Identification of novel targets of azithromycin activity against Pseudomonas aeruginosa grown in physiologically relevant media

Pseudomonas aeruginosa causes severe multi-drug resistant infections that often lead to bacteremia and sepsis. Physiologically relevant conditions can increase the susceptibility of pathogens to antibiotics, such as azithromycin (AZM). When compared to minimal inhibitory concentrations (MIC) in lab media, AZM had a 16-fold lower MIC in tissue culture medium with 5% MHB, and a 64-fold lower MIC in this tissue culture medium with 20% human serum. AZM also demonstrated increased synergy in combination with synthetic host defence peptides DJK-5 and IDR-1018 under host-like conditions and in a murine abscess model. To mechanistically study the altered effects of AZM under physiologically relevant conditions, global transcriptional analysis was performed on P. aeruginosa with and without effective concentrations of AZM. This revealed that the arn operon, mediating arabinosaminylation of lipopolysaccharides, and related regulatory systems, were downregulated in host-like media when compared to MHB. Inactivation of genes within the arn operon led to increased susceptibility of P. aeruginosa to AZM and great increases in synergy between AZM and other antimicrobial agents, indicating that dysregulation of the arn operon might explain increased AZM uptake and synergy in host-like media. Furthermore, genes involved in central and energy metabolism, and ribosome biogenesis were dysregulated more in physiologically relevant conditions treated with AZM, likely due to general changes in cell physiology as a result of the increased effectiveness of AZM in these conditions. These data suggest that, in addition to the arn operon, there are multiple factors in host-like environments that are responsible for observed changes in susceptibility. RNA-Seq of P. aeruginosa grown in MHB, RPMI+5% MHB, or RPMI+5% MHB+20% human serum with or without AZM treatment

铜绿假单胞菌（Pseudomonas aeruginosa）可引发严重的多重耐药感染，常导致菌血症与脓毒症。生理相关培养条件可增强病原体对抗生素的敏感性，例如阿奇霉素（azithromycin, AZM）。与实验室培养基中的最低抑菌浓度（minimal inhibitory concentrations, MIC）相比，阿奇霉素在含5%穆勒-辛顿肉汤（MHB）的组织培养基中的MIC降低16倍，在含20%人血清的该组织培养基中的MIC更是降低64倍。在宿主模拟环境及小鼠脓肿模型中，阿奇霉素与合成宿主防御肽DJK-5、IDR-1018联用时的协同抗菌活性显著增强。为从机制层面探究生理相关条件下阿奇霉素效应的改变，研究人员对暴露于有效浓度阿奇霉素与否的铜绿假单胞菌开展了全转录组分析。结果显示，相较于穆勒-辛顿肉汤培养基，宿主模拟培养基中介导脂多糖阿拉伯糖基化修饰的arn操纵子（arn operon）及其相关调控系统均出现转录水平下调。arn操纵子内基因的失活可提升铜绿假单胞菌对阿奇霉素的敏感性，且大幅增强阿奇霉素与其他抗菌药物的协同抗菌活性，这表明arn操纵子的表达失调或可解释宿主模拟环境中阿奇霉素摄取量增加及协同活性增强的现象。此外，在生理相关条件下经阿奇霉素处理后，参与中枢代谢与能量代谢及核糖体生物发生的基因失调程度更为显著，这可能源于该环境中阿奇霉素效力提升所引发的细胞生理整体变化。上述数据表明，除arn操纵子外，宿主模拟环境中还存在多种因素共同导致了所观测到的敏感性变化。本数据集包含在穆勒-辛顿肉汤（MHB）、RPMI+5%穆勒-辛顿肉汤（MHB）或RPMI+5%穆勒-辛顿肉汤（MHB）+20%人血清中培养，且分别给予或不给予阿奇霉素（azithromycin, AZM）处理的铜绿假单胞菌的RNA测序（RNA-Seq）数据。