Characterization and Quantitation of a Novel β-Lactamase Gene Found in a Wastewater Treatment Facility and the Surrounding Coastal Ecosystem

Wastewater treatment plants (WWTPs) are engineered structures that collect, concentrate, and treat human waste, ultimately releasing treated wastewater into local environments. While WWTPs efficiently remove most biosolids, it has been shown that many antibiotics and antibiotic-resistant bacteria can survive the treatment process. To determine how WWTPs influence the concentration and dissemination of antibiotic-resistant genes into the environment, a functional metagenomic approach was used to identify a novel antibiotic resistance gene within a WWTP, and quantitative PCR (qPCR) was used to determine gene copy numbers within the facility and the local coastal ecosystem. From the WWTP metagenomic library, the fosmid insert contained in one highly resistant clone (MIC, similar to 416 mu g ml(-1) ampicillin) was sequenced and annotated, revealing 33 putative genes, including a 927-bp gene that is 42% identical to a functionally characterized beta-lactamase from Staphylococcus aureus PC1. Isolation and subcloning of this gene, referred to as bla(M-1), conferred ampicillin resistance to its Escherichia coli host. When normalized to volume, qPCR showed increased concentrations of bla(M-1) during initial treatment stages but 2-fold-decreased concentrations during the final treatment stage. The concentration ng(-1) DNA increased throughout the WWTP process from influent to effluent, suggesting that bla(M-1) makes up a significant proportion of the overall genetic material being released into the coastal ecosystem. Average discharge was estimated to be 3.9 x 10(14) copies of the bla(M-1) gene released daily into this coastal ecosystem. Furthermore, the gene was observed in all sampled coastal water and sediment samples surrounding the facility. Our results suggest that WWTPs may be a pathway for the dissemination of novel antibiotic resistance genes into the environment.