Building up efflux-mediated antimicrobial resistance in Staphylococcus pseudintermedius

Last of the resistance mechanisms to be identified, the overexpression of membrane efflux pumps (EPs) allows bacteria to extrude noxious compounds, including antimicrobial agents, influencing multiple stages of resistance development [1]. The study of EPs in Staphylococcus pseudintermedius has been neglected to date. This microorganism is among the most relevant antimicrobial-resistant bacteria worldwide due to its frequent implication in clinical diseases of dogs and cats, increasing adaptation to the human organism, and the high levels of resistance to clinically relevant antibiotics [2, 3]. Here, we performed an in silico prediction of drug EPs in S. pseudintermedius, and we investigated their role in conferring resistance to antibiotic and biocidal agents and biofilm formation. A S. pseudintermedius efflux mutant was obtained by stimulating the isogenic strain ATCC 49444 with increasing concentrations of the efflux system substrate ethidium bromide (EtBr). The efflux activity of S. pseudintermedius ATCC 49444 and its derivative was quantified by fluorometry. Changes in antimicrobial susceptibility were evaluated in the presence/absence of the EP inhibitors thioridazine (TZ) and reserpine (RES) by checkerboard assays. The biofilm-forming capability of the original strain and its derivative and the biofilm inhibition ability of TZ and RES were evaluated by microtiter-plate test. Homologues of EPs of Staphylococcus aureus and Staphylococcus epidermidis were searched by exploratory GenBank investigations. Gene expression analyses and sequencing were then conducted on selected genes. S. pseudintermedius efflux mutant showed a higher efflux activity than the parent strain and increased resistance to gentamicin, 2% chlorhexidine digluconate and ciprofloxacin, which is the major active metabolite of the veterinary fluoroquinolone enrofloxacin [4]. On the contrary, enrofloxacin efficacy was only slightly affected by the augmented efflux. TZ restored the bacterial susceptibility to gentamicin and ciprofloxacin. Biofilm production was greatly increased in S. pseudintermedius efflux mutant and it was significantly inhibited by TZ and RES at MIC/2 (p=0.04). EtBr pressure induced a transient overexpression of multiple transporters of the Major Facilitator Superfamily (MFS). However, only the multidrug EP gene norA remained highly expressed in S. pseudintermedius efflux mutant, which had an 11 bp-deletion in norA promoter region. icaA gene, encoding for extracellular polymeric substances of staphylococcal biofilm, was up-regulated as well. How the overexpression of MFS EPs influences biofilm production at the transcript level is not clear. However, the physiological role of MFS EPs, particularly NorA, appears to be far more complex than merely that of an antibiotic export protein. Combinations composed of an antibiotic EP substrate and an EP inhibitor might be an attractive strategy to combat staphylococcal infections in the context of veterinary and human medicine