Drug-resistant bacteria are an increasingly serious threat to the aquaculture industry. In order to develop new methods to combat these bacteria, it is necessary to first understand their mechanisms of drug resistance. We previously isolated a potential probiotic strain, Streptomyces sp. NHF165, which produced cytotoxic actinonin against the aquaculture pathogen Vibrio anguillarum. In this study, a spontaneous V. anguillarum mutant with an eight-fold increased MIC for actinonin was characterized for elucidation of actinonin resistance mechanisms. Transcriptomic analysis revealed significant up-regulation of genes related to anaerobic respiratory (nap and tor) and down-regulation of genes involved in anaerobic respiration (nqr, sdh and frd) in mutant strains. Meanwhile, no mutations in bacterial respiration genes or translation initiation genes (the proposed actinonin targeted pathway) were detected by whole genome resequencing analysis. A series of experiments confirmed that actinonin-resistance in the mutant strain was associated with respiration. When aerobic respiration was chemically disrupted by CCCP, the growth of wild-type V. anguillarum changed significantly, with a 11.74-fold increase in cell density under actinonin treatment. For anaerobic respiration, an increased nitrate reductase activity was detected in mutant cells by addition of an exogenous electron donor. Noteworthy was the finding that nitrate inhibited the growth of the wild-type strain but promoted the growth of the mutant. Futhermore, the product of nitrate-dependent anaerobic respiration, nitrite, did not arrest the growth of mutant V. anguillarum, which was contrary to previous reports and might exacerbate the formation of vibriosis. Finally, we showed that chemical inhibition of nitrate reductase could restore the susceptibility of mutant V. anguillarum to actinonin. Together these data demonstrate that the combined use of an anaerobic-respiratory inhibitor and an antibiotic might be a promising approach for the treatment of drug-resistant bacteria in aquaculture.

耐药细菌对水产养殖业构成越来越严重的威胁。为了开发对抗这些细菌的新方法，有必要首先了解它们的耐药性机制。我们之前曾分离出一种潜在的益生菌菌株，链霉菌。NHF165，可产生针对水产养殖病原体鳗弧菌的细胞毒肌动蛋白。在这项研究中，自发性鳗弧菌突变体的肌动蛋白MIC增加了八倍，其特征在于阐明了肌动蛋白抵抗机制。转录组学分析显示与厌氧呼吸有关的基因（小睡和r）和下调参与厌氧呼吸（基因的NQR，SDH和FRD在突变株）。同时，通过全基因组重测序分析未检测到细菌呼吸基因或翻译起始基因（提出的肌动蛋白靶向途径）中的突变。一系列实验证实，突变菌株中的肌动蛋白抗性与呼吸有关。当有氧呼吸被CCCP化学破坏时，野生型鳗弧菌的生长变化显着，在肌动蛋白处理下细胞密度增加了11.74倍。对于无氧呼吸，通过添加外源电子供体在突变细胞中检测到硝酸盐还原酶活性的增加。值得注意的发现是硝酸盐抑制了野生型菌株的生长，但促进了突变体的生长。此外，硝酸盐依赖性厌氧呼吸的产物亚硝酸盐不能阻止突变体鳗弧菌的生长，这与先前的报道相反，并且可能加剧弧菌病的形成。最后，我们表明化学抑制硝酸盐还原酶可以恢复突变体V. anguillarum的敏感性。肌动蛋白。这些数据加在一起表明，厌氧呼吸抑制剂和抗生素的组合使用可能是治疗水产养殖中耐药细菌的一种有前途的方法。