PDF(Pubmed)
Abstract：
Bacterial persisters are a transient subpopulation of non-growing, antibiotic-tolerant cells. There is increasing evidence that bacterial persisters play an important role in treatment failure leading to recurring infections and promoting the development of antibiotic resistance. Current research reveals that recurring legionellosis is often the result of relapse rather than reinfection and suggests that the mechanism of bacterial persistence may play a role. The development of single-cell techniques such as the Timerbac system allows us to identify potential persister cells and investigate their physiology. Here, we tested the persister forming capacity of 7 pairs of Legionella pneumophila (Lp) clinical isolates, with isolate pairs corresponding to two episodes of legionellosis in the same patient. We distinguished non-growing subpopulations from their replicating counterparts during infection in an amoeba model. Imaging flow cytometry allowed us to identify single non-growing bacteria within amoeba cells 17 h post-infection, thus corresponding to this subpopulation of potential persister cells. Interestingly the magnitude of this subpopulation varies between the 7 pairs of Lp clinical isolates. Biphasic killing kinetics using ofloxacin stress confirmed the persister development capacity of ST1 clinical isolates, highlighting enhanced persister formation during the host cell infection. Thus, persister formation appears to be strain or ST (sequence type) dependent. Genome sequence analysis was carried out between ST1 clinical isolates and ST1 Paris. No genetic microevolution (SNP) linked to possible increase of persistence capacity was revealed among all the clones tested, even in clones issued from two persistence cycle experiments, confirming the transient reversible phenotypic status of persistence. Treatment failure in legionellosis is a serious issue as infections have a 5-10% mortality rate, and investigations into persistence in a clinical context and the mechanisms involved may allow us to combat this issue.
摘要：
细菌持久性是一个短暂的亚群，不生长，耐抗生素细胞。越来越多的证据表明，细菌持久性在导致反复感染和促进抗生素耐药性发展的治疗失败中起着重要作用。目前的研究表明，反复发作的军团菌病通常是复发而不是再感染的结果，并表明细菌持续存在的机制可能起作用。诸如Timerbac系统之类的单细胞技术的发展使我们能够识别潜在的持久细胞并研究其生理学。这里,我们测试了7对嗜肺军团菌（Lp）临床分离株的持久性形成能力，与分离对对应于同一患者的两次军团菌病发作。在变形虫模型中，我们在感染期间将非生长亚群与复制亚群区分开。成像流式细胞术使我们能够在感染后17小时鉴定变形虫细胞内的单个非生长细菌，因此对应于这个潜在持久细胞的亚群。有趣的是，该亚群的大小在7对Lp临床分离株之间变化。使用氧氟沙星应激的双相杀伤动力学证实了ST1临床分离株的持久性发育能力，强调在宿主细胞感染期间增强的持久性形成。因此，持久性形成似乎是菌株或ST(序列类型)依赖性的。在ST1临床分离株和ST1Paris之间进行基因组序列分析。在所有测试的克隆中，没有发现与持久性能力可能增加有关的遗传微进化（SNP）。即使是在两个持续周期实验的克隆中，确认持久性的瞬时可逆表型状态。军团菌病的治疗失败是一个严重的问题，因为感染的死亡率为5-10％，对临床背景下的持久性和所涉及的机制的调查可能使我们能够解决这个问题。
