Stationary-phase Pseudomonas aeruginosa Levofloxacin tolerance

The ubiquitous opportunistic pathogen, Pseudomonas aeruginosa, is highly adaptive and refractory to several different classes of antibiotics. However, we found in this study that stationary-phase P. aeruginosa cultures exhibit greater sensitivity to the fluoroquinolone Levofloxacin (Levo) than other bactericidal antibiotics, including an aminoglycoside (Tobramycin) and beta-lactam (Aztreonam). To understand the basis of this sensitivity, we conducted time-lapse fluorescence microscopy experiments of cells during Levo treatment. We discovered that stationary-phase P. aeruginosa cells die rapidly during treatment and undergo heterogenous morphological changes, including explosive lysis, filamentation, and gradual loss of membrane integrity as evidenced by propidium iodide uptake. These morphologies are reminiscent of how the model organism Escherichia coli appears when recovering from fluoroquinolone treatment, a period when activation of the DNA damage-induced SOS response is crucial. Accordingly, we monitored the morphologies and survival of P. aeruginosa recA mutants and found that the SOS response is not involved in P. aeruginosa Levo sensitivity like it is for E. coli. We hypothesized that Levo sensitivity may be due to P. aeruginosa maintaining active metabolism in stationary phase. We determined that stationary-phase P. aeruginosa cells transcribe, maintain reductase activity, and accumulate reactive metabolic species which contribute to Levo-mediated death. By elucidating how P. aeruginosa cells sustain metabolic activity during the stationary phase, we can design strategies to sensitize these persistent subpopulations to Levo and maintain the efficacy of this clinically important fluoroquinolone antibiotic.

无处不在的条件致病菌铜绿假单胞菌（Pseudomonas aeruginosa）适应性极强，且对多类抗生素均具有耐药性。然而本研究发现，静止期（stationary phase）铜绿假单胞菌培养物对氟喹诺酮类（fluoroquinolone）药物左氧氟沙星（Levofloxacin，Levo）的敏感性，高于包括氨基糖苷类（aminoglycoside）药物妥布霉素（Tobramycin）与β-内酰胺类（beta-lactam）药物氨曲南（Aztreonam）在内的其他杀菌类抗生素。为阐明该敏感性的分子基础，我们开展了左氧氟沙星处理过程中细菌细胞的延时荧光显微镜实验。研究发现，静止期铜绿假单胞菌在药物处理期间会快速死亡，并出现异质性形态变化，包括爆炸性裂解、丝状化以及膜完整性的逐步丧失——该现象可通过碘化丙啶（propidium iodide）摄取实验得以验证。上述形态变化与模式生物大肠杆菌（Escherichia coli）在氟喹诺酮类药物处理后恢复期的形态特征高度相似，而该时期DNA损伤诱导的SOS应答（SOS response）激活发挥关键作用。据此，我们对铜绿假单胞菌recA突变体的形态变化与存活率进行了监测，结果发现，与大肠杆菌不同，SOS应答并未参与铜绿假单胞菌对左氧氟沙星的敏感性调控。我们推测，左氧氟沙星敏感性的产生，可能源于静止期铜绿假单胞菌仍维持活跃的代谢状态。经实验证实，静止期铜绿假单胞菌仍可进行转录过程、维持还原酶活性，并积累活性代谢产物（reactive metabolic species）——这些物质均参与了左氧氟沙星介导的细菌死亡过程。通过阐明静止期铜绿假单胞菌如何维持代谢活性，我们可设计相应策略，使这类持留亚群体对左氧氟沙星更为敏感，从而维持这一临床重要氟喹诺酮类抗生素的抗菌疗效。