ABSTRACT Broad-host-range plasmids are frequently associated with antibiotic resistance genes and can quickly spread antibiotic resistant phenotypes among diverse bacterial populations. Wastewater treatment plants have been identified as reservoirs for broad-host-range plasmids carrying resistance genes. The threat of broad-host-range plasmids released into the environment from wastewater treatment plants has identified the need for disinfection protocols to target broad-host- range plasmid destruction. Here we evaluate the efficacy of dissolved ozone at 2 and 8 mg·L–1 as a primary means for the destruction of broad-host-range plasmid and chromosomal DNA in simulated effluent. Pilot-scale tests using an experimental unit were carried out in municipal wastewater treatment plant effluent and compared with ultraviolet (UV)-irradiation and chlorination methodologies. Genes specific to Escherichia coli (uidA) and IncP broad-host-range plasmids (trfA) were monitored using real-time quantitative polymerase chain reaction (qPCR), and total DNA was monitored using absorbance spectroscopy. In wastewater treatment plant experiments, E. coli qPCR results were compared to a recognized culture-based method (Colilert?) for E. coli. In laboratory experiments, dissolved ozone at 8 mg·L–1 significantly destroyed 93% total, 98% E. coli, and 99% of broad-host-range plasmid DNA. Ozonation, UV-irradiation, and chlorination significantly reduced DNA concentrations and culturable E. coli in wastewater treat- ment plant effluent. Chlorination and UV disinfection resulted in 3-log decreases in culture-based E. coli concentrations in wastewater treatment plant effluent while changes were not significant when measured with qPCR. Only ozonation significantly decreased the IncP broad-host-range plasmid trfA gene, although concentrations of 2.2 × 105 copies trfA·L–1 remained in effluent. Disinfection processes utilizing high dissolved ozone concentrations for the destruction of emerging contaminants such as broad-host-range plasmid and total DNA may have utility as methods to ensure downstream environmental health and safe water reuse become more important.
Cite this paper
K. L. Asfahl and M. C. Savin, "Destruction of Escherichia coli and Broad-Host-Range Plasmid DNA in Treated Wastewater by Dissolved Ozone Disinfection under Laboratory and Field Conditions," Advances in Microbiology, Vol. 2 No. 1, 2012, pp. 1-7. doi: 10.4236/aim.2012.21001.
 T. Akiyama and M. C. Savin, “Populations of Antibiotic-Resistant Coliform Bacteria Change Rapidly in a Wastewater Effluent Dominated Stream,” Science of the Total Environment, Vol. 408, No. 24, 2010, pp. 6192- 6201. doi:10.1016/j.scitotenv.2010.08.055
 T. Akiyama, K. L. Asfahl and M. C. Savin, “Broad-Host- Range Plasmids in Treated Wastewater Effluent and Receiving Streams,” Journal of Environmental Quality, Vol. 39, No. 6, 2010, pp. 2211-2215. doi:10.2134/jeq2010.0228
 M. F. da Silva, I. Tiago, A. Veríssimo, R. A. R. Boaventura, O. C. Nunes and C. M. Manaia, “Antibiotic Resistance of Enterococci and Related Bacteria in an Urban Wastewater Treatment Plant,” FEMS Microbiology Ecology, Vol. 55, No. 2, 2005, pp. 322-329.
 B. E. Haggard, J. M. Galloway, W. R. Green and M. T. Meyer, “Pharmaceuticals and other Organic Chemicals in Selected North-Central and Northwestern Arkansas Streams,” Journal of Environmental Quality, Vol. 35, No. 4, 2006, pp. 1078-1087. doi:10.2134/jeq2005.0248
 S. Richardson, “Disinfection By-Products and Other Emerging Contaminants in Drinking Water,” Trends in Analytical Chemistry, Vol. 22, No. 10, 2003, pp. 666-684.
 S. Richardson and T. A. Ternes, “Water Analysis: Emerging Contaminants and Current Issues,” Analytical Chemistry, Vol. 77, No. 12, 2005, pp. 3807-3838.
 A. Pruden, R. T. Pei, H. Storteboom and K. H. Carlson, “Antibiotic Resistance Genes as Emerging Contaminants: Studies in Northern Colorado,” Environmental Science & Technology, Vol. 40, No. 23, 2006, pp. 7445-7450.
 World Health Organization (WHO), “Guidelines for the Safe Use of Wastewater, Excreta and Greywater. Volume 3: Wastewater and Excreta Use in Aquaculture,” WHO, Geneva, 2006.
 M. Droge, A. Pühler and W. Selbitschka, “Phenotypic and Molecular Characterization of Conjugative Antibiotic Resistance Plasmids Isolated from Bacterial Communities of Activated Sludge,” Molecular and General Genetics, Vol. 263, No. 3, 2000, pp. 471-482.
