Eated groups.doi: 10.1371/journal.pone.0073376.ggene acquisition events [80?2]. In contrast to S. aureus, it has been shown that biofilm formation and dispersal by a number of S. epidermidis strains is not sensitive to Proteinase K or other proteases [76,77]. Similar to these results, we found biofilm formation by S. epidermidis strains 1457 and NJ9709 to be insensitive to Proteinase K inhibition and Proteinase K caused little to no detachment in mature biofilms of these strains as well. Extracellular DNA (eDNA) is another component of the biofilm matrix and the structural role of eDNA in promoting biofilm stability is highly variable and dependent on the bacterial species, growth conditions, and age of the biofilm [61,83?6]. We found DNaseI treatment to have a varying effect on both biofilm inhibition and dispersal. Specifically, when DNaseI was added at the time of inoculation, all of the strains tested displayed a range of sensitivity, from little to no effect to strong, nearly complete inhibition of biofilm formation. DNaseI was observed to have varying effects on the dispersal as well, with some strains showing a much higher degree ofsensitivity to this PD150606MedChemExpress PD150606 enzyme than others. Both inhibition and dispersal by DNaseI seem to vary among S. aureus strains and MLST types indicating that eDNA may be a more significant component in some MLST types of S. aureus than in others. The ST398 strains in particular were the most sensitive to both inhibition of biofilm formation and dispersal of pre-formed biofilms by DNaseI, with a greater reduction in biofilm biomass than other non-ST398 strains, including other swine-origin isolates. The polysaccharide PNAG has been extensively studied as a biofilm matrix component and is a target for the enzyme DspB [52]. PNAG is the product of the icaADBC operon, which is highly conserved among Staphylococcus isolates [87]. Many studies have shown the importance of this polysaccharide in S. epidermidis biofilms, where it is proposed to be the major component of the biofilm matrix, as DspB can inhibit biofilm formation and disperse pre-formed biofilms [59,76,77,88]. However, the role of PNAG in S. aureus biofilms is less clear, as studies have shown that some strains of S. aureus producePLOS ONE | www.plosone.orgSwine MRSA Isolates form Robust BiofilmsFigure 5. Dispersal of established biofilms by Proteinase K. Strains tested are shown along the x-axis and grouped based on methicillin-sensitivity and isolation source. The indicated strains were grown statically for 24 hours to allow biofilm formation. Wells were washed and treated with buffer alone (- Prot. K) or 100 /ml Proteinase K (+ Prot. K) 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.ghigh levels of PNAG, while others VesnarinoneMedChemExpress OPC-8212 produce little to no PNAG [60]. Additionally, some strains have been shown to be sensitive to biofilm dispersal by DspB whereas other S. aureus strains are unaffected by this enzyme [59] or the compound sodium metaperiodate, which breaks down PNAG via an oxidation reaction [60,89]. Our results show that DspB has little effect on both biofilm formation and dispersal in the S. aur.Eated groups.doi: 10.1371/journal.pone.0073376.ggene acquisition events [80?2]. In contrast to S. aureus, it has been shown that biofilm formation and dispersal by a number of S. epidermidis strains is not sensitive to Proteinase K or other proteases [76,77]. Similar to these results, we found biofilm formation by S. epidermidis strains 1457 and NJ9709 to be insensitive to Proteinase K inhibition and Proteinase K caused little to no detachment in mature biofilms of these strains as well. Extracellular DNA (eDNA) is another component of the biofilm matrix and the structural role of eDNA in promoting biofilm stability is highly variable and dependent on the bacterial species, growth conditions, and age of the biofilm [61,83?6]. We found DNaseI treatment to have a varying effect on both biofilm inhibition and dispersal. Specifically, when DNaseI was added at the time of inoculation, all of the strains tested displayed a range of sensitivity, from little to no effect to strong, nearly complete inhibition of biofilm formation. DNaseI was observed to have varying effects on the dispersal as well, with some strains showing a much higher degree ofsensitivity to this enzyme than others. Both inhibition and dispersal by DNaseI seem to vary among S. aureus strains and MLST types indicating that eDNA may be a more significant component in some MLST types of S. aureus than in others. The ST398 strains in particular were the most sensitive to both inhibition of biofilm formation and dispersal of pre-formed biofilms by DNaseI, with a greater reduction in biofilm biomass than other non-ST398 strains, including other swine-origin isolates. The polysaccharide PNAG has been extensively studied as a biofilm matrix component and is a target for the enzyme DspB [52]. PNAG is the product of the icaADBC operon, which is highly conserved among Staphylococcus isolates [87]. Many studies have shown the importance of this polysaccharide in S. epidermidis biofilms, where it is proposed to be the major component of the biofilm matrix, as DspB can inhibit biofilm formation and disperse pre-formed biofilms [59,76,77,88]. However, the role of PNAG in S. aureus biofilms is less clear, as studies have shown that some strains of S. aureus producePLOS ONE | www.plosone.orgSwine MRSA Isolates form Robust BiofilmsFigure 5. Dispersal of established biofilms by Proteinase K. Strains tested are shown along the x-axis and grouped based on methicillin-sensitivity and isolation source. The indicated strains were grown statically for 24 hours to allow biofilm formation. Wells were washed and treated with buffer alone (- Prot. K) or 100 /ml Proteinase K (+ Prot. K) 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.ghigh levels of PNAG, while others produce little to no PNAG [60]. Additionally, some strains have been shown to be sensitive to biofilm dispersal by DspB whereas other S. aureus strains are unaffected by this enzyme [59] or the compound sodium metaperiodate, which breaks down PNAG via an oxidation reaction [60,89]. Our results show that DspB has little effect on both biofilm formation and dispersal in the S. aur.
