Phenotypically amplified assay for rapid, automated determination of beta-lactam susceptibility

PROJECT SUMMARY/ABSTRACT β-lactams (penicillins, cephalosporins, and carbapenems) are the most commonly utilized class of antibiotics.1 Enterobacterales (e.g., E. coli, Klebsiella) with resistance to β-lactams via the production of β-lactamases are continually evolving and expanding, threatening the effectiveness of β-lactams.2–7 Although these β-lactamase- producing Enterobacterales (βL-E) present an immediate danger to patients, overly aggressive treatments further drive resistance. Thus, the CDC urges antibiotic stewardship “to effectively treat infections, protect patients from harms caused by unnecessary antibiotic use, and combat antibiotic resistance.”8 As opposed to defaulting to broad spectrum antibiotics,9 clinicians should identify the antibiotic susceptibility of the infecting pathogen and select the narrowest-spectrum drug that would be effective. However, this is especially challenging for bloodborne bacterial infections (bacteremia) because they can lead to sepsis and death within a shorter time frame than the 2-4 days required for antibiotic susceptibility testing (AST).10 Thus, there is an urgent need for a tool that can determine the susceptibility of bacteremia within hours instead of days. We propose a new approach that combines the phenotypic detection of βL activity with the detection power of DNA amplification. We have developed a Phenotypic Amplified β-Lactamase (PhAβL) assay for the rapid identification of βL-producing pathogens in trace quantities, thus eliminating extensive culture durations. The PhAβL assay detects βL activity using a triggered amplification scheme in which βLs cleave cephalosporin molecules from custom DNA molecules (Cep-DNA), enabling amplification of the cleaved DNA. In the proposed work, our global aim is to leverage the PhAβL assay to develop a fully automated approach for rapid (< 4 hours) discrimination of bloodborne βL-producing pathogens to enable clinicians to balance effective treatment of sepsis with antimicrobial stewardship. .