1534 ©IWA Publishing2013 water Science & Technology | 67.7 | 2013 Pyrosequencing estimates of the diversity of antibiotic resistant bacteria in a wastewater system Jesus Sigala and Adrian Une ABSTRACT 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 £ 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. Key words | 454 pyrosequencing, antibiotic resistance, Proteobacteria, wastewater collection and treatment system J. sígala A. une (corresponding auUioi) Plant and Environmental Sciences. New Mexico State University. BOX 30003 MSC 3Q. Skeen Hall Room N 127, Las Cruces, NM 88003-8003, USA E-mail: aunc@nmsu.edu INTRODUCTION Standard measures of assessing the quality of treated waste- water focus on Escherichia coli and fecal coliform counts. Technological advances (Aw & Rose 2012) have made it possible to go beyond the current set of indicators for a more extensive assessment of total microbial population diversity in the estimates of wastewater treatment (WWT) efficacy. Although not regulated, antibiotic resistance in bac- teria is a critical parameter that may be used in an integrated assessment of the risks associated with receiving surface waters (Une et al 2012) or with wastewater effluent, and thus in relevant decision support systems. Pyrosequencing is an essential tool in assessing complex microbial diversity in diverse habitats. It has been used to detect pathogenic bacteria in wastewater and biosolids treat- ment (Bibby et al 2010; Ye & Zhang 2on) and to reveal total bacterial diversity in fixed-film reactors (Kwon et al. 2010). Pyrosequencing of the plasmid metagenome has shov^oi antibiotic resistance genes to be prominent in activated sludge samples (Sanapareddy et al. 2009; Zhang et al 2on). These studies show wastewater bacteria to harbor anti- biotic resistance genes and to also be carriers of all accessory genes and mobile genetic elements necessary for dissemination of resistance gehes, thus establishing the potential for resistance genes to be carried forth and disse- minate via wastewater. What is less understood is the proximate source of these resistance genes and what factors favor their resilience in the microbial population exposed to WWT stresses. Hence the importance of understanding wastewater source types as potential differential sources of resistance genes, and the role of biotic and abiotic stresses on the persistence or possibly selection of antibiotic resistance. To understand the significance of variable sotirces of wastewater and the extent of treatment on microbial populations, we assessed doi: 10.2166/wst.2013.026