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.

项目总结/摘要 由产超广谱β-内酰胺酶（ESBL）细菌驱动的感染对大多数或所有 青霉素和头孢菌素。然而，如果不使用碳青霉烯类来治疗任何疾病， 可能由广谱或超广谱β-内酰胺酶产生菌引起的感染。使用 碳青霉烯类（最后的药物）可能会导致更危险的抗菌素耐药性， 必须立即具体确认ESBL耐药，以确保 选定的治疗同时最大限度地减少对病人和公众的风险。目前的方法依赖于 病原微生物的培养，需要24小时以上进行鉴定。我们的团队旨在 创建一个快速（1小时）和无培养的工具，以表型筛选产ESBL的病原体。我们提出 合成一种分子触发剂，其中头孢菌素共价连接到单链DNA上， 在3'末端附近的寡核苷酸。没有ESBL酶活性，寡核苷酸不能被DNA延伸 在ESBL活性时，头孢菌素被释放，使DNA能够沿着DNA延伸 检测中存在的模板，这意味着DNA扩增可以由ESBL活性特异性触发。 因此，我们建议联合收割机ESBL活性的表型检测与 DNA扩增所提出的方法导致立即区分ESBL驱动的耐药性， 因此能够快速治疗而不牺牲抗生素管理。