Increased Activity of Efflux Pumps in Tigecycline-Resistant Staphylococcus aureus Selected in Vitro

HERRERA M; DI GREGORIO; FERNÁNDEZ S; POSSE G; MOLLERACH M; DI CONZA J

Denver

Conferencia; 53rd Interscience Conference on Antimicrobial Agents and Chemotherapy; 2013

American Society for Microbiology

Background: Staphylococcus aureus is able to develop resistance under selective pressure to different antibiotics and efflux activity appears to be one of the mechanisms involved in the acquired resistance. The aim of this study was the in vitro selection of S. aureus mutants with reduced susceptibility to tigecycline (TIG) and the evaluation of the efflux activity as a possible resistance mechanism in these mutants. Methods: Twenty unrelated S. aureus clinical isolates were studied (10 MRSA and 10 MSSA). The in vitro selection of mutants was performed by serial passage in increasing concentrations of TIG. Susceptibility testing to different antibiotics was assayed following CLSI recommendations. TIG MIC was determined by epsilometric method considering FDA breakpoints. The clonality between the parental and derived mutants was confirmed by PFGE. Efflux activity was evaluated by determining the MICs of TIG and ethidium bromide (EB) in presence and absence of reserpine (RS) and PaβN (20 µg /ml). An EB MIC ≥ 32 µg/ml coupled with a RS or PaβN mediated MIC reduction of at least 4 twofold dilutions (TFD) was considered indicative of efflux activity enhancement. Results: Twelve TIG-resistant mutants were obtained, which showed an increase in MICs values between 32 to 128 fold (MIC range: 1-16 µg/ml) higher than its parental strain. Two of these mutants also showed a modification in susceptibility to other antibiotics. In one of them, MIC of oxacillin decreased 8 fold while the other strain increased 4 fold the MIC values of both vancomycin and oxacillin. The MIC range of EB was 32-128 µg/ml. A decrease of ≥ 4 TFD in TIG MIC in presence of RS was observed in 10/12 mutants, moreover, 9/10 these mutants exhibited the same behavior with EB. The remaining mutants decreased 3 TFD the MIC values for both agents. The MICs of oxacillin and vancomycin in the two co-selected mutants were not affected by the presence of inhibitors. Conclusion: this study demonstrates the ability of S.aureus to develop resistance to tigecycline under selective pressure, being the increase of efflux activity one of the possible resistance mechanisms involved. The acquisition of this resistance may eventually be associated by changes in susceptibility to other antibiotics such oxacillin and vancomycin.