Survey of antibiotic resistome in a mixed-use watershed in Western Newfoundland, Canada

Abstract

Antibiotic production is a natural phenomenon employed by microorganisms to control their environment in stress situations. Thus, antibiotic resistance has evolved in parallel to counteract naturally produced antibiotics. On the other hand, excessive use of antibiotics by humans, either for protecting human or animal health, may induce unnaturally high antibiotic stresses leading to enhanced antibiotic resistance, the so-called acquired resistance. This study is the first to report an initial exploration into the distribution and quantification of antibiotic resistance genes, i.e. resistome, in a mixed-use watershed in Western Newfoundland, along the Humber river. The river was sampled along a gradient of increased human impact, mainly associated with the discharge of municipal wastewaters. The goals of my thesis were to (1) understand the relationship between human impact in the Humber river and the occurrence of antibiotic resistance genes in the environment, and (2) understand the role of the river in mitigating the abundance of these resistance genes, if any role is present. Total DNA was extracted from the river water samples, sequenced by shotgun sequencing on an Illumina Hiseq platform with sequencing data quality controlled and cleaned at the sequencing facility via a QIIME pipeline, followed by identification and quantification of antibiotic resistance markers through a computational pipeline carried out in ShortBRED against a curated Antibiotic Resistance Genes Database (ARDB). About 400 antibiotic resistance genes, of variable abundance, were identified, distributed across the tested systems, an indication of the wide distribution of antibiotic resistance in the environment. This analysis revealed the distribution and abundance of Antibiotic Resistance Genes (ARGs) within the microbial population in the Humber River. Quantitative real-time PCR (qRT-PCR) was used to quantify absolute (copies L⁻¹) and relative abundances (copies /16S rRNA) for TetO, TetM and AdeC genes. Relative abundance of ARGs (copies / 16S rRNA) was higher in the upstream locations versus downstream locations. The results showed evidence that anthropogenic impacts, especially associated with the use of antibiotics, led to an increase in the diversity and total abundance of antibiotic resistance markers in the Humber River.