Techniques for the Assessment of Growth of Micro-organisms on Plumbing Materials Used in Contact with Potable Water Supplies

Water samples from plumbing installations have often been of poorer microbiological quality than samples collected from the distribution main. This deterioration is often associated with a musty taste or visible turbidity in the water and slimes have sometimes been observed on water fittings. A technique to assess the ability of plumbing materials to support microbial growth is described and the most common categories of materials capable of supporting growth are identified. The most significant micro-organisms frequently found to grow on unsuitable materials were coliform organisms, especially members of the genus Citrobacter. Aeromonas hydrophila, Pseudomonas aeruginosa and fungi.     

Detection of Protozoan Hosts for Legionella pneumophila in Engineered Water Systems by Using a Biofilm Batch Test

Legionella pneumophila proliferates in aquatic habitats within free-living protozoa, 17 species of which have been identified as hosts by using in vitro experiments. The present study aimed at identifying protozoan hosts for L. pneumophila by using a biofilm batch test (BBT). Samples (600 ml) collected from 21 engineered freshwater systems, with added polyethylene cylinders to promote biofilm formation, were inoculated with L. pneumophila and subsequently incubated at 37°C for 20 days. Growth of L. pneumophila was observed in 16 of 18 water types when the host protozoan Hartmannella vermiformis was added. Twelve of the tested water types supported growth of L. pneumophila or indigenous Legionella anisa without added H. vermiformis. In 12 of 19 BBT flasks H. vermiformis was indicated as a host, based on the ratio between maximum concentrations of L. pneumophila and H. vermiformis, determined with quantitative PCR (Q-PCR), and the composition of clone libraries of partial 18S rRNA gene fragments. Analyses of 609 eukaryotic clones from the BBTs revealed that 68 operational taxonomic units (OTUs) showed the highest similarity to free-living protozoa. Forty percent of the sequences clustering with protozoa showed ