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Elucidating repair processes central to fluoroquinolone persistence in growth-inhibited populations

阐明对生长抑制人群中氟喹诺酮持久性至关重要的修复过程

基本信息

批准号：
10574520
负责人：
Allison Herzfeld
金额：
$ 2.89万
依托单位：
RUTGERS BIOMEDICAL AND HEALTH SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-03-01 至 2023-05-12
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10574520
关键词：
Adjuvant Antibiotics Bacteria Bacterial Infections Biological Assay Cells Chemicals Chromosomes Chronic Complement DNA DNA Damage DNA Markers DNA Repair DNA Replication Timing DNA Topoisomerases DNA biosynthesis Data Development Dose Drug Metabolic Detoxication Drug Tolerance Dyes Enzymes Escherichia coli Excision Exhibits Fluorescence Fluorescence Microscopy Fluorescence-Activated Cell Sorting Fluoroquinolones Genetic Goals Gram-Negative Bacteria Growth Individual Infection Kinetics Levaquin Mediator Methods Microbial Biofilms Microscopy Minimum Inhibitory Concentration measurement Modeling Modern Medicine Moxifloxacin Nucleic Acids Nucleotides Nutrient Pathway interactions Pharmaceutical Preparations Phase Phenotype PicoGreen Population Predisposition Process Pseudomonas aeruginosa Recovery Regulation Relapse Replication Origin Reporter Resolution Rifampin SOS Response Site Sorting Stains Starvation System Temperature Testing Time Treatment Failure Visualization Work antibiotic tolerance bactericide burden of illness chronic infection experience experimental study homologous recombination inducible gene expression mutant novel prevent prophylactic recombinational repair recurrent infection repaired soft drink temperature sensitive mutant treatment duration

项目摘要

Project Summary Many antibiotics rapidly kill growing populations of bacteria but struggle to kill non-growing populations. Even for drugs that can kill the majority of growth-inhibited bacteria, such as fluoroquinolones (FQs), the presence of persisters can lead to treatment failure. While current paradigms suggest that persisters survive due to limited antibiotic-induced damage, for FQs this is not the case. In non-growing populations, FQ persisters experience the same amount of antibiotic-induced DNA damage as their genetically identical kin and require the homologous recombination repair machinery during the post-antibiotic recovery period in order to survive. Currently, the mechanism underlying why persisters can survive FQ-induced damage while their clonal kin cannot remains ill-defined. We hypothesize that, i) chromosome number, and ii) the relative timing of DNA synthesis and repair during the post-antibiotic period, are phenotypic variables that govern the likelihood a bacterium will be an FQ persister. Since our first hypothesis is based on the importance of homologous recombination to FQ persistence in growth-inhibited populations, we will use fluorescence-activated cell sorting (FACS) to sort live wild-type and mutant populations of Escherichia coli based on chromosome number as determined by staining with cell-permeant nucleic acid dyes, and will subject the isolated populations to tolerance assays and quantitative PCR for chromosome number verification. To complement these assays, we will use time-lapse microscopy of an FQ-treated E. coli strain that harbors an origin of replication reporter in order to visualize the chromosome content of persisters and nonpersisters during the post-FQ recovery period. Our second hypothesis is based on a recent study from our group that showed that starvation following FQ treatment increased persister levels in non-growing populations in a RecA- and time-dependent manner. To test whether the timing of DNA replication vs. DNA repair during recovery impacts FQ persistence, we will conduct time-lapse fluorescence microscopy of single cells harboring reporters for DNA repair or DNA replication both in the presence and absence of nutrients. We will then conduct bulk culture experiments by employing temperature sensitive mutants and inducible systems of the DNA replication and DNA repair machinery. We will first investigate levofloxacin, a representative FQ, and stationary-phase E. coli cultures, because non-growing infections are the most difficult to eradicate, before establishing the generality of any findings by using other FQs (e.g., moxifloxacin) and bacterial species (e.g., Pseudomonas aeruginosa). Data from these experiments will assess whether chromosome number and the relative timing of DNA synthesis vs. DNA repair during recovery from FQ treatment are phenotypic variables important for FQ persistence. Increased understanding of persister survival tactics will open the door for the development of anti-persister strategies, which would reduce the burden of chronic and relapsing infections.

项目总结