The Controversial Effect of Antibiotics on Methicillin-Sensitive S. aureus: A Comparative In Vitro Study

Abstract

Methicillin-sensitive Staphylococcus (S.) aureus (MSSA) bacteremia remains a global challenge, despite the availability of antibiotics. Primary treatments include β-lactam agents such as cefazolin and flucloxacillin. Ongoing discussions have focused on the potential synergistic effects of combining these agents with rifampicin or fosfomycin to combat infections associated with biofilm formation. Managing staphylococcal infections is challenging due to antibacterial resistance, biofilms, and S. aureus's ability to invade and replicate within host cells. Intracellular invasion shields the bacteria from antibacterial agents and the immune system, often leading to incomplete bacterial clearance and chronic infections. Additionally, S. aureus can assume a dormant phenotype, known as the small colony variant (SCV), further complicating eradication and promoting persistence. This study investigated the impact of antibiotic combinations on the persistence of S. aureus 6850 and its stable small colony variant (SCV strain JB1) focusing on intracellular survival and biofilm formation. The results from the wild-type strain 6850 demonstrate that β-lactams combined with RIF effectively eliminated biofilms and intracellular bacteria but tend to select for SCVs in planktonic culture and host cells. Higher antibiotic concentrations were associated with an increase in the zeta potential of S. aureus, suggesting reduced membrane permeability to antimicrobials. When using the stable SCV mutant strain JB1, antibiotic combinations with rifampicin successfully cleared planktonic bacteria and biofilms but failed to eradicate intracellular bacteria. Given these findings, it is reasonable to report that β-lactams combined with rifampicin represent the optimal treatment for MSSA bacteremia. However, caution is warranted when employing this treatment over an extended period, as it may elevate the risk of selecting for small colony variants (SCVs) and, consequently, promoting bacterial persistence.

Keywords: Staphylococcus aureus; bacteremia; bacterial persistence; cefazolin; flucloxacillin; fosfomycin; rifampicin; small colony variants.

Figure 1

Efficacy of antibiotic monotherapy or combinations for the treatment of intracellular S. aureus 6850 (A) and JB1 (B), expressed as mean log CFU versus untreated control ± SD. Statistical analyses were performed using two-way ANOVA followed by Dunnett’s multiple comparison test. The significance levels obtained in the post hoc analyses are indicated by asterisks. According to the results of the analyses, the differences are either not significant (p > 0.05) or significant (* p < 0.05; ** p < 0.01). Results are from 5 independent experiments.

Figure 2

Ratio of small colony variants to wild-type colonies (SCV/WT) shown as mean ± SD of S. aureus 6850 in planktonic culture (A) and intracellular (B). No SCVs were found in the controls (=0). Statistical analyses were performed using two-way ANOVA followed by Dunnett’s multiple comparison test with respect to the untreated control. Significance levels of post hoc analyses are indicated by asterisks. Significance levels of post-hoc analyses are indicated by asterisks. According to the results of the analyses, the differences are either significant (p > 0.05) or not significant (* p < 0.05; ** p < 0.01; and **** p < 0.0001). The results were obtained from 5 independent experiments.

Figure 3

Biofilm inhibition reported as the mean for S. aureus 6850 (A) and JB1 (B) relative to the untreated control (OD_{(treated sample)} × 100/OD_{(untreated sample)}). The untreated control was set to 100%. Statistical analyses were performed using two-way ANOVA followed by Dunnett’s multiple comparison test. The significance of the post-hoc analyses is indicated by asterisks when the biofilm mass was reduced and by hash marks when it was induced. According to the results of the analyses, the differences are either not significant (p > 0.05) and **** p < 0.0001). Results are from 5 independent experiments.

Figure 4

Biofilm eradication for S. aureus 6850 (A) and JB1 (B) expressed as the mean of the OD relative to the untreated control ± SD (OD_{(treated sample)} × 100/OD_{(untreated sample)}). All values represent at least five independent experiments (n = 5). The untreated control was set to 100%. Statistical analyses were performed using two-way ANOVA followed by Dunnett’s multiple comparison test. Significance levels of post-hoc analyses are indicated by asterisks when biofilm mass was reduced or hash marks when it was induced. According to the results of the analyses, the differences are either not significant (p > 0.05) or significant (* p < 0.05; ** p < 0.01; *** p < 0.001 and **** p < 0.0001).

Figure 5

Zeta potential in mV ± SD. A: Zeta potential of S. aureus wild-type strain 6850 and SCV strain JB1 without antibiotic pre-treatment. The SCV strain shows significantly less negative zeta potential than the wild-type strains. n = 3. B: Zeta potential of 6850 at 10 × MIC. All treatments resulted in a significant increase in zeta potential. n = 4. Each point in the graph represents an independent experiment. Statistical analyses were performed with unpaired t-test (A) or one-way ANOVA followed by Dunnett’s multiple comparison test (B). According to the results of the post-hoc analyses, the significance is indicated by asterisks. (** p < 0.01; *** p < 0.001; **** p < 0.0001).