Semester

Fall

Date of Graduation

2008

Document Type

Thesis

Degree Type

MS

College

Statler College of Engineering and Mineral Resources

Department

Civil and Environmental Engineering

Committee Chair

Benoit Van Aken.

Abstract

2-(2,5-Dichlorophenoxy)-5-chlorophenol or Triclosan is a widely used antimicrobial agent included in a multitude of products, such as soap, toothpaste, and personal care products.;The objective of this research was to investigate the effects of Triclosan on the resistance of activated sludge bacteria to conventional disinfection processes applied in wastewater treatment processes, such as chlorination and UV.;Specific Aim 1. A complex bacterial community collected from an active sludge was acclimated over several sub-cultures in the presence of different concentrations of Triclosan (from 0.0 to 20 mg L-1) and then exposed to sub-lethal doses of chlorine and UV irradiation. Determination of bacterial viability by direct counting and flow-cytometry showed that microbial communities exposed to Triclosan exhibited an increased susceptibility to chlorination and UV by comparison to non-exposed bacteria.;Specific Aim 2. In order to determine the origin of the change of susceptibility observed in Specific Aim 1, we characterized the microbial communities by construction of gene library and restriction fragment length polymorphism (RFLP). Results showed that acclimation on Triclosan resulted in a significant reduction of the bacterial diversity, suggesting that the change of susceptibility to disinfection could be explained, at least partly, by a change of the bacterial community structure. To investigate whether exposure to Triclosan could select bacterial species with a lower susceptibility to chlorination, two highly resistant strains were isolated from Triclosan-acclimated cultures and identified as Pseudomonas fluorescens and Serratia marcescens, which are opportunistic pathogens previously documented as highly resistant to Triclosan. This time, flow cytometry results showed that one of the strains, S. marcescens, exhibited a significant decrease of susceptibility to chlorination after acclimation in the presence of high concentration of Triclosan.;Specific Aim 3. In order to investigate further the relative susceptibility to chlorination of different species in Triclosan-exposed activated sludge bacteria, the microbial community structure was characterized by terminal-restriction fragment length polymorphism (T-RFLP) after sorting dead and living bacteria by flow-cytometry. Our preliminary results showed again that acclimation on Triclosan resulted in a significant reduction of the bacterial diversity, and, more importantly, that several species in Triclosan-acclimated bacterial suspensions exhibited a much higher resistance to chlorination.;Our results provide the first evidence that acclimation on sub-lethal doses of the antimicrobial agent Triclosan can result in decreasing susceptibility of opportunistic pathogens, which could have potential important implication for public health. In addition, our experiments combining flow-cytometry for dead-living bacteria sorting with T-RFLP for microbial community structure characterization opens the way for further research on the effect of antimicrobial agents on the relative susceptibility of different pathogens to disinfection systems.

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