Abstract
The dissemination of antibiotic resistant bacteria from anthropogenic sources into the environment poses an emerging public health threat. Antibiotic resistance genes (ARGs) and gene-capturing systems such as integron-associated integrase genes (intI) play a key role in alterations of microbial communities and the spread of antibiotic resistant bacteria into the environment. In order to assess the effect of anthropogenic activities on watersheds in southwestern British Columbia, the presence of putative antibiotic resistance and integrase genes was analyzed in the microbiome of agricultural, urban influenced, and protected watersheds. A metagenomics approach and high-throughput quantitative PCR (HT qPCR) were used to screen for elements of resistance including ARGs and intI. Metagenomic sequencing of bacterial genomic DNA was used to characterize the resistome of microbial communities present in watersheds over a 1-year period. There was a low prevalence of ARGs relative to the microbial population (<1%). Analysis of the metagenomic sequences detected a total of 60 elements of resistance including 46 ARGs, intI1, and groEL/intI1 genes and 12 quaternary ammonium compounds (qac) resistance genes across all watershed locations. The relative abundance and richness of ARGs was found to be highest in agriculture impacted watersheds compared to urban and protected watersheds. A downstream transport pattern was observed in the impacted watersheds (urban and agricultural) during dry months. Similar to other reports, this study found a strong association between intI1 and ARGs (e.g., sul1), an association which may be used as a proxy for anthropogenic activities. Chemical analysis of water samples for three major groups of antibiotics was below the detection limit. However, the high richness and gene copy numbers (GCNs) of ARGs in impacted sites suggest that the effects of effluents on microbial communities are occurring even at low concentrations of antimicrobials in the water column. Antibiotic resistance and integrase genes in a year-long metagenomic study showed that ARGs were driven mainly by environmental factors from anthropogenized sites in agriculture and urban watersheds. Environmental factors such as land-use and water quality parameters accounted for 45% of the variability observed in watershed locations.
Highlights
Antibiotic resistance is recognized as a major emerging health threat worldwide
Each sampling study site represented different land-use: agriculture (Agricultural Upstream or AUP site located in a forested area with minimal housing, Agricultural Polluted or agricultural impacted watersheds included streptomycin phosphotransferase (APL) site receiving effluents from multiple farms, and Agricultural Downstream or ADS site, 2.5 km from APL and fed by waters from APL and another tributary with both agricultural and some urban influence); urban (Urban Polluted or UPL site characterized by residential development in a mountainous forest area, and urban downstream or UDS site, 1 km from UPL and running through residential, forested, and park areas); and non-impacted (Protected Upstream or PUP site, in a protected watershed that provides drinking water for a large community, and Protected Downstream or PDS site, 16 km from PUP)
Eighty-nine surface water samples among seven sampling sites located in three watersheds of differing land-use in southwestern British Columbia were analyzed
Summary
Antibiotic resistance is recognized as a major emerging health threat worldwide. While not a new phenomenon, factors such as the global population growth, overuse, and limited development of novel antibiotics have increased overall the morbidity and mortality as well as the costs of treating of bacterial diseases (Spellberg et al, 2008; Colomer-Lluch et al, 2011). Integrons are considered the main agents of bacterial evolution due to their role in the dissemination of ARGs, development of multiple drug resistance, and their ability to add gene structures into bacterial genomes (Mazel, 2006; Joss et al, 2009; Cambray et al, 2010; Gillings, 2014). These assembly platforms containing gene cassettes with a variety of functions, mainly antimicrobial resistance, are composed of a gene encoding integrase (intI), a recombination site (attI), and a promoter (PC) located upstream of the gene cassette (Fluit and Schmitz, 2004; Gillings, 2014). Understanding the interplay between land use, watersheds, and the dissemination of ARGs or genetic elements associated to resistance, is important to understanding and planning to prevent future impacts to both the public health and the environment
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