Adaptation of Staphylococcus aureus in a Medium Mimicking a Diabetic Foot Environment

Abstract

Staphylococcus aureus is the most prevalent pathogen isolated from diabetic foot infections (DFIs). The purpose of this study was to evaluate its behavior in an in vitro model mimicking the conditions encountered in DFI. Four clinical S. aureus strains were cultivated for 16 weeks in a specific environment based on the wound-like medium biofilm model. The adaptation of isolates was evaluated as follows: by Caenorhabditis elegans model (to evaluate virulence); by quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) (to evaluate expression of the main virulence genes); and by Biofilm Ring test^® (to assess the biofilm formation). After 16 weeks, the four S. aureus had adapted their metabolism, with the development of small colony variants and the loss of β-hemolysin expression. The in vivo nematode model suggested a decrease of virulence, confirmed by qRT-PCRs, showing a significant decrease of expression of the main staphylococcal virulence genes tested, notably the toxin-encoding genes. An increased expression of genes involved in adhesion and biofilm was noted. Our data based on an in vitro model confirm the impact of environment on the adaptation switch of S. aureus to prolonged stress environmental conditions. These results contribute to explore and characterize the virulence of S. aureus in chronic wounds.

Keywords: EDIN; Panton–Valentin leukocidin; Staphylococcus aureus; adaptation; biofilm; diabetic foot infection; in vitro model; nematode; virulence.

Figure 1

Effects of a pre-culture in a wound-like medium (WLM) and 10% glucose combined with sub-minimum inhibitory concentration (MIC) of vancomycin (0.5× MIC) and linezolid (0.5× MIC) on S. aureus biofilm formation after 16 weeks of culture. The kinetics of the early phase of biofilm formation were determined on (A) NSA739; (B) NSA1077; (C) NSA7475; and (D) NSA1385 by the BioFilm ring test^® (BioFilm Control, France). The control corresponds to the evaluation of biofilm formation of strains in Brain Heart Infusion (BHI) medium alone. Dotted horizontal lines: >7, no biofilm; <2, fixed biofilm. Means ± standard errors of the mean of biofilm indexes (BFIs) for at least three independent replicates are presented. Statistical differences between the different culture conditions at each time were obtained using two-way analysis of variance (ANOVA), followed by Dunnett’s multiple-comparison test.

Figure 1

Effects of a pre-culture in a wound-like medium (WLM) and 10% glucose combined with sub-minimum inhibitory concentration (MIC) of vancomycin (0.5× MIC) and linezolid (0.5× MIC) on S. aureus biofilm formation after 16 weeks of culture. The kinetics of the early phase of biofilm formation were determined on (A) NSA739; (B) NSA1077; (C) NSA7475; and (D) NSA1385 by the BioFilm ring test^® (BioFilm Control, France). The control corresponds to the evaluation of biofilm formation of strains in Brain Heart Infusion (BHI) medium alone. Dotted horizontal lines: >7, no biofilm; <2, fixed biofilm. Means ± standard errors of the mean of biofilm indexes (BFIs) for at least three independent replicates are presented. Statistical differences between the different culture conditions at each time were obtained using two-way analysis of variance (ANOVA), followed by Dunnett’s multiple-comparison test.

Figure 2

Relative mRNA expression levels of toxinogenic genes of S. aureus NSA1077 strain cultivated in WLM supplemented with 10% glucose, vancomycin (0.5× MIC), linezolid (0.5× MIC), 10% glucose + vancomycin (0.5× MIC), and 10% glucose + linezolid (0.5× MIC) after 24 h (H24) and 16 weeks (W16). The log-transformed averages of relative fold changes of NSA1077 in different environmental media compared with NSA1077 in Luria-Bertani (LB) medium for 24 h and 16 weeks are presented. The error bars represent the standard deviations from the three independent RNA preparations. Significant differences from the NSA1077 in LB medium for 24 h and 16 weeks using Dunnett’s test are indicated. *, p < 0.01; ***, p < 0.001.

Figure 3

Relative mRNA expression levels of virulence genes and a regulator gene of S. aureus NSA739 strain cultivated in WLM supplemented with 10% glucose, vancomycin (0.5× MIC), linezolid (0.5× MIC), 10% glucose + vancomycin (0.5× MIC), and 10% glucose + linezolid (0.5× MIC) after a long exposure (16 weeks). The log-transformed averages of relative fold changes of NSA739 in different environmental media compared with NSA739 in LB medium are presented. The error bars represent the standard deviations from the three independent RNA preparations. Significant differences from the NSA739 in LB medium using Dunnett’s test are indicated. ***, p < 0.001.