Characterization and quantitation of a novel β-lactamase gene found in a wastewater treatment facility and the surrounding coastal ecosystem

Abstract

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, ≈ 416 μg ml(-1) ampicillin) was sequenced and annotated, revealing 33 putative genes, including a 927-bp gene that is 42% identical to a functionally characterized β-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 × 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.

Fig. 1.

Sampling site locations. (A) WWTP and Charleston Harbor sites. The WWTP site 1 (principal effluent) is located in Charleston, SC. Environmental collection sites were labeled as follows: site 2, outfall/discharge site; site 3, Ashley River; site 4, Ashley and Cooper River mixing zone; site 5, Cooper River; site 6, Charleston Harbor mouth. (B) Site 7, North Inlet control site. Dark-gray-shaded zones represent areas of urban land cover.

Fig. 2.

Neighbor-joining consensus tree for representative class A to D β-lactamases. Sequences used for comparison between the bla_M-1 sequence found in the present study versus those of different β-lactamase classes were retrieved from the National Center for Biotechnology Information (NCBI) database. Numbers in the branches represent bootstrap values from 1,000 replications. Numbers in parentheses are the GenBank accession identification numbers.

Fig. 3.

Copy number of the bla_M-1 gene ml⁻¹ of sample (A) and ng⁻¹ of DNA (B). Copy numbers were quantified by qPCR using metagenomic DNA extracted from 3 stages in the WWTP: raw sewage (RS), activated sludge (AS), and principal effluent (PE). Black/gray bars represent GCN means for each treatment (n = 3) during years 2007 and 2009, respectively. Error bars indicate standard deviations. Means with different upper- and lowercase letters indicate significant differences across treatment for years 2007 and 2009, respectively.

Fig. 4.

Copy number of the bla_M-1 gene in sediments g⁻¹ of sample (A) and ng⁻¹ of DNA (B). Copy numbers were quantified by qPCR using metagenomic DNA extracted from 5 different sites in the Charleston Harbor area: site 2 (WWTP outfall), site 3 (Ashley River), site 4 (Ashley and Cooper River mixing zone), site 5 (Cooper River), site 6 (Charleston Harbor mouth), and site 7 (NI control site). Bars represent the mean for each treatment (n = 3). Error bars indicate standard deviations. Means with different letters indicate statistical significance at the 0.05 level.