Evaluation of chlorogenic acid and carnosol for anti-efflux pump and anti-biofilm activities against extensively drug-resistant strains of Staphylococcus aureus and Pseudomonas aeruginosa.

Efflux pumps and biofilm play significant roles in bacterial antibiotic resistance. This study investigates the potential of chlorogenic acid (CGA) and carnosol (CL), as phenolic and diterpene compounds, respectively, for their inhibitory effects on efflux pumps. Among the 12 multidrug-resistant (MDR) strains of Staphylococcus aureus and Pseudomonas aeruginosa isolated from nosocomial skin infections, eight strains were identified as extensively drug resistant (XDR) using the disc diffusion method. The presence of efflux pumps in MDR strains of S. aureus and P. aeruginosa was screened using carbonyl cyanide-m-chlorophenylhydrazone. Between the 12 MDR strains of S. aureus and P. aeruginosa, 80% (4 out of 5) of the S. aureus strains and 85.7% (6 out of 7) of the P. aeruginosa strains exhibited active efflux pumps associated with gentamicin resistance. The checkerboard assay results, in combination with gentamicin, demonstrated that CGA exhibited a reduction in the minimum inhibitory concentration (MIC) for XDR S. aureus strain. Similarly, CL showed a synergistic effect and reduced the MIC for both XDR strains of S. aureus and P. aeruginosa. Flow cytometry was used to examine efflux pump activity at sub-MIC concentrations of 1/8, 1/4, and 1/2 MIC in comparison to the control. In XDR S. aureus, CGA demonstrated 39%, 70%, and 19% inhibition, while CL exhibited 74%, 73.5%, and 62% suppression. In XDR P. aeruginosa, CL exhibited inhibition rates of 25%, 10%, and 15%. The inhibition of biofilm formation was assessed using the microtiter plate method, resulting in successful inhibition of biofilm formation. Finally, the MTT assay was conducted, and it confirmed minimal cytotoxicity. Given the significant reduction in efflux pump activity and biofilm formation observed with CGA and CL in this study, these compounds can be considered as potential inhibitors of efflux pumps and biofilm formation, offering potential strategies to overcome antimicrobial resistance.

Importance: In summary, CGA and CL demonstrated promising potentiating antimicrobial effects against XDR strains of Staphylococcus aureus and Pseudomonas aeruginosa, suggesting their probably potential as candidates for addressing nosocomial pathogens. They exhibited significant suppression of efflux pump activity, indicating a possible successful inhibition of this mechanism. Moreover, all substances effectively inhibited biofilm formation, while showing minimal cytotoxicity. However, further advancement to clinical trials is needed to evaluate the feasibility of utilizing CGA and CL for reversing bacterial XDR efflux and determining their efficacy against biofilms. These trials will provide valuable insights into the practical applications of these compounds in combating drug-resistant infections.