Investigation of Chloramphenicol Antibiotic Resistance and Its Interplay with Biofilm Formation in Escherichia coli via CRISPR Interference

Presenter

Sarthak Narain Srivastava

Campus

UMass Amherst

Sponsor

Lauren B. Andrews, Department of Chemical Engineering, UMass Amherst

Schedule

Session 3, 1:30 PM - 2:15 PM [Schedule by Time][Poster Grid for Time/Location]

Location

Poster Board C15, Poster Showcase Room (163), Row 2 (C11-C20) [Poster Location Map]

Abstract

Antimicrobial resistance (AMR) in bacteria poses a significant global health threat, linked to 4.95 million deaths annually. Pathogenic strains of Escherichia coli, in particular, represent a critical concern in the context of bacterial AMR, contributing significantly to the mortality associated and attributed with this global health challenge. We aimed to employ a functional genomic approach to identify genetic determinants of antibiotic resistance against a bacteriostatic antibiotic, chloramphenicol, and to explore its interactions with biofilm formation, a significant virulence factor in the pathogenesis of E. coli. Our approach utilizes targeted gene silencing using CRISPR interference (CRISPRi) and a genome-wide CRISPRi library with gRNAs targeting >99.9% of protein and RNA gene products in the Escherichia coli genome with 8 gRNAs for 92.0% of gene clusters. To identify genotypes conferring improved antibiotic resistance, the library was exposed to selective conditions, specifically grown with a sub-inhibitory concentration of the antibiotic. Thereafter, we identified enriched gRNAs post-selection. To investigate the interactions of AMR and biofilm formation, pooled screens with antibiotics were also performed with two medically relevant biomaterials, hydrophobic polydimethylsiloxane (PDMS) and hydrophilic polyethylene glycol dimethacrylate (PEG), and cells adhered to the biomaterials were identified. Our initial efforts established the experimental framework and determined the conditions for subsequent analyses. We expect our work to deepen our understanding of antimicrobial resistance and guide the development of new strategies to counteract this issue in pathogenic Escherichia coli strains.

Keywords

gene regulation, antibiotic resistance, biofilm formation, CRISPR interference, Escherichia coli

Research Area

Genetics

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