Role and regulation of membrane efflux pumps in Staphylococcus pseudintermedius clinical isolates

The overexpression of efflux pumps (EPs) is one of the strategies through which Staphylococcus spp. survive antibiotic treatment. This process is regulated by intricate and still not fully understood mechanisms. This study aimed to evaluate the efflux capability of Staphylococcus pseudintermedius clinical isolates, investigate its implications in multidrug resistance, and elucidate the molecular mechanisms underlying EP expression. Additionally, potential therapeutic approaches for inhibiting staphylococcal EPs were assessed. Phenotypic characterization of efflux activity included ethidium bromide (EtBr) MIC screening, fluorometry-based accumulation and efflux assays, and synergy tests with EP inhibitors (EPIs). The genetic basis of high efflux phenotypes was explored using WGS and qPCR. Based on EtBr MIC, nine out of 109 isolates (8.3 %) progressed to fluorometry tests, confirming high phenotypic efflux in six isolates (Spearman rs= 0.886). These were classified into distinct multilocus sequence types (ST71, ST496, ST551 and ST1062), with two isolates representing novel STs, designated ST2837 and ST2838. Synergism was observed when combining an EPI with EtBr, ciprofloxacin, clindamycin, and chloramphenicol; however, no EPI restored full susceptibility to these antibiotics. All isolates contained at least one plasmid-encoded EP gene, with qacG being the most common (6/6), followed by qacH (3/6), tetK (3/6), smr (2/6), qacJ (1/6), and msrA/B (1/6). Variable expression of EP genes was noted, with MFS-JC286_RS07290 overexpressed in all isolates, while MFS-JC286_RS11100 and MFS-JC286_RS11015 showed the highest expression levels. Comparative alignments suggested that the observed high efflux is unlikely due to mutational events upstream. These findings imply a potential role for transcriptional regulators, highlighting new research directions in this area.