ESBL-triggered exponential amplification for culture-free phenotypic detection of MDR pathogens

ESBL 触发指数扩增，用于 MDR 病原体的无培养表型检测

• 批准号: 10242638
• 负责人: Ian M White
• 金额: $ 19.05万
• 依托单位: UNIV OF MARYLAND, COLLEGE PARK
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-20 至 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10242638
• 关键词: Active Sites; Amino Acid Substitution; Antibiotics; Antimicrobial Resistance; Biological Assay; Carbapenems; Cephalosporins; Chromogenic Substrates; Colony-forming units; Complementary DNA; DNA; DNA Sequence; DNA amplification; DNA-Directed DNA Polymerase; Dangerousness; Detection; Diagnostic; Discrimination; Ensure; Enterobacteriaceae; Enzyme Kinetics; Enzymes; Escherichia coli; Extended-spectrum β-lactamase; Generations; Goals; Hour; Hydrolysis; Infection; Klebsiella; Link; Measures; Methods; Molecular; Oligonucleotides; Organism; Outcome; Pathogenicity; Patients; Penicillins; Performance; Pharmaceutical Preparations; Phenotype; Public Health; Reaction; Resistance; Resort; Risk; Screening procedure; Side; Single-Stranded DNA; Specific qualifier value; Specificity; Structure; Sulfur; System; Testing; Therapeutic; Translating; Variant; Vertebral column; Work; bacterial resistance; base; beta-Lactamase; beta-Lactams; design; detection limit; enzyme activity; insight; multi-drug resistant pathogen; pathogen; polymerization; risk minimization; tool

PROJECT SUMMARY/ABSTRACT Infections driven by extended-spectrum β-lactamase (ESBL)-producing bacteria are resistant to most or all penicillins and cephalosporins. However, there is significant risk in defaulting to carbapenems to treat any infection potentially resulting from broad- or extended-spectrum β-lactamase-producers. The use of carbapenems (a drug of last resort) may contribute to even more dangerous antimicrobial resistance and thus it is imperative to immediately and specifically confirm ESBL-based resistance in order to ensure that the selected therapeutic simultaneously minimizes risk to the patient and to the public. Current methods rely on culture of the pathogenic organisms, which requires more than 24 hours for identification. Our team aims to create a rapid (1 hr) and culture-free tool to phenotypically screen for ESBL-producing pathogens. We propose to synthesize a molecular trigger in which a cephalosporin is covalently attached to a single-stranded DNA oligonucleotide near the 3’ end. Without ESBL enzyme activity, the oligo cannot be extended by DNA polymerase; upon ESBL activity, the cephalosporin is released, enabling the DNA to be extended along DNA templates present in the assay, implying that DNA amplification can be triggered specifically by ESBL activity. Hence, we are proposing to combine the phenotypic detection of ESBL activity with the detection power of DNA amplification. The proposed approach results in an immediate discrimination of ESBL-driven resistance, thus enabling rapid treatment without sacrificing antibiotic stewardship.

项目摘要/摘要 由延伸谱β-内酰胺酶（ESBL）产生的细菌驱动的感染对大多数或全部具有抗性 青霉素和头孢菌素。但是，违约到碳青霉烯的风险很大 可能由广泛或扩展的β-内酰胺酶产生剂引起的感染。使用 碳青霉烯（最后一种手段）可能会导致更危险的抗菌素耐药性，从而导致 必须立即，特别明确确认基于ESBL的阻力，以确保 选定的治疗方法只会最大程度地减少对患者和公众的风险。当前方法依赖 病原生物的培养物需要超过24小时才能识别。我们的团队的目标是 创建一个快速（1小时）和无培养工具，以表型筛选产生ESBL的病原体。我们建议 合成一个分子触发，其中头孢菌素共同附着在单链DNA上 3'端附近的寡核苷酸。没有ESBL酶活性，寡核不能通过DNA扩展 聚合酶； ESBL活性后，释放头孢菌素，使DNA沿DNA扩展 测定中存在的模板，这意味着可以通过ESBL活性专门触发DNA扩增。 因此，我们提议将ESBL活性的表型检测与检测能力相结合 DNA扩增。提出的方法导致立即歧视ESBL驱动的抗性， 因此，无需牺牲抗生素管理即可快速治疗。