He part of any particular strain was not due to degradation of the inhibitor enzyme. Detection of extracellular proteases at a much higher level in the planktonic culture medium is consistent with prior reports that expression of extracellular proteases is maximal instationary phase cultures in vitro [90,91]. In S. aureus, production of the extracellular proteases is tightly controlled, subject to positive regulation by agr and negative regulation by SarA [73]. Numerous reports have shown that OPC-8212 web biofilm production in S. aureus is regulated by these same pathways, with SarA stimulating biofilm formation and agr promoting biofilm dispersal [50,57,58,92]. High levels of extracellular protease production have been shown to reduce biofilm formation [73,93], possibly by degrading cell (R)-K-13675 web wall-associated proteins such as fibronectin-binding proteins (FnBPA and FnBPB) [79,94]. Our results demonstrating minimal extracellular protease production during growth as a biofilm coupled with higher protease production during planktonic growth are consistent with proposed mechanisms whereby the extracellular proteases participate in the biofilm dispersal process through induction of the agr system, promoting dissemination of the bacteria and aiding in the transition from an attached to an invasive phenotype in vivo [58,73,78,95].PLOS ONE | www.plosone.orgSwine MRSA Isolates form Robust BiofilmsFigure 7. Dispersal of established biofilms by DspB. S. aureus strains tested are shown along the x-axis and grouped based on methicillin-sensitivity and isolation source. S. epidermidis (S. epi) strains tested are shown along the x-axis and grouped together. Wells were washed and treated with buffer alone (- DspB) or 40 /ml DspB (+ DspB) for 2 hours. Biofilm formation was then quantified by standard microtiter assays and measuring the absorbance at 538 nm, plotted along the y-axis. Bars represent the average absorbance obtained from at least 3 independent plates representing biological replicates; error bars represent the SEM. Asterisks (*) denote a p-value less than 0.05 between the treated and untreated groups.doi: 10.1371/journal.pone.0073376.gInterestingly, the magnitude of the protease activity detected in the planktonic culture medium varied across the strains tested, and this variation appears to correlate with MLST type: the ST398 strains tended to have higher levels of protease activity and the ST5 (USA100, MN06, MRS1008, MRS879, MRS935), ST8 (USA300, TCH1516) and ST9 (MN55, MN56) strains had lower activity. Protease activity was undetected in the laboratory strains Newman, 29213, SH1000 and 43300. Variation in extracellular protease activity in different laboratory strains and clinical isolates has been reported previously, ascribed to differences in the levels of expression of global regulators such as sarA, agr and saeRS [58,96,97]. Investigation of the ability of the various strains to produce functional extracellular nuclease revealed that the majority of strains tested had detectable nuclease activity during both planktonic and biofilm growth. In our assays, there appears to be an association of nuclease production with sensitivity to biofilm inhibition and dispersal by DNaseI, as the strains that demonstrated little or no nuclease activity were also among theleast sensitive to DNaseI-mediated inhibition of biofilm formation and dispersal of established biofilms. The ST398 strains, which were the most sensitive to DNaseI inhibition and dispersal, produc.He part of any particular strain was not due to degradation of the inhibitor enzyme. Detection of extracellular proteases at a much higher level in the planktonic culture medium is consistent with prior reports that expression of extracellular proteases is maximal instationary phase cultures in vitro [90,91]. In S. aureus, production of the extracellular proteases is tightly controlled, subject to positive regulation by agr and negative regulation by SarA [73]. Numerous reports have shown that biofilm production in S. aureus is regulated by these same pathways, with SarA stimulating biofilm formation and agr promoting biofilm dispersal [50,57,58,92]. High levels of extracellular protease production have been shown to reduce biofilm formation [73,93], possibly by degrading cell wall-associated proteins such as fibronectin-binding proteins (FnBPA and FnBPB) [79,94]. Our results demonstrating minimal extracellular protease production during growth as a biofilm coupled with higher protease production during planktonic growth are consistent with proposed mechanisms whereby the extracellular proteases participate in the biofilm dispersal process through induction of the agr system, promoting dissemination of the bacteria and aiding in the transition from an attached to an invasive phenotype in vivo [58,73,78,95].PLOS ONE | www.plosone.orgSwine MRSA Isolates form Robust BiofilmsFigure 7. Dispersal of established biofilms by DspB. S. aureus strains tested are shown along the x-axis and grouped based on methicillin-sensitivity and isolation source. S. epidermidis (S. epi) strains tested are shown along the x-axis and grouped together. Wells were washed and treated with buffer alone (- DspB) or 40 /ml DspB (+ DspB) for 2 hours. Biofilm formation was then quantified by standard microtiter assays and measuring the absorbance at 538 nm, plotted along the y-axis. Bars represent the average absorbance obtained from at least 3 independent plates representing biological replicates; error bars represent the SEM. Asterisks (*) denote a p-value less than 0.05 between the treated and untreated groups.doi: 10.1371/journal.pone.0073376.gInterestingly, the magnitude of the protease activity detected in the planktonic culture medium varied across the strains tested, and this variation appears to correlate with MLST type: the ST398 strains tended to have higher levels of protease activity and the ST5 (USA100, MN06, MRS1008, MRS879, MRS935), ST8 (USA300, TCH1516) and ST9 (MN55, MN56) strains had lower activity. Protease activity was undetected in the laboratory strains Newman, 29213, SH1000 and 43300. Variation in extracellular protease activity in different laboratory strains and clinical isolates has been reported previously, ascribed to differences in the levels of expression of global regulators such as sarA, agr and saeRS [58,96,97]. Investigation of the ability of the various strains to produce functional extracellular nuclease revealed that the majority of strains tested had detectable nuclease activity during both planktonic and biofilm growth. In our assays, there appears to be an association of nuclease production with sensitivity to biofilm inhibition and dispersal by DNaseI, as the strains that demonstrated little or no nuclease activity were also among theleast sensitive to DNaseI-mediated inhibition of biofilm formation and dispersal of established biofilms. The ST398 strains, which were the most sensitive to DNaseI inhibition and dispersal, produc.
