Pyrosequencing estimates of the diversity of antibiotic resistant bacteria in a wastewater system

Standard protocols for monitoring wastewater treatment efficacy target Escherichia coli and fecal coliforms. This might not accurately describe risks associated with antibiotic resistance in the bacterial population of treated wastewaters. We modified a standard agar recovery method by amending it with various antibiotics. The resulting bacterial colonies were submitted to 454 pyrosequencing; thus we identified the diversity of culturable antibiotic resistant bacteria from treated and raw wastewaters. This approach produced 209,706 high quality reads of >300 bp. Operational taxonomic units (OTUs) within Proteobacteria dominated the system. The Shannon–Wiener H′ index showed bacterial populations recovered on ciprofloxacin amended agars to be the least diverse. Principal component analysis of OTU distribution at phylum level showed that Proteobacteria accounted for most of the variability. The same analysis revealed most of the samples to have similar diversities at phylum level being dominated by Proteobacteria, though a few samples, typically recovered from ciprofloxacin or doxycycline amended agars were often dissimilar. Arcobacter spp. or E. coli were dominant in the bacterial communities recovered on agars amended with ciprofloxacin or doxycycline, respectively. Genera containing putative pathogens were mostly representatives of Gamma and Epsilon proteobacteria. Bacterial populations containing multiple antibiotic resistance (MAR) in the final treated effluent was a possibility.