AMR Surveillance and Discovery Using Functional Metagenomics in Ontario Wastewater

Antimicrobial resistance (AMR) is an increasing challenge in health care. Wastewater sampling provides a chance to survey regional AMR in a non-invasive way for the contributing population. In this study, functional metagenomic libraries were constructed from total DNA using the mosaic ends tagmentation approach (METa), and the cosmid library construction method, yielding average insert sizes of 2kb and 20-30kb respectively. Shotgun metagenomics is performed in parallel on the wastewater samples to screen for known AMR genes. This approach seeks to determine the current antimicrobial resistance gene (ARG) landscape in the populations reflected by the regional wastewater and, in combination with functional metagenomics, to potentially discover new ARGs. Our research looks at ARG resistance towards some common antibiotics such as Kanamycin, Ampicillin, and Tetracycline while also targeting two carbapenems, Meropenem (MP) and Imipenem (IP). Beta-Lactam antibiotics are a well prescribed and diverse family of antibiotics. IP and MP specifically are used as final interventions for Gram-negative bacteria with other beta-lactam resistance that cause pulmonary infections in cystic fibrosis patients, meningitis, sepsis, and others. Therefore, the initial hosts of choice for these libraries were E. coli and P. putida. In further studies, using other hosts of interest, namely Moraxella catarrhalis and Bacillus subtilis, different ARGs not expressed in P. putida and E. coli can be identified. Clones which contain no known resistance genes will be sub-cloned using transposon mutagenesis to determine the location of the resistance gene and ORFs will be predicted as a first step in investigating potentially novel ARGs.