The Critical Challenge of Antibiotic Resistance: Are Wastewater Treatment Plants a Concern? Kourtney Brown 1, Stefan Walston 2, Channah Rock 2, & Jean E. McLain 3 1 Sonoran Science Academy-Tucson, Tucson AZ 2 Department of Soil, Water, and Environmental Science, University of Arizona, Tucson AZ 3 Water Resources Research Center, University of Arizona, Tucson AZ Preliminary Data Methods Each Isolate was grown in EC broth (Becton, Dickinson and Company, Franklin Lakes, NJ) and incubated at 37ºC for 24 hours. Sample Collection Stages After growth, the isolates were spread plated on MacConkey agar (Becton, Dickinson and Company,). This process was completed twice, each time followed by a 24 hour incubation at 37ºC. 5 colonies from each plate were selected for plating on 96-well antibiotic resistance plates (Trek Diagnostics, Cleveland, OH). Plates were incubated for 24 hours before being assessed for growth using fluorescence assay in accordance with manufacturer instructions. TREK antibiotic resistance plates. test cultures for resistance against 25 antibiotics where resistance is indicated by culture growth beyond an inhibitory concentration identified by the Clinical and Laboratory Standards Institute (CLSI, 2010). Abstract In 2000, a World Health Organization (WHO) report identified antibiotic resistance as the most critical health challenge of the next century. With this, the WHO has stated that "antibiotic resistance is rapidly becoming a major public health risk and is threatening to undo decades of advances in our ability to treat disease.” Wastewater entering treatment plants from hospitals and clinics may contain trace levels of antibiotics. During the treatment process, bacteria are exposed to these residual chemicals, with the result that antibiotic resistant strains might be selectively enriched in the final recycled water product. Little is known of treatment plant processes that may contribute to, or reduce, the development of antibiotic resistance. Such knowledge is critical to municipalities that increasingly use recycled water for irrigation of sport fields and playgrounds in Arizona. 48 Gram negative bacteria previously isolated from treatment plants across Arizona were used (Walston, 2013); these isolates were selectively enriched from recycled water samples taken from the primary influent, and the secondary basin along the treatment process (Figure 1). Primary treatment exposes bacteria to trace levels of antibiotics in wastewater under ideal growth conditions, and we hypothesized that this may be a site where increases in resistance could occur. Identification of treatment technologies that may exacerbate or reduce antibiotic resistance will contribute to improved wastewater treatment technologies. Preliminary Results and Future Directions Figures 2 indicates lower levels of antimicrobial resistance in secondary effluents, contrary to our initial hypothesis that initial treatment would INCREASE resistance levels. This trend is also seen in Figure 3, which indicates a decrease in the number of organisms showing resistance after secondary treatment; these decrease occurs in 13 antibiotics Future work planned for Summer 2014: Screening of Gram positive isolates for resistance to antibiotics; utilization of real-time quantitative PCR to enumerate resistance genes in DNA extracted from raw wastewater (primary and secondary) samples. Acknowledgements This research is funded through the AZ Partners in Science by the Research Cooperation. This research was made possible by the collaboration of Dr. Jean McLain’s lab in the Department of Soil, Water and Environmental Science. Support of the research was made possible by McLain laboratory technicians Elissa Malott and Victoria Obergh. Figure 1. Example of water treatment process. Clinical and Laboratory Standards Institute (CLSI) (2010) Performance standards for antimicrobial susceptibility testing, 20 th informational supplement. Document M100-S20, CLSI, Wayne, PA. Walston, S. (2013). Does Increasing Solids Retention Time During the Wastewater Treatment Process Affect the Persistence of Antibiotic Resistance Genes?, Master’s Thesis, School of Soil, Water and Environmental Science, University of Arizona, Tucson. Works Cited