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.

项目概要/摘要 由产生超广谱 β-内酰胺酶 (ESBL) 的细菌引起的感染对大多数或所有细菌都具有耐药性 然而，如果不使用碳青霉烯类药物来治疗任何药物，则存在很大的风险。 可能由广谱或超广谱 β-内酰胺酶生产者引起的感染。 卡青霉烯类药物（最后手段）可能会导致更危险的抗菌素耐药性，因此 必须立即具体确认 ESBL 耐药性，以确保 同时选择的治疗方法可以最大限度地降低对患者和公众的风险。 病原微生物的培养，需要 24 小时以上才能进行鉴定。 我们建议创建一种快速（1 小时）且无需培养的工具来对产生 ESBL 的病原体进行表型筛选。 合成分子触发器，其中头孢菌素共价连接到单链 DNA 3’端附近的寡核苷酸如果没有 ESBL 酶活性，则寡核苷酸无法通过 DNA 延伸。 聚合酶；在 ESBL 活性下，头孢菌素被释放，使 DNA 能够沿着 DNA 延伸 检测中存在模板，这意味着 ESBL 活性可以特异性触发 DNA 扩增。 因此，我们建议将 ESBL 活性的表型检测与 DNA 扩增可立即区分 ESBL 驱动的耐药性， 从而在不牺牲抗生素管理的情况下实现快速治疗。