Department of Veterinary Medicine, University of Perugia, via San Costanzo 4, Perugia 06126, Italy.
Department of Pharmaceutical Sciences, University of Perugia, via del Liceo 1, Perugia 06123, Italy.
J Antimicrob Chemother. 2022 Nov 28;77(12):3283-3290. doi: 10.1093/jac/dkac314.
To perform an in silico prediction of drug efflux pumps (EPs) in Staphylococcus pseudintermedius and investigate their role in conferring resistance to antibiotic and biocidal agents and biofilm formation.
A S. pseudintermedius efflux mutant was obtained by stimulating an isogenic line (ATCC 49444) with increasing concentrations of an efflux system substrate. Changes in antimicrobial susceptibility and biofilm-forming capability were evaluated in the presence/absence of the EP inhibitors (EPIs) thioridazine and reserpine and the efflux activity was assayed by fluorometry. 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.
Susceptibility to chlorhexidine, gentamicin and ciprofloxacin, but not enrofloxacin, was affected by the increased efflux and it was variably restored by the EPIs. The efflux mutant showed much greater biofilm formation that the original strain, which was significantly inhibited by thioridazine and reserpine at MIC/2. A high expression of norA, which was mgrA-independent, was found in the S. pseudintermedius efflux mutant, apparently regulated by an 11 bp deletion in its promoter region, whilst lmrB was transitorily overexpressed. icaA, which encodes the polysaccharide intercellular adhesin forming the extracellular matrix of staphylococcal biofilm, was also up-regulated.
EPs, particularly NorA, are supposed to have complex involvement in multiple stages of resistance development. Overexpression of EPs appears to be correlated with a remarkable increase of S. pseudintermedius biofilm production; however, the regulatory mechanisms remain to be explored.
对中间葡萄球菌中的药物外排泵（EPs）进行计算机预测，并研究其在赋予抗生素和杀生物剂耐药性以及生物膜形成中的作用。
通过用递增浓度的外排系统底物刺激同源系（ATCC 49444）获得中间葡萄球菌外排突变体。在存在/不存在外排泵抑制剂（EPIs）硫利哒嗪和利血平的情况下，评估抗菌药物敏感性和生物膜形成能力的变化，并通过荧光法测定外排活性。通过探索性 GenBank 调查搜索金黄色葡萄球菌和表皮葡萄球菌的 EP 同源物。然后对选定的基因进行基因表达分析和测序。
对氯己定、庆大霉素和环丙沙星的敏感性受到增加的外排的影响，但对恩诺沙星没有影响，并且 EPIs 可使其耐药性恢复。与原始菌株相比，外排突变体的生物膜形成能力更强，而噻氯哒嗪和利血平在 MIC/2 时可显著抑制其生物膜形成。发现中间葡萄球菌外排突变体中存在高表达的 norA，该基因独立于 mgrA，显然受到其启动子区域 11bp 缺失的调节，而 lmrB 则短暂过表达。icaA 编码形成葡萄球菌生物膜细胞外基质的多糖细胞间黏附素，也被上调。
EPs，特别是 NorA，可能在多种耐药性发展阶段中具有复杂的作用。EPs 的过度表达似乎与中间葡萄球菌生物膜产生的显著增加相关；然而，调控机制仍有待探索。
