Comparative evaluation of small molecules reported to be inhibitors of Staphylococcus aureus biofilm formation

Abstract

Because biofilm formation is such a problematic feature of Staphylococcus aureus infections, much effort has been put into identifying biofilm inhibitors. However, the results observed with these compounds are often reported in isolation, and the methods used to assess biofilm formation vary between labs, making it impossible to assess relative efficacy and prioritize among these putative inhibitors for further study. The studies we report address this issue by directly comparing putative biofilm inhibitors using a consistent in vitro assay. This assay was previously shown to maximize biofilm formation, and the results observed with this assay have been proven to be relevant in vivo. Of the 19 compounds compared using this method, many had no impact on biofilm formation under these conditions. Indeed, only one proved effective at limiting biofilm formation without also inhibiting growth.

Keywords: Staphylococcus aureus; antibiotic resistance; antimicrobial activity; antimicrobial agents; biofilms; infectious disease.

Fig 1

Inhibition of biofilm formation by celastrol requires concentrations that also inhibit growth. Biofilm formation and growth inhibition were assessed for UAMS-1 (A, B) and LAC (C, D) with the designated concentrations of celastrol. Biofilm formation is presented as the percent of biofilm relative to the dimethylsulfoxide (DMSO) control without celastrol. Significance was assessed by one-way analysis of variance (ANOVA) with Dunnett’s correction, where **** indicates P > 0.0001 against the DMSO control and #### indicates significance against the isogenic sarA mutant (ΔsarA). Growth in the presence of celastrol is presented as log₁₀(colony-forming units) [log(CFU)], where bars above the black line indicate growth above the log(CFU) at the time of inoculation (T0) and bars at or below the black line indicate a lack of growth.

Fig 2

Telithromycin can inhibit biofilm formation at sub–minimum inhibitory concentrations (MICs). Biofilm formation and growth inhibition were assessed for UAMS-1 (A, B) and LAC (C, D) with the designated concentrations of telithromycin. Biofilm formation is presented as the percent (%) of biofilm mass relative to the telithromycin untreated DMSO-only control. Significance was assessed by one-way ANOVA with Dunnett’s correction, where **** indicates P > 0.0001 against the DMSO control and #### indicates significance against the isogenic sarA mutant (ΔsarA). Growth in the presence of telithromycin is presented as log₁₀(colony-forming units) [log(CFU)], where bars above the black line indicate growth by comparison to the log(CFU) at T0 and bars at or below the black line indicate a lack of growth or reduction in cell viability.

Fig 3

Thermal shift assay with telithromycin and purified SarA. Glomelt dye was used to determine the thermal stability of SarA in the presence of telithromycin. Analysis of melting temperature (Tm) was performed using the Boltzmann method from a plot of fluorescence intensity versus temperature.

Fig 4

Impact of telithromycin on protease production. Overall protease activity was assessed using CM from overnight cultures of LAC and UAMS-1 with and without 0.49-µM telithromycin. CM from an overnight culture of the isogenic sarA mutant grown without telithromycin was included as a control. Results are shown after 1-h (left) and 24-h incubation of the EnzChek gelatinase/collagenase protease assay.

Fig 5

Impact of telithromycin on extracellular protein profiles. CM from duplicate cultures of UAMS-1 and LAC grown overnight with and without 0.49-µM telithromycin was examined by SDS-PAGE. MW indicates molecular size markers in kDa as shown.

Fig 6

Impact of telithromycin on autolysis activity. Autolysis was assessed with the indicated mutants and conditions for LAC. The results are representative of two independent biological replicates with at least three experimental replicates each and presented as the percent (%) of autolysis at 30-min intervals for 4 h.

Fig 7

Impact of telithromycin on an established biofilm. Biofilms were allowed to form under our standardized experimental conditions before removing media and non-adherent cells and adding media containing the indicated amount of telithromycin in DMSO. The parent strain assayed with media containing an equivalent concentration of DMSO without telithromycin and the isogenic sarA mutant assayed with media that did not contain DMSO or telithromycin were included as controls. Results are reported as the percent (%) of biofilm mass remaining relative to the parent strain.