Biofilm Composition and Threshold Concentration for Growth of Legionella pneumophila on Surfaces Exposed to Flowing Warm Tap Water without Disinfectant

Abstract

Legionella pneumophila in potable water installations poses a potential health risk, but quantitative information about its replication in biofilms in relation to water quality is scarce. Therefore, biofilm formation on the surfaces of glass and chlorinated polyvinyl chloride (CPVC) in contact with tap water at 34 to 39°C was investigated under controlled hydraulic conditions in a model system inoculated with biofilm-grown L. pneumophila. The biofilm on glass (average steady-state concentration, 23 Ϯ 9 pg ATP cm Ϫ2 ) exposed to treated aerobic groundwater (0.3 mg C liter Ϫ1 ; 1 g assimilable organic carbon [AOC] liter Ϫ1 ) did not support growth of the organism, which also disappeared from the biofilm on CPVC (49 Ϯ 9 pg ATP cm Ϫ2 ) after initial growth. L. pneumophila attained a level of 4.3 log CFU cm Ϫ2 in the biofilms on glass (1,055 Ϯ 225 pg ATP cm Ϫ2 ) and CPVC (2,755 Ϯ 460 pg ATP cm Ϫ2 ) exposed to treated anaerobic groundwater (7.9 mg C liter Ϫ1 ; 10 g AOC liter Ϫ1 ). An elevated biofilm concentration and growth of L. pneumophila were also observed with tap water from the laboratory. The Betaproteobacteria Piscinibacter and Methyloversatilis and amoeba-resisting Alphaproteobacteria predominated in the clones and isolates retrieved from the biofilms. In the biofilms, the Legionella colony count correlated significantly with the total cell count (TCC), heterotrophic plate count, ATP concentration, and presence of Vermamoeba vermiformis. This amoeba was rarely detected at biofilm concentrations of Ͻ100 pg ATP cm Ϫ2 . A threshold concentration of approximately 50 pg ATP cm Ϫ2 (TCC ϭ 1 ϫ 10 6 to 2 ϫ 10 6 cells cm Ϫ2 ) was derived for growth of L. pneumophila in biofilms.IMPORTANCE Legionella pneumophila is the etiologic agent in more than 10,000 cases of Legionnaires' disease that are reported annually worldwide and in most of the drinking water-associated disease outbreaks reported in the United States. The organism proliferates in biofilms on surfaces exposed to warm water in engineered freshwater installations. An investigation with a test system supplied with different types of warm drinking water without disinfectant under controlled hydraulic conditions showed that treated aerobic groundwater (0.3 mg liter Ϫ1 of organic carbon) induced a low biofilm concentration that supported no or very limited growth of L. pneumophila. Elevated biofilm concentrations and L. pneumophila colony counts were observed on surfaces exposed to two types of extensively treated groundwater, containing 1.8 and 7.9 mg C liter Ϫ1 and complying with the microbial water quality criteria during distribution. Control measures in warm tap water installations are therefore essential for preventing growth of L. pneumophila.

Primary Colonizing Betaproteobacteriales Play a Key Role in the Growth of Legionella pneumophila in Biofilms on Surfaces Exposed to Drinking Water Treated by Slow Sand Filtration

“…The logarithmic means of the LpCs did not correlate with the BFPs on glass and CPVC (Table 3). However, in the biofilms exposed to samples from SSFs I and III, the high LpC values with high CFU/pg ATP ratios fitted with the relationship (solid line) derived in a previous study (9). The LpC/BfC ratios of a number of colony counts in the biofilms exposed to samples from SSFs II and IVD exceeded the relationship presented by the solid line ( Fig.…”

“…In 1986, the Health Council of the Netherlands recommended notification of legionellosis cases, as well as temperature (T) management (T Ͻ 25°C and T Ͼ 60°C) to prevent growth of L. pneumophila in premise plumbing systems (5). Following the large LD outbreak at a flower show in 1999 (6), measures for control of Legionella were incorporated in drinking water regulations, and investigations into the relationship between drinking water quality, biofilm formation potential (BFP), and the multiplication of Legionella pneumophila bacteria in tap water installations were conducted (7)(8)(9). The use of a model system, the boiler biofilm monitor (BBM), representing worst-case conditions (intermittent flow at 37°C), revealed that L. pneumophila can multiply at low biofilm concentrations (BfCs) due to its ability to proliferate within protozoan hosts consuming oligotrophic bacteria that grow on drinking water-exposed surfaces (9).…”

Online characterization of bacterial processes in drinking water systems

“…However, with increasing bacterial cell numbers in the drinking water during distribution, (i) more biofilm on the inside of the pipes 5,6 , and (ii) a larger chance of elevated numbers of higher organisms such as aquatic sow bugs (e.g., Asellidae, 1-12 mm) and oligochaete worms 6 , a (iii) higher risk of occurrence of Aeromonas 5 , total coliforms 7 , Escherichia coli 8 , and opportunistic pathogens such as Pseudomonas sp. 9 and NTB mycobacteria 10 , as well as Legionella pneumophila 11 will occur in the network. Moreover, there is higher risk for consumer complaints (e.g., brown water, smell) and higher costs (e.g., corrosion, misreading of water meters).…”

Photocatalytic biocidal effect of copper doped TiO2 nanotube coated surfaces under laminar flow, illuminated with UVA light on Legionella pneumophila

“…The tearing of biofilms is dependent on materials, speed and type of water flow. In the water systems turbulent water discharged the biofilm and intensify the spread of bacteria in comparison to laminar, or stagnant water which does not stimulate the spread, but allows the unobstructed growth of biofilm [6,11,12] L. pneumophila bacteria is capable of surviving under high humidity conditions and a relatively large range of ambient temperature between 20˚C-50˚C; however, it survives even at temperatures below freezing, due its protection in the biofilm and amebas. In addition, its ability to survive is also associated with symbiotic and parasitic interactions with other microorganisms.…”