Presence and survival of Legionella in drinking water distribution systems

Legionella are a relevant example of a poorly understood water-born pathogen often associated with water biofilms. These bacteria are ubiquitous in natural and artificial water supplies where protozoan host endoparasitization and biofilm association are involved in their life cycle. Humans can become incidental hosts potentially resulting in the deadly pneumonia Legionnaires' Disease. Current Legionella knowledge is severely lacking in several key areas, particularly concerning the interactions between the bacteria and the biofilms they colonize. To further elucidate the ecology and survival of Legionella in distribution systems, three approaches were taken: 1) examining biofilm samples collected from water meters, 2) inoculating a model distribution system with Legionella, and 3) assessing the survival of Legionella in tap water at varying temperatures. Private residence drinking water meters were collected from two general locations within Central Arizona, 40 from area A and 30 from area B. Upon removal for repair by local city personnel, the meters were sampled within two hours. Biofilms were collected by scraping the inlet of each meter using nylon brushes. PCR was performed on samples using Legionella spp. 16s RNA primers and L. pneumophila mip gene primers, followed by sequencing for confirmation. PCR positive samples were then cultured onto BCYE-GPCV media. Of the meter samples, 9 tested positive for Legionella (all from Area A) and 5 of these samples also tested positive for L. pneumophila. A model water distribution system was used to examine the dissemination and survival of Legionella into water system. This 50L model consists of PVC and copper tubing attached to a reservoir holding coupons for biofilm formation and circulates tap water from the city of Tempe, AZ. After several weeks of biofilm development, a culture of L. pneumophila was inoculated into the system. Through the course of 131 days, water and biofilm samples were collected from the system and analyzed for the presence of Legionella using the techniques previously described. Throughout the entire course of the experiment, Legionella remained culturable in both water and biofilm samples. Furthermore, complete association of Legionella with the system's biofilms was not observed. A bench scale study was performed in which L. pneumophila cells were inoculated into tubes containing tap and incubated at 4 and 25° C, with concentrations measured over time. Both temperatures showed a steady decrease in concentrations, followed by stabilization. This occurred almost 30 days earlier at 25° C with final population levels nearly 3-fold higher than at 4° C. The results provided by this study document the survival of Legionella under different environmental conditions and provide relevant knowledge in identifying conditions which promote survival and potential growth of Legionella, information that could prove useful for maintenance and monitoring procedures in drinking water distribution systems.