Exploring Efflux as a Mechanism of Reduced Susceptibility towards Biocides and Fluoroquinolones in Staphylococcus pseudintermedius

SIMPLE SUMMARY: Staphylococcus pseudintermedius is the main bacterial agent of skin and soft tissue infections in companion animals. The rising antimicrobial resistance in this species is a public health concern. Efflux activity is a resistance mechanism poorly characterized for this bacterium. This study aimed to evaluate efflux as contributor of biocide and fluoroquinolone resistance in S. pseudintermedius. Determination and application of cut-off values detected a non-wild type population against the biocide tetraphenylphosphonium bromide, linked to increased efflux activity. Further characterization of this efflux activity demonstrated that it is strain-specific and glucose-dependent. Fluoroquinolone resistance was mainly related to target mutations, which may be masking the contribution of efflux. This study highlights the relevance of efflux-mediated resistance in S. pseudintermedius, particularly to biocides, and provides a methodological basis for further studies on the efflux activity on this important veterinary pathogen. ABSTRACT: Staphylococcus pseudintermedius is the main bacterial cause of skin and soft tissue infections (SSTIs) in companion animals, particularly dogs. The emergence of methicillin-resistant S. pseudintermedius (MRSP) strains, frequently with multidrug resistance phenotypes is a public health concern. This study aimed to evaluate efflux, a resistance mechanism still poorly characterized in S. pseudintermedius, as a contributor to biocide and fluoroquinolone resistance. Susceptibility to the efflux pump substrates ethidium bromide (EtBr), tetraphenylphosphonium bromide (TPP) and ciprofloxacin (CIP) was evaluated by minimum inhibitory concentration (MIC) determination for 155 SSTIs-related S. pseudintermedius in companion animals. EtBr and TPP MIC distributions were analyzed to estimate cut-off (CO(WT)) values. The effect of the efflux inhibitors (EIs) thioridazine and verapamil was assessed upon MICs and fluorometric EtBr accumulation assays, performed with/without glucose and/or EIs. This approach detected a non-wild type population towards TPP with increased efflux, showed to be strain-specific and glucose-dependent. Resistance to fluoroquinolones was mainly linked to target gene mutations, yet a contribution of efflux on CIP resistance levels could not be ruled out. In sum, this study highlights the relevance of efflux-mediated resistance in clinical S. pseudintermedius, particularly to biocides, and provides a methodological basis for further studies on the efflux activity on this important pathogen of companion animals.