Diversity of Multidrug-Resistant Bacteria in an Urbanized River: A Case Study of the Potential Risks from Combined Sewage Overflows

Wastewater contamination and urbanization contribute to the spread of antibiotic resistance in aquatic environments. This is a particular concern in areas receiving chronic pollution of untreated waste via combined sewer overflow (CSO) events. The goal of this study was to expand knowledge of CSO impacts, with a specific focus on multidrug resistance. We sampled a CSO-impacted segment of the James River (Virginia, USA) during both clear weather and an active overflow event and compared it to an unimpacted upstream site. Bacteria resistant to ampicillin, streptomycin, and tetracycline were isolated from all samples. Ampicillin resistance was particularly abundant, especially during the CSO event, so these isolates were studied further using disk susceptibility tests to assess multidrug resistance. During a CSO overflow event, 82% of these isolates were resistant to five or more antibiotics, and 44% were resistant to seven or more. The latter statistic contrasts starkly with the upstream reference site, where only 4% of isolates displayed resistance to more than seven antibiotics. DNA sequencing (16S rRNA gene) revealed that similar to 35% of our isolates were opportunistic pathogens, comprised primarily of the genera Stenotrophomonas, Pseudomonas, and Chryseobacterium. Together, these results demonstrate that CSOs can be a significant source of viable clinically-relevant bacteria to the natural environment and that multidrug resistance is an important understudied component of the environmental spread of antibiotic resistance.