 E. Kristiansson, J. Fick, A. Janzon, R. Grabic, C. Rutgersson, B. Weijdegard, H. Soderstrom and D. G. J. Larsson, “Pyrosequencing of Antibiotic-Contaminated River Sediments Reveals High Levels of Resistance and Gene Transfer Elements,” Public Library of Science, Vol. 6, No. 2, 2011, Article ID: e17038.
 A. Schlüter, R. Szczepanowski, A. Pühler and E. M. Top, “Genomics of IncP-1 Antibiotic Resistance Plasmids Isolated from Wastewater Treatment Plants Provides Evidence for a Widely Accessible Drug Resistance Gene Pool,” FEMS Microbiology Reviews, Vol. 31, No. 4, 2007, pp. 449-477. doi:10.1111/j.1574-6976.2007.00074.x
 K. Smalla, H. Heuer, A. Gotz, D. Niemeyer, E. Kroger- recklenfort and E. Tietze, “Exogenous Isolation of Antibiotic Resistance Plasmids from Piggery Manure Slurries Reveals a High Prevalence and Diversity of IncQ-Like Plasmids,” Applied and Environmental Microbiology, Vol. 66, No. 11, 2000, pp. 4854-4862.
 A. Siddique and D. H. Figurski, “The Active Partition Gene incC of IncP Plasmids Is Required for Stable Maintenance in a Broad Range of Hosts,” Journal of Bacteriology, Vol. 184, No. 6, 2002, pp. 1788-1793.
 R. I. Aminov and R. I. Mackie, “Evolution and Ecology of Antibiotic Resistance Genes,” FEMS Microbiology Letters, Vol. 271, No. 2, 2007, pp. 147-161.
 J. Sikorski, N. Teschner and W. Wackernagel, “Highly Different Levels of Natural Transformation Are Associated with Genomic Subgroups within a Local Population of Pseudomonas stutzeri from Soil,” Applied and Environmental Microbiology, Vol. 68, No. 2, 2002, pp. 865- 873. doi:10.1128/AEM.68.2.865-873.2002
 A. Kontana, C. A. Papadimitriou, P. Samaras, A. Zdragas and M. Yiangou, “Effectiveness of Ozonation and Chlorination on Municipal Wastewater Treatment Evaluated by a Battery of Bioassays and Biomarkers,” Water Science and Technology, Vol. 60, No. 6, 2009, pp. 1497-1505.
 J. Kuo and L. Abustan, “Disinfection and Antimicrobial Processes,” Water Environment Research, Vol. 81, No. 10, 2009, pp. 1361-1375.
 M. A. Oneby, C. O. Bromley, J. H. Borchardt and D. S. Harrison, “Ozone Treatment of Secondary Effluent at U.S. Municipal Wastewater Treatment Plants,” Ozone Science & Engineering, Vol. 32, No. 1, 2010, pp. 43-55.
 G. B. Wickramanayake, A. J. Rubin and O. J. Sproul, “Inactivation of Giardia lamblia Cysts with Ozone,” Applied and Environmental Microbiology, Vol. 48, No. 3, 1984, pp. 671-672.
 G. R. Finch, E. K. Black, L. Gyürék and M. Belosevic, “Ozone Inactivation of Cryptosporidium parvum in Demand Free Phosphate Buffer Determined by in Vitro Excystation and Animal Infectivity,” Applied and Environmental Microbiology, Vol. 59, No. 12, 1993, pp. 4203-4210.
 N. K. Hunt and B. J. Marinas, “Kinetics of Escherichia coli Inactivation with Ozone,” Water Research, Vol. 31, No. 6, 1997, pp. 1355-1362.
 J. L. Rennecker, B. J. Marinas, J. H. Owens and E. W. Rice, “Inactivation of Cryptosporidium parvum Oocysts with Ozone,” Water Research, Vol. 33, No. 11, 1999, pp. 2481-2488. doi:10.1016/S0043-1354(99)00116-5
 A. Driedger, E. Staub, U. Pinkernell, B. Marinas, W. Koster and U. von Gunten, “Inactivation of Bacillus subtilis Spores and Formation of Bromate during Ozonation,” Water Research, Vol. 15, No. 12, 2001, pp. 2950-2960.
 U. von Gunten, “Ozonation of Drinking Water: Part I. Oxidation Kinetics and Product Formation,” Water Re- search, Vol. 37, No. 7, 2003, pp. 1443-1467.
