Actinobacillus pleuropneumoniae, the etiological agent of porcine pleuropneumonia, is increasingly resistant to antibiotics. However, little is known about the mechanisms of antibiotic resistance in this pathogen. In this study, we experimentally evolved the reference strain of both A. pleuropneumoniae serovar 1 and serovar 7, the most prevalent serovars worldwide, to quinolone resistance by sequential exposure to subinhibitory concentrations of ciprofloxacin. The adaptive ciprofloxacin-resistant mutants of A. pleuropneumoniae serovar 1 and serovar 7 had a minimum inhibitory concentration (MIC) increment from 0.004 to 1 or 2 μg/mL, respectively. Adaptation to ciprofloxacin was shown to confer quinolone resistance with a 32- to 512-fold increase (serovars 1 and 7, respectively) as well as cross-resistance to ampicillin with an increased MIC by 16,384- and 64-fold (serovars 1 and 7, respectively). The genetic analysis of quinolone resistance-determining region mutations showed that substitutions occurred in gyrA (S83A) and parC (D84N) of serovar 1, and gyrA (D87N) of serovar 7. The ciprofloxacin-resistant mutants showed significantly reduced bacterial fitness. The mutants also showed changes in efflux ability and biofilm formation. Notably, the transcription and secretion levels of Apx toxins were dramatically reduced in ciprofloxacin-resistant mutants compared with their wild-type strains. Altogether, these results demonstrated marked phenotypic changes in ciprofloxacin-resistant mutants of A. pleuropneumoniae. The results stress the need for further studies on the impact of both the genotypic and phenotypic characteristics of A. pleuropneumoniae following exposure to subinhibitory concentrations of antibiotics.

中文翻译：

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暴露于亚致死环丙沙星可诱导胸膜肺炎放线杆菌对环丙沙星和交叉抗生素的耐药性、适应性、生物膜形成和 Apx 毒素分泌

猪胸膜肺炎的病原体胸膜肺炎放线杆菌对抗生素的耐药性越来越强。然而，人们对这种病原体的抗生素耐药机制知之甚少。在这项研究中，我们通过连续暴露于亚抑制浓度的环丙沙星，实验性地将全球最流行的血清型A. pleuropneumoniae血清型 1 和血清型 7的参考菌株进化为对喹诺酮类药物的耐药性。胸膜肺炎A的适应性环丙沙星耐药突变体血清型 1 和血清型 7 的最小抑菌浓度 (MIC) 增量分别为 0.004 至 1 或 2 μg/mL。对环丙沙星的适应显示使喹诺酮类药物耐药性增加 32 至 512 倍（血清型分别为 1 和 7）以及对氨苄青霉素的交叉耐药性增加 16,384 和 64 倍（血清型 1 和 7 ， 分别）。喹诺酮类耐药决定区突变的遗传分析表明，血清型 1 的gyrA (S83A) 和parC (D84N) 发生了取代，而gyrA(D87N) 血清型 7。耐环丙沙星的突变体显示出显着降低的细菌适应性。突变体还表现出外排能力和生物膜形成的变化。值得注意的是，与野生型菌株相比，环丙沙星耐药突变体中 Apx 毒素的转录和分泌水平显着降低。总之，这些结果表明胸膜肺炎放线菌的环丙沙星耐药突变体有显着的表型变化。结果强调需要进一步研究暴露于亚抑制浓度抗生素后胸膜肺炎放线菌的基因型和表型特征的影响。