Impact of extracellular nuclease production on the biofilm phenotype of Staphylococcus aureus under in vitro and in vivo conditions

Abstract

Recent studies suggest that extracellular DNA promotes biofilm formation in Staphylococcus aureus and, conversely, that extracellular nucleases limit the ability to form a biofilm. S. aureus produces at least two extracellular nucleases, and in the study described in this report, we examined the impact of each of these nucleases on biofilm formation under both in vitro and in vivo conditions. Our results demonstrate that both nucleases impact biofilm formation in the clinical isolate UAMS-1. Under certain in vitro conditions, this impact is negative, with mutation of either or both of the nuclease genes (nuc1 and nuc2) resulting in an enhanced capacity to form a biofilm. However, this effect was not apparent in vivo in a murine model of catheter-associated biofilm formation. Rather, mutation of either or both nuclease genes appeared to limit biofilm formation to a degree that could be correlated with increased susceptibility to daptomycin.

Fig 1

In vitro biofilm formation in sarA and nuclease mutants with and without plasma coating. Biofilm formation was assessed using an in vitro model of catheter-associated biofilm formation with and without first coating the catheters with human plasma (23). Results are shown for UAMS-1 (wild type [WT]), its isogenic derivatives carrying mutations in nuc1, nuc2, or both (nuc12) with and without concomitant mutation of sarA (sarA). Strain designations: s+n1, sarA nuc1 double mutant; s+n2, sarA nuc2 double mutant; s+n12, sarA nuc1 nuc2 triple mutant. *, statistical significance (P < 0.05) by comparison to the results observed with the sarA mutant under the same assay condition; **, statistical significance (P < 0.05) by comparison to the wild-type strain.

Fig 2

Production of extracellular nuclease. Nuclease production was assessed using a FRET-based assay with supernatants from cultures grown in TSB with (right) and without (left) supplementation of the medium with glucose and salt. Strain designations are the same as described in the Fig. 1 legend.

Fig 3

Impact of sarA and nuc mutations on daptomycin susceptibility in vitro. Relative susceptibility to daptomycin was assessed by Etest using Mueller-Hinton agar as the growth medium.

Fig 4

Impact of sarA and nuclease production on susceptibility of UAMS-1 to daptomycin in vivo. Biofilm formation and relative susceptibility to daptomycin were assessed using an in vivo model of catheter-associated biofilm formation (24). Results are shown for UAMS-1 (wild type [WT]) and its isogenic derivatives carrying mutations in sarA and/or SA0746 (nuc1). Asterisks indicate statistical significance (P < 0.05) by comparison to the results observed with the wild-type strain under the same assay condition. The NS above the bracket indicates the lack of statistical significance between the sarA and sarA nuc1 mutants. Solid dots represent outlying observations.

Fig 5

Impact of sarA and nuclease production on susceptibility of UAMS-1 to daptomycin in vivo. Biofilm formation and relative susceptibility to daptomycin were assessed using an in vivo model of catheter-associated biofilm formation (24). Asterisks indicate statistical significance (P < 0.05) by comparison to the results observed with the wild-type strain under the same assay condition. The NS above the bracket indicates the lack of statistical significance between the sarA and sarA nuc12 mutants. Solid dots represent outlying observations.