Examining fluoroquinolone-induced DNA damage in persisters and its contributions to antibiotic resistance.

摘要 含有生物膜的感染特别难治疗。这些感染通常对抗生素有反应。 治疗但很快复发，导致慢性和复发性感染。生物被膜感染的顽固性 被认为是由细菌持续体的存在引起的。持久者是耐受抗生素的细胞， 在基因上与所有死于抗生素的人群相同，但具有良好的表型 治疗时的利基。一般来说，这些幸存者被认为处于休眠状态，在那里 抗生素主要靶点活性降低，抗生素损伤程度严重 有限的。目前的生物被膜感染周期模型包括通过抗生素清除正常细胞和 豁免权，无论是在电影中还是从电影中脱落，以及清除从电影中脱落的持久者 豁免权。免疫细胞被阻止访问生物膜内的持续体，当抗生素 水平下降，坚持者继续重新填充胶片，导致复发感染。每一次复发， 发生抗生素耐药突变的机会增加，而且由于坚持者被认为几乎不会受到 抗生素治疗不会造成伤害，耐药突变株从持久繁殖的后代中产生的速率 培养物被认为是与正常细菌种群相同的。最近，我们发现 在生长抑制人群中坚持使用氟喹诺酮类(FQ)的人会经历FQ诱导的DNA损伤 这相当于对因治疗而死亡的细菌造成的损害。这些意想不到的结果表明 这些持久者可能会被FQ诱变，而从持久者成长起来的种群，如那些 复发感染，将是遗传多样性的，并以高比率产生抗药性突变，这 我们发现这是真的。这些数据表明，在持久性和抗生素耐药性之间存在着一条高速公路。 其入口坡道是用一种常用的处方类抗生素FQS治疗的。我们假设 增加对持久者的FQ伤害以及他们如何在伤害中生存的理解将有助于 减少复发感染和阻碍抗生素耐药性发展的战略。为了检验我们的假设， 我们将量身定做一种在基因组水平上量化DNA双链断裂(DSB)的方法，以适应FQ诱导的 DSB，并用它来研究具有不同持久水平的菌株；使用延时显微镜来询问 SOS反应、DNA修复、延长和隔膜在FQ后持续体恢复中的作用 并使用基因突变、谱系追踪和全基因组测序来确定 氟喹诺酮诱导的突变有助于提高氟喹诺酮衍生群体的抗生素耐药性 坚持者。总的来说，这些实验和我们将用来分析结果数据的统计方法 将提供有关FQ持久者的机制知识，以及他们如何增加抗生素的发生率 耐药性，这可能为顽固性感染的治疗干预提供新的途径。

Summary Infections that contain biofilms are exceptionally difficult to treat. These infections often respond to antibiotic therapy but quickly relapse, resulting in chronic and recurrent infections. The recalcitrance of biofilm infections is thought to arise from the presence of bacterial persisters. Persisters are antibiotic-tolerant cells that are genetically identical to the overall population that succumbs to antibiotics, but occupy a favorable phenotypic niche at the time of treatment. In general, these survivors are thought to be in a state of dormancy, where the activities of antibiotic primary targets are reduced and the extent of antibiotic-induced damage is severely limited. The current model of a biofilm infection cycle includes clearance of normal cells by antibiotics and immunity, both within the film and shed from it, and clearance of persisters that are shed from the film by immunity. Immune cells are hindered from accessing persisters within the biofilm, and when the antibiotic levels drop, persisters proceed to repopulate the film, causing a relapse infection. With every relapse, the chance for an antibiotic-resistant mutation to occur increases, and since persisters are thought to suffer little to no injury from antibiotic treatment, the rate at which resistant mutants should arise from persister-spawned cultures has been assumed to be the same as that of normal bacterial populations. Recently, we discovered that persisters to fluoroquinolones (FQ) in growth-inhibited populations experience FQ-induced DNA damage that is equivalent to damage in bacteria that die from treatment. These unexpected results suggested that those persisters might be mutagenized by FQ and that populations grown up from persisters, such as those of relapse infections, would be genetically diverse and produce antibiotic-resistant mutants at high rates, which we found to be true. These data suggest that there is a highway between persistence and antibiotic resistance whose entrance ramp is treatment with a commonly prescribed class of antibiotics, FQs. We hypothesize that increased understanding of FQ damage in persisters and how they survive that damage will illuminate strategies to reduce relapse infections and hinder antibiotic resistance development. To test our hypothesis, we will tailor a method that quantifies DNA double-strand breaks (DSBs) at the genome-scale to FQ-induced DSBs and employ it to study strains with different persister levels; use time-lapse microscopy to interrogate the roles of the SOS response, DNA repair, elongation, and septation to the recovery of persisters following FQ treatment; and use genetic mutants, lineage tracking, and whole-genome sequencing to determine whether FQ-induced mutagenesis contributes to heightened antibiotic resistance in populations derived from FQ persisters. Collectively, these experiments and the statistical methods we will use to analyze the resulting data will provide mechanistic knowledge of FQ persisters and how they enhance the incidence of antibiotic resistance, which could illuminate new avenues of therapeutic intervention for recalcitrant infections.