 M. M. Huber, A. Gobel, A. Joss, N. Hermann, D. Loffler, S. Mcardell, A. Ried, H. Siegrist, T. A. Ternes and U. von Gunten, “Oxidation of Pharmaceuticals during Ozonation of Municipal Wastewater Effluents: A Pilot Study,” Environmental Science and Technology, Vol. 39, No. 11, 2005, pp. 4290-4299. doi:10.1021/es048396s
 N. James, “Soil Extract in Soil Microbiology,” Canadian Journal of Microbiology, Vol. 4, No. 4, 1958, pp. 363- 370. doi:10.1139/m58-038
 R. S. Jobanputra and N. Datta, “Trimethoprim R Factors in Enterobacteria from Clinical Specimens,” Journal of Medical Microbiology, Vol. 7, No. 2, 1974, pp. 169-177.
 L. Heijnen and G. Medema, “Quantitative Detection of E. coli, E. coli O157 and Other Shiga Toxin Producing E. coli in Water Samples Using a Culture Method Combined with Real-Time PCR,” Journal of Water & Health, Vol. 4, No. 4, 2006, pp. 487-498.
 A. Gotz, R. Pukall, E. Smit, E. Tietze, R. Prager, H. Tschope, J. D. van Elsas and K. Smalla, “Detection and Characterization of Broad-Host-Range Plasmids in Environmental Bacteria by PCR,” Applied and Environmental Microbiology, Vol. 62, No. 7, 1996, pp. 2621-2628.
 S. Tripathi, V. Pathak, D. M. Tripathi and B. D. Tripathi, “Application of Ozone Based Treatments of Secondary Effluents,” Bioresource Technology, Vol. 102, No. 3, 2011, pp. 2481-2486. doi:10.1016/j.biortech.2010.11.028
 P. Servais, J. Prats, J. Passerat and T. Garcia-Armisen, “Abundance of Culturable versus Viable Escherichia coli in Freshwater,” Canadian Journal of Microbiology, Vol. 55, No. 7, 2009, pp. 905-909. doi:10.1139/W09-043
 I. Arana, P. Santorum, A. Muela and I. Barcina, “Chlori- nation and Ozonation of Waste-Water: Comparative Analysis of Efficacy through the Effect on Escherichia coli Membranes,” Journal of Applied Microbiology, Vol. 86, No. 5, 1999, pp. 883-888.
 R. L. Whitman, Z. Ge, M. B. Nevers, A. B. Boehm, E. C. Chern, R. A. Haugland, A. M. Lukasik, M. Molina, K. Przybyla-Kelly, D. A. Shively, E. M. White, R. G. Zepp and M. N. Byappanahalli, “Relationship and Variation of qPCR and Culturable Enterococci Estimates in Ambient Surface Waters Are Predictable,” Environmental Science & Technology, Vol. 44, No. 13, 2010, pp. 5049-5054.
 I. R. Komanapalli and B. H. S. Lau, “Ozone-Induced Damage of Escherichia coli K-12,” Applied Environmental and Biotechnology, Vol. 46, No. 5-6, 1996, pp. 610-614.
 F. Zuma, J. Lin and S. B. Jonnalagadda, “Ozone-Initiated Disinfection Kinetics of Escherichia coli in Water,” Journal of Environmental Science and Health, Vol. 44, No. 1, 2009, pp. 48-56. doi:10.1080/10934520802515335
 R. L. Wolfe, “Ultraviolet Disinfection of Potable Water— Current Technology and Research Needs,” Envrionmental Science and Technology, Vol. 24, No. 6, 1990, pp. 768- 773. doi:10.1021/es00076a001
 J. Y. Maillard, “Bacterial Target Sites for Biocide Action,” Journal of Applied Microbiology, Vol. 92, No. 1, 2002, pp. 16S-27S. doi:10.1046/j.1365-2672.92.5s1.3.x
 A. Babic, M. B. Berkmen, C. A. Lee and A. D. Grossman, “Efficient Gene Transfer in Bacterial Cell Chains,” mBio, Vol. 2, No. 2, 2011, doi:10.1128/mbio.00027-11.
 M. Sota, H. Yano, J. M. Hughes, G. W. Daughdrill, Z. Abdo, L. J. Forney and E. M. Top, “Shifts in the Host Range of a Promiscuous Plasmid through Parallel Evolution of Its Replication Initiation Protein,” The ISME Journal, Vol. 4, No. 12, 2010, pp. 1568-1580.
 R. Szczepanowski, I. Krahn, N. Bohn, A. Pühler and A. Schlüter, “Novel Macrolide Resistance Module Carried by the IncP-1β Resistance Plasmid pRSB111, Isolated from a Wastewater Treatment Plant,” Antimicrobial Agents and Chemotherapy, Vol. 51, No. 2, 2005, pp. 673-678.