A Treatment Plant Receiving Waste Water from Multiple Bulk Drug Manufacturers Is a Reservoir for Highly Multi-Drug Resistant Integron-Bearing Bacteria Nachiket P. Marathe 1. , Viduthalai R. Regina 1.¤ , Sandeep A. Walujkar 1 , Shakti Singh Charan 1 , Edward R. B. Moore 2,3 , D. G. Joakim Larsson 3 , Yogesh S. Shouche 1 * 1 Microbial Culture Collection (MCC), National Center for Cell Science, Pune, Maharashtra, India, 2 Culture Collection University of Gothenburg (CCUG), Gothenburg, Sweden, 3 Department of Infectious Diseases, Institute of Biomedicine, The Sahlgrenska Academy at the University of Gothenburg, Gothenburg, Sweden Abstract The arenas and detailed mechanisms for transfer of antibiotic resistance genes between environmental bacteria and pathogens are largely unclear. Selection pressures from antibiotics in situations where environmental bacteria and human pathogens meet are expected to increase the risks for such gene transfer events. We hypothesize that waste-water treatment plants (WWTPs) serving antibiotic manufacturing industries may provide such spawning grounds, given the high bacterial densities present there together with exceptionally strong and persistent selection pressures from the antibiotic- contaminated waste. Previous analyses of effluent from an Indian industrial WWTP that processes waste from bulk drug production revealed the presence of a range of drugs, including broad spectrum antibiotics at extremely high concentrations (mg/L range). In this study, we have characterized the antibiotic resistance profiles of 93 bacterial strains sampled at different stages of the treatment process from the WWTP against 39 antibiotics belonging to 12 different classes. A large majority (86%) of the strains were resistant to 20 or more antibiotics. Although there were no classically-recognized human pathogens among the 93 isolated strains, opportunistic pathogens such as Ochrobactrum intermedium, Providencia rettgeri, vancomycin resistant Enterococci (VRE), Aerococcus sp. and Citrobacter freundii were found to be highly resistant. One of the O. intermedium strains (ER1) was resistant to 36 antibiotics, while P. rettgeri (OSR3) was resistant to 35 antibiotics. Class 1 and 2 integrons were detected in 74/93 (80%) strains each, and 88/93 (95%) strains harbored at least one type of integron. The qPCR analysis of community DNA also showed an unprecedented high prevalence of integrons, suggesting that the bacteria living under such high selective pressure have an appreciable potential for genetic exchange of resistance genes via mobile gene cassettes. The present study provides insight into the mechanisms behind and the extent of multi- drug resistance among bacteria living under an extreme antibiotic selection pressure. Citation: Marathe NP, Regina VR, Walujkar SA, Charan SS, Moore ERB, et al. (2013) A Treatment Plant Receiving Waste Water from Multiple Bulk Drug Manufacturers Is a Reservoir for Highly Multi-Drug Resistant Integron-Bearing Bacteria. PLoS ONE 8(10): e77310. doi:10.1371/journal.pone.0077310 Editor: Zhi Zhou, National University of Singapore, Singapore Received July 17, 2013; Accepted September 4, 2013; Published October 29, 2013 Copyright: ß 2013 Marathe et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The work was funded by the Swedish International Development Agency (SIDA) project nr. 348-2008-6146; this funding enabled the exchange of NPM between India and Sweden. The work was also funded by the Swedish Research Council (VR), the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (FORMAS) and The Swedish Foundation for Strategic Environmental Research (MISTRA). Nachiket Marathe held a fellowship from Council of Scientific and Industrial Research (CSIR), New Delhi, during the completion of this work. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: yogesh@nccs.res.in . These authors contributed equally to this work. ¤ Current address: The Interdisciplinary Nanoscience Centre (iNANO), Aarhus University, Aarhus, Denmark Introduction The emergence of multi-drug resistance in bacterial human pathogens is one of the most serious challenges for health care globally. Pathogens that earlier were sensitive to antibiotics are becoming resistant by mutations in their preexisting DNA or by acquisition of DNA containing resistance genes [1]. Most of the antibiotic resistance genes carried by pathogens have their origins in environments other than the clinical world and the normal bacterial flora of disparate environments are thought to be the reservoirs of these resistance genes [2–7]. The ability of bacteria to exchange genes across species boundaries is an important factor in the spread of acquired antibiotic resistance. This often is facilitated by mobile genetic elements such as plasmids, transposons, integrons and genomic islands that harbor antibiotic resistance genes. Recent studies have shown that antibiotic resistance genes are ancient and were present in the environment long before the antibiotic era [8,9]. The overuse and misuse of antibiotics has led to increased selection pressures, also in the environment. In bacterial communities exposed to sufficient selection pressure from exposure to antibiotics, resistance genes may increase radically in abundance within the populations [10–12]. Such increases may also be accompanied by increased frequencies in genetic elements facilitating their mobility [12,13]. Thus, exposure to antibiotics is expected to increase the risk for the transfer of resistance between bacterial species. This is true not only during antibiotic treatment of humans and animals, but also when a sufficiently high level of PLOS ONE | www.plosone.org 1 October 2013 | Volume 8 | Issue 10 | e77310