Impact of sarA on antibiotic susceptibility of Staphylococcus aureus in a catheter-associated in vitro model of biofilm formation

Abstract

Mutation of the staphylococcal accessory regulator (sarA) in Staphylococcus aureus limits but does not abolish the capacity of the organism to form a biofilm. As a first step toward determining whether this limitation is therapeutically relevant, we carried out in vitro studies comparing the relative susceptibility of an S. aureus clinical isolate (UAMS-1) and its isogenic sarA mutant (UAMS-929) in the specific context of a catheter-associated biofilm. The antibiotics tested were daptomycin, linezolid, and vancomycin, all of which were evaluated by using concentrations based on the MIC defined as the breakpoint for a susceptible strain of S. aureus (< or = 1.0, < or = 2.0, and < or = 4.0 microg/ml for daptomycin, vancomycin, and linezolid, respectively). Mutation of sarA had no significant impact on the MIC of UAMS-1 for any of the targeted antibiotics, as defined by Etest antimicrobial susceptibility testing. However, mutation of sarA did result in a significant increase in antimicrobial susceptibility to all targeted antibiotics when they were tested in the specific context of a biofilm. Additionally, whether susceptibility was assessed by using UAMS-1 or its sarA mutant, daptomycin was found to be more effective against established S. aureus biofilms than either linezolid or vancomycin.

FIG. 1.

Impact of sarA on biofilm formation. Catheters colonized with UAMS-1 (white bars) or its isogenic sarA mutant (gray bars) were harvested at daily intervals. Biofilm formation was assessed as described in the text. Horizontal lines indicate the median from each group. Each box defines the interquartile range between the 25th and 75th percentiles. Bars extending upward from each box indicate the boundary defined by values 1.5 times higher than the 75th percentile, while those extending downward represent the boundary defined by values 1.5 times lower than the 25th percentile. Open circles represent individual data points outside these two extremes. The numbers are P values obtained in comparisons of the count data for catheters colonized with UAMS-1 or its sarA mutant on each day in the absence of antibiotic exposure.

FIG. 2.

Impact of sarA on overall antibiotic susceptibility. Graphs illustrate the overall distribution of count data obtained with individual catheters colonized with UAMS-1 (wild type [WT]) or its sarA mutant and exposed to daptomycin (A), vancomycin (B), or linezolid (C), irrespective of the antibiotic concentration or the time of exposure. The fact that all curves are shifted to the left demonstrates that the sarA mutant was more susceptible to all three antibiotics at all concentrations (5×, 10×, and 20×) and at all time points tested (1, 2, and 3 days).

FIG. 3.

Concentration- and time-dependent impacts of sarA on antibiotic susceptibility. The results illustrate the count data obtained with daptomycin (A), vancomycin (B), and linezolid (C) as a function of the antibiotic concentration and the time of exposure with UAMS-1 (white bars) and its isogenic sarA mutant (gray bars). Each box defines the interquartile range between the 25th and 75th percentiles. Bars extending upward from each box indicate the boundary defined by values 1.5 times higher than the 75th percentile, while those extending downward represent the boundary defined by values 1.5 times lower than the 25th percentile. Open circles represent individual data points outside these two extremes. Statistical analysis confirmed that the sarA mutant was more susceptible than the parent strain to all three antibiotics to an extent that cannot be fully explained by the reduced capacity of the sarA mutant to form a biofilm (see the text). d1, d2, and d3, days 1, 2, and 3, respectively.

FIG. 4.

Relative overall efficacy of individual antibiotics. Graphs illustrate the overall distribution of the count data obtained with individual catheters colonized with UAMS-1 (A) or its sarA mutant (B) and exposed to daptomycin (Dap), vancomycin (Van), or linezolid (Lin), irrespective of the antibiotic concentration or the time of exposure. The fact that curves obtained with daptomycin are shifted to the left by comparison to the curves obtained with both vancomycin and linezolid is indicative of the greater efficacy of daptomycin in the context of biofilms formed by both UAMS-1 and its sarA mutant.

FIG. 5.

Time- and concentration-dependent effects with the wild-type strain. The results illustrate the count data obtained when catheters colonized with UAMS-1 were exposed to each antibiotic at each concentration over time. Each box defines the interquartile range between the 25th and 75th percentiles. Bars extending upward from each box indicate the boundary defined by values 1.5 times higher than the 75th percentile, while those extending downward represent the boundary defined by values 1.5 times lower than the 25th percentile. Open circles represent individual data points outside these two extremes. The numbers in each box are P values based on pairwise comparisons between different antibiotics at each concentration and each time point. Dap, daptomycin; Van, vancomycin; Lin, linezolid.

FIG. 6.

Comparisons across antibiotic concentrations with the wild-type strain. The count data obtained on days 1 and 2 for catheters colonized with UAMS-1 were exposed to daptomycin at a 10× concentration (Dap10) were compared with those for catheters exposed to linezolid or vancomycin at a 20× concentration (Lin20 and Van20, respectively). Each box defines the interquartile range between the 25th and 75th percentiles. Bars extending upward from each box indicate the boundary defined by values 1.5 times higher than the 75th percentile, while those extending downward represent the boundary defined by values 1.5 times lower than the 25th percentile. Open circles represent individual data points outside these two extremes. The numbers above each bar are P values obtained for comparisons made between daptomycin and each of the other antibiotics.

FIG. 7.

Time- and concentration-dependent effects with the sarA mutant. The results illustrate the count data obtained when catheters colonized with the UAMS-1 sarA mutant were exposed to each antibiotic at each concentration over time. Each box defines the interquartile range between the 25th and 75th percentiles. Bars extending upward from each box indicate the boundary defined by values 1.5 times higher than the 75th percentile, while those extending downward represent the boundary defined by values 1.5 times lower than the 25th percentile. Open circles represent individual data points outside these two extremes. The numbers in each box are P values obtained from pairwise comparisons between different antibiotics at each concentration and each time point. Dap, daptomycin; Van, vancomycin; Lin, linezolid.

FIG. 8.

Comparisons across antibiotic concentrations with the sarA mutant. The count data obtained on day 1 when catheters colonized with the UAMS-1 sarA mutant were exposed to daptomycin at a 5× concentration (Dap5) were compared with those for catheters exposed to linezolid at a 10× or a 20× concentration (Lin10 and Lin20, respectively) or vancomycin at a 10× or a 20× concentration (Van10 and Van20, respectively). Each box defines the interquartile range between the 25th and 75th percentiles. Bars extending upward from each box indicate the boundary defined by values 1.5 times higher than the 75th percentile, while those extending downward represent the boundary defined by values 1.5 times lower than the 25th percentile. Open circles represent individual data points outside these two extremes. The numbers above each bar are P values for comparisons made between daptomycin and each of the other antibiotics.