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, 3 (4), e00198-12

Low Levels of β-Lactam Antibiotics Induce Extracellular DNA Release and Biofilm Formation in Staphylococcus Aureus

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Low Levels of β-Lactam Antibiotics Induce Extracellular DNA Release and Biofilm Formation in Staphylococcus Aureus

Jeffrey B Kaplan et al. mBio.

Abstract

Subminimal inhibitory concentrations of antibiotics have been shown to induce bacterial biofilm formation. Few studies have investigated antibiotic-induced biofilm formation in Staphylococcus aureus, an important human pathogen. Our goal was to measure S. aureus biofilm formation in the presence of low levels of β-lactam antibiotics. Fifteen phylogenetically diverse methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) strains were employed. Methicillin, ampicillin, amoxicillin, and cloxacillin were added to cultures at concentrations ranging from 0× to 1× MIC. Biofilm formation was measured in 96-well microtiter plates using a crystal violet binding assay. Autoaggregation was measured using a visual test tube settling assay. Extracellular DNA was quantitated using agarose gel electrophoresis. All four antibiotics induced biofilm formation in some strains. The amount of biofilm induction was as high as 10-fold and was inversely proportional to the amount of biofilm produced by the strain in the absence of antibiotics. MRSA strains of lineages USA300, USA400, and USA500 exhibited the highest levels of methicillin-induced biofilm induction. Biofilm formation induced by low-level methicillin was inhibited by DNase. Low-level methicillin also induced DNase-sensitive autoaggregation and extracellular DNA release. The biofilm induction phenotype was absent in a strain deficient in autolysin (atl). Our findings demonstrate that subminimal inhibitory concentrations of β-lactam antibiotics significantly induce autolysin-dependent extracellular DNA release and biofilm formation in some strains of S. aureus.

Importance: The widespread use of antibiotics as growth promoters in agriculture may expose bacteria to low levels of the drugs. The aim of this study was to investigate the effects of low levels of antibiotics on bacterial autoaggregation and biofilm formation, two processes that have been shown to foster genetic exchange and antibiotic resistance. We found that low levels of β-lactam antibiotics, a class commonly used in both clinical and agricultural settings, caused significant autoaggregation and biofilm formation by the important human pathogen Staphylococcus aureus. Both processes were dependent on cell lysis and release of DNA into the environment. The effect was most pronounced among multidrug-resistant strains known as methicillin-resistant S. aureus (MRSA). These results may shed light on the recalcitrance of some bacterial infections to antibiotic treatment in clinical settings and the evolution of antibiotic-resistant bacteria in agricultural settings.

Figures

FIG 1
FIG 1
Bacterial growth and biofilm formation by three MRSA strains (strains 11490, Mu50, and FPR3757) in the presence of sub-MICs of four β-lactam antibiotics. Bacterial growth (A490) is indicated on the left-hand y axes, biofilm formation (A595) is indicated on the right-hand y axes, and antibiotic concentration is indicated on the x axes. Values show average absorbance values for duplicate wells. Error bars were omitted for clarity.
FIG 2
FIG 2
Biofilm formation by S. aureus strains FPR3757 (USA300) and 11490 (USA500) in the presence of sub-MICs of methicillin. (A) Photographs of crystal violet-stained microtiter plate wells. Duplicate wells are shown. (B) Average biofilm CFU/well values for biofilms cultured in 0 or 2 µg/ml methicillin. Error bars indicate ranges. Values that were significantly different (P < 0.05) from those of the no-methicillin control are indicated by an asterisk.
FIG 3
FIG 3
Effect of rhDNase on biofilm formation by S. aureus in sub-MICs of methicillin. Strains FPR3757 (USA300), 11490 (USA500), and 383 were cultured in broth supplemented with 10 µg/ml rhDNase (open circles) or no enzyme (filled circles). Values show average absorbance values for duplicate wells. Error bars were omitted for clarity.
FIG 4
FIG 4
Sub-MICs of methicillin induce autoaggregation in S. aureus. Strains FPR3757 (USA300) and 11490 (USA500) were cultured for 18 h in broth supplemented with 0 or 2 µg/ml methicillin and then treated with 0 or 10 µg/ml rhDNase. The cells were rinsed with saline and transferred to a microcentrifuge tube. The tubes were incubated statically for 10 min and then photographed.
FIG 5
FIG 5
Sub-MICs of methicillin induce eDNA release in S. aureus. Biofilms of strains FPR3757 (USA300) and 11490 (USA500) were cultured for 18 h in broth supplemented with increasing concentrations of methicillin. Biofilm supernatants were analyzed by agarose gel electrophoresis. The sizes (in kilobases) of DNA molecular size markers electrophoresed in an adjacent lane are shown to the right of the gels.
FIG 6
FIG 6
Biofilm formation by S. aureus strain LAC in sub-MICs of methicillin. (A) Biofilm formation by wild-type S. aureus strain LAC and isogenic nuc, agr, and atl mutant strains in sub-MICs of methicillin. (B) Biofilm formation by strain LAC in sub-MICs of methicillin in the presence or absence of 500 µg/ml of the autolysis inhibitor polyanethole sulfonate (PAS). Values in both panels show average absorbance for duplicate wells. Error bars were omitted for clarity.

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