Inhibition of staphylococcal biofilm formation by nitrite

Abstract

Several environmental stresses have been demonstrated to increase polysaccharide intercellular adhesin (PIA) synthesis and biofilm formation by the human pathogens Staphylococcus aureus and Staphylococcus epidermidis. In this study we characterized an adaptive response of S. aureus SA113 to nitrite-induced stress and show that it involves concomitant impairment of PIA synthesis and biofilm formation. Transcriptional analysis provided evidence that nitrite, either as the endogenous product of respiratory nitrate reduction or after external addition, causes repression of the icaADBC gene cluster, mediated likely by IcaR. Comparative microarray analysis revealed a global change in gene expression during growth in the presence of 5 mM sodium nitrite and indicated a response to oxidative and nitrosative stress. Many nitrite-induced genes are involved in DNA repair, detoxification of reactive oxygen and nitrogen species, and iron homeostasis. Moreover, preformed biofilms could be eradicated by the addition of nitrite, likely the result of the formation of toxic acidified nitrite derivatives. Nitrite-mediated inhibition of S. aureus biofilm formation was abrogated by the addition of nitric oxide (NO) scavengers, suggesting that NO is directly or indirectly involved. Nitrite also repressed biofilm formation of S. epidermidis RP62A.

FIG. 1.

Biofilm formation of S. aureus and S. epidermidis is impaired by sodium nitrate and sodium nitrite. Biofilm formation of S. aureus SA113 (upper panel) and S. epidermidis RP62A (lower panel) was assayed using the microtiter plate adherence assay. The cells were grown in the absence or initial presence of 1 to 5 mM sodium nitrate (NO₃⁻) or sodium nitrite (NO₂⁻). After 24 h, the residual nitrite concentrations in the culture supernatants were determined by colorimetric detection (24 h NO₂⁻). Adherent cells were stained with safranin.

FIG. 2.

Respiratory nitrate reduction resulting in the accumulation of nitrite is required to inhibit biofilm formation. (A) S. aureus wild type (WT) and its isogenic narG deletion (ΔnarG) mutant were grown under biofilm conditions in microtiter plate wells in the absence or presence of 2 to 10 mM sodium nitrate (NO₃⁻). After 24 h, the residual nitrite concentrations in the culture supernatants were determined (NO₂⁻ after 24 h). Biofilm formation of the S. aureus WT and the ΔnarG mutant in response to nitrate (NO₃⁻) (B) or nitrite (NO₂⁻) (C) was quantified by spectrophotometric measurement of the absorbance of adherent cells after safranin staining at 450 nm. Data represent the means ± standard errors of the means (n = 8) of one representative experiment.

FIG. 3.

Effect of nitrate and nitrite on icaA transcription and PIA synthesis. S. aureus wild type (WT) and its isogenic narG deletion (ΔnarG) mutant were grown under biofilm conditions in 6-well cell culture plate wells in the absence or presence of 20 mM sodium nitrate (NO₃⁻) (A) or 5 and 10 mM sodium nitrite (NO₂⁻) (B). Cells were harvested after 6 h, and total RNA was isolated. The relative abundance levels of icaA mRNAs normalized to gyrB signals were assessed by semiquantitative RT-PCR and densitometric analysis. The levels of icaA expression in the presence of nitrate or nitrite were compared with normalized levels obtained from their matched controls set to 100% (bars). (C) PIA was extracted from cells grown for 24 h under biofilm conditions in 6-well cell culture plates in the absence (−) or presence (+) of 5 mM sodium nitrite (NO₂⁻). PIA was detected by dot blot analysis using wheat germ agglutinin coupled to horseradish peroxidase. The PIA-deficient S. aureus SA113 ica (Δica) mutant was used as a negative control. PIA quantification using rabbit antiserum raised against S. epidermidis PIA yielded comparable results (not shown).

FIG. 4.

Phenotypic validation of microarray data by mapping physiological states of the cells. (A) Cells were grown in 6-well cell culture plates under biofilm conditions for 6 h. SDS-PAGE analysis of crude protein extracts followed by Coomassie blue staining showed induction of an approximately 35-kDa protein in response to 20 mM nitrate (NO₃⁻) or 10 mM nitrite (NO₂⁻) (arrowheads), which was identified by MS analysis as the class I fructose-bisphosphate aldolase FdaB. Crude protein extracts from cells grown under biofilm conditions for 6 h in the absence (Control) or presence of 5 mM nitrite (NO₂⁻) were assayed for fructose-bisphosphate aldolase (FBA)- and catalase-specific activities (panels B and C, respectively). Data represent the means of two independent experiments.

FIG. 5.

Nitrite toxicity toward S. aureus requires acidification. (A) S. aureus wild type (WT) and its isogenic narG deletion (ΔnarG) mutant were seeded onto TSB agar plates buffered at the indicated pH values. Five millimolar sodium nitrate (NO₃⁻) was included (+) or omitted (−) from the plates. After the placement of a filter disc soaked with 20 μl of a 2 M NaNO₂ solution, the plates were incubated under conditions of oxygen limitation for 48 h and monitored for growth inhibition zones (indicated by dotted circles). (B) Nitrite- and SNAP-mediated inhibition of biofilm formation are abrogated in the presence of NO scavengers. Biofilm formation of S. aureus SA113 in the absence (C, control) or presence of 5 mM nitrite (NO₂⁻) or the NO donor SNAP (0.1 mM) was quantified after 24 h by spectrophotometric measurement of the absorbance of adherent cells after safranin staining at 450 nm. The NO scavengers carboxy-PTIO (PTIO; 2.5 mM) and hemoglobin (Hb; 0.5 mM) were initially added as indicated or omitted (−). Data are means ± standard error of the means of two to four experiments performed at least in triplicate.