Lms that were much less Aprotinin supplement sensitive to dispersal by DNaseI (Figure 6 and Table 3). These data suggest that the role of eDNA in the development of late stage biofilms varies between S. aureus strains and LA-MRSA STs. Figure 7 shows the results after a 2-hour treatment of preformed biofilms with DspB. Treatment with DspB did notdisperse the biofilms formed by any of the S. aureus strains tested (Figure 7). In contrast, the S. epidermidis strains 1457 and NJ9709 formed biofilms that were highly sensitive to this enzyme and showed significant dispersal. The swine isolates showed no difference in sensitivity to DspB from the human isolates or laboratory strains. Consistent with previous findings, these results demonstrate a differential sensitivity to DspB in S. aureus compared to S. epidermidis.Gene expression in biofilmsTo further characterize the biofilms formed by the LA-MRSA strains, we performed quantitative real-time PCR (qPCR) using RNA isolated from mature biofilms. Specifically, we were interested in evaluating expression of genes potentially involved in the production of extracellular matrix components. The polysaccharide PNAG is the product of four enzymes encoded by the icaADBC operon; expression of this locus is highly regulated by numerous transcription factors, including the negative regulator IcaR [68]. The icaR gene is locatedPLOS ONE | www.plosone.orgSwine MRSA Isolates form Robust BiofilmsFigure 3. Inhibition of biofilm formation by DNaseI. Strains tested are shown along the x-axis and grouped based on methicillinsensitivity and isolation source. The indicated strains were grown statically for 24 hours in media alone (- DNaseI) or in media supplemented with 140 U/ml DNaseI (+ DNaseI). Biofilm formation was 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.gimmediately upstream of icaADBC, however it is divergently transcribed [69]. Extracellular nucleases, encoded by the nuc1 and nuc2 genes [70], have been proposed to impact the accumulation of extracellular DNA in the biofilm matrix [61,71,72]. Expression of genes involved in PNAG production (icaA, icaR) and extracellular nuclease (nuc1, nuc2) was measured in biofilms of the S. aureus strains and compared to expression in strain Pan-RAS-IN-1MedChemExpress Pan-RAS-IN-1 USA300 (Figure 8). No statistically significant difference in expression of icaA, icaR, nuc1, or nuc2 was seen across the panel of strains.Extracellular protease activityThere are 10 extracellular proteases produced by S. aureus, which have been proposed to act on microbial surface proteins as well as on host proteins [73,74]. We measured extracellular protease activity in the conditioned medium from biofilm and planktonic cultures, to determine if there were any differences between strains. As shown in Figure 9, in the majority ofstrains, little to no protease activity was detected in the biofilm culture medium. In contrast, for most strains, the planktonic culture medium had measurably higher levels of protease activity. The magnitude of this activity varied across the strains tested, and among the S. aureus strains, generally correlated with MLST type: the ST5, ST8 and ST9 strains had lower levels of protease activity than th.Lms that were much less sensitive to dispersal by DNaseI (Figure 6 and Table 3). These data suggest that the role of eDNA in the development of late stage biofilms varies between S. aureus strains and LA-MRSA STs. Figure 7 shows the results after a 2-hour treatment of preformed biofilms with DspB. Treatment with DspB did notdisperse the biofilms formed by any of the S. aureus strains tested (Figure 7). In contrast, the S. epidermidis strains 1457 and NJ9709 formed biofilms that were highly sensitive to this enzyme and showed significant dispersal. The swine isolates showed no difference in sensitivity to DspB from the human isolates or laboratory strains. Consistent with previous findings, these results demonstrate a differential sensitivity to DspB in S. aureus compared to S. epidermidis.Gene expression in biofilmsTo further characterize the biofilms formed by the LA-MRSA strains, we performed quantitative real-time PCR (qPCR) using RNA isolated from mature biofilms. Specifically, we were interested in evaluating expression of genes potentially involved in the production of extracellular matrix components. The polysaccharide PNAG is the product of four enzymes encoded by the icaADBC operon; expression of this locus is highly regulated by numerous transcription factors, including the negative regulator IcaR [68]. The icaR gene is locatedPLOS ONE | www.plosone.orgSwine MRSA Isolates form Robust BiofilmsFigure 3. Inhibition of biofilm formation by DNaseI. Strains tested are shown along the x-axis and grouped based on methicillinsensitivity and isolation source. The indicated strains were grown statically for 24 hours in media alone (- DNaseI) or in media supplemented with 140 U/ml DNaseI (+ DNaseI). Biofilm formation was 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.gimmediately upstream of icaADBC, however it is divergently transcribed [69]. Extracellular nucleases, encoded by the nuc1 and nuc2 genes [70], have been proposed to impact the accumulation of extracellular DNA in the biofilm matrix [61,71,72]. Expression of genes involved in PNAG production (icaA, icaR) and extracellular nuclease (nuc1, nuc2) was measured in biofilms of the S. aureus strains and compared to expression in strain USA300 (Figure 8). No statistically significant difference in expression of icaA, icaR, nuc1, or nuc2 was seen across the panel of strains.Extracellular protease activityThere are 10 extracellular proteases produced by S. aureus, which have been proposed to act on microbial surface proteins as well as on host proteins [73,74]. We measured extracellular protease activity in the conditioned medium from biofilm and planktonic cultures, to determine if there were any differences between strains. As shown in Figure 9, in the majority ofstrains, little to no protease activity was detected in the biofilm culture medium. In contrast, for most strains, the planktonic culture medium had measurably higher levels of protease activity. The magnitude of this activity varied across the strains tested, and among the S. aureus strains, generally correlated with MLST type: the ST5, ST8 and ST9 strains had lower levels of protease activity than th.