adherence (PA) by representative E. faecalis clinical isolates (A) and mutants (B). Median and IQR values are shown. Values for the biofilm assays are from 12 determinations (three independent experiments, each performed in quadruplicate). All readings for TX1393 and TX0291 were 3.5, the maximum OD detectable by our microplate reader. Primary adherence was assessed after incubation on a polystyrene surface for 30 min for clinical isolates and 2 h for mutants. Bacteria in five different fields from two independent plates were subjected to light microscopy (HPF, high-power field; magnification, ×1,000) and counted after Gram staining. TX10275, TX10276, TX10292, TX10298, TX37200, and etaR are two-component regulatory system mutants. The other 10 mutants which showed no change in biofilm were not tested for primary adherence.

FIG. 3.

Phase-contrast photomicrographs of biofilms on a polystyrene surface. Images are representative of what was observed in multiple fields (magnification, ×600).