Consumer exposure to opportunistic pathogens in potable water systems poses a significant challenge to public health as manifested by numerous cases of pneumonia, non-tuberculosis lung disease, and keratitis eye infections. Water utilities have extensive understanding in control of heterotrophic and coliform bacteria re-growth in water distribution systems via disinfection, control of assimilable organic carbon (AOC), and biologically degradable organic carbon (BDOC). However, much little is known about the effect of AOC on the proliferation of heterotrophic bacteria and pathogens within premise plumbing. This thesis is the first systematic examination of opportunistic pathogen persistence and amplification in simulated glass water heaters (SGWH) as a function of influent organic matter concentration. The role of plumbing conditions that may internally generate AOC is critically examined as part of this evaluation. Strong correlations were often observed between influent organic matter and heterotrophic bacteria in effluent of SGWH as indicated by 16S rRNA gene abundance (average R2 value of 0.889 and 0.971 for heterotrophic organisms and 16S rRNA respectively). The correlation was strongest if water turnover was more frequent (every 48-72 hours) and decreased markedly when water changes were less frequent (stagnation up to 7 days). No simple correlations were identified between the concentration of pathogenic bacteria (L. pneumophila, M. avium, A. polyphaga, and H. vermiformis) and AOC, although correlations were observed between M. avium and TOC over a limited range (and only for a subset of experiments). Indeed, there was little evidence that Legionella and Acanthamoeba proliferated under any of the conditions tested in this work. Parallel experiments were conducted to examine the extent to which factors present in premise plumbing (e.g. sacrificial magnesium anode rods, cross-linked polyethylene, nitrifying bacteria, and iron) could influence water chemistry and influence growth of bacteria or specified pathogens. Although these factors could strongly influence pH, dissolved oxygen concentrations, and levels of organic matter (e.g. iron, magnesium, nitrifying), there was no major impact on effluent concentrations of either heterotrophic bacteria or premise plumbing pathogens under the conditions investigated. While additional research is needed to confirm these findings, at present, there is no evidence of correlations between organic matter and pathogen concentrations from SGWH under conditions tested. Substantial effort was also invested in attempting to identify SGWH and oligotrophic nutrient conditions that would consistently support L. pneumophila and A. polyphaga amplification. A review of the literature indicates no prior examples of large scale amplification of these microorganisms at nutrient levels commonly found in synthesized potable water. It is likely that a complex combination of abiotic and biotic factors (i.e. micronutrients, necrotrophic growth, ambient water temperature, disinfectant type and dose, plumbing materials, water usage patterns), which are not yet fully understood, control the amplification and viability of these pathogenic organisms in premise plumbing systems. / Master of Science

Mycobacterium avium and Mycobacterium intracellulare are environmental opportunistic pathogens whose source for human infection is water and soil. M. avium and M. intracellulare cause pulmonary infections (tuberculosis) in immunocompetent individuals and bacteremia in immunodeficient individuals (e.g. AIDS). One factor likely influencing the lack of success of antibiotic therapy in patients would be their ability to form biofilms. Growth in biofilms might result in antimicrobial resistance because (1) cells are protected by layers of other cells and extracellular material (2) and differences in physiologic state of cells as a consequence of growing on surfaces. The objectives of the work were to (1) establish methods for reproducible growth of mycobacterial biofilms (2) measure the formation of biofilms on surfaces by cells of M. avium and M. intracellulare (3) measure the antibiotic- and chlorine- susceptibility of M. avium and M. intracellulare strain TMC1406T in cell grown in suspension, cells grown in biofilms and suspended and of cells grown in biofilms (4) measure the hydrophobicity of M. avium and M. intracellulare grown in suspension and in biofilms. Methods were developed for growing mycobacteria in biofilms in polystyrene flasks and on glass beads. Although both strains formed biofilms, M. intracellulare strain TMC 1406T more readily formed biofilms than M. avium strain A5 in polystyrene flasks. The majority of M. intracellulare strain TMC 1406T cells grew on the walls of the flasks rather than in suspension like M. avium strain A5. The susceptibility of M7H9 medium-grown cells of M. avium strain A5 and M. intracellulare strain TMC 1406T cells grown in suspension, cells grown in biofilms and suspended and cells grown in biofilms was measured against clarithromycin, ethambutol, kanamycin, rifampicin and streptomycin. Cells grown in biofilms and exposed to antibiotics in biofilms were five-fold resistant to antibiotics than were cells grown in biofilms and exposed in suspension. Cells grown and exposed in suspension were ten-fold more sensitive to antibiotics than were cells grown in biofilms and exposed in suspension. The chlorine susceptibility of cells grown in medium and water was also measured. Cells grown in biofilms were more resistant to chlorine than cells grown in biofilms and suspended. Cells grown in suspension were more sensitive to chlorine than cells grown in biofilms and suspended. The hydrophobicity data (i.e., hexadecane adherence and contact angle measurements) showed that cells grown in biofilms are more hydrophobic than cells grown in biofilms and suspended and cells grown in suspension. It is clear that there are physiological changes between cells grown in suspension, cells grown in biofilms and suspended and cells in biofilms. / Master of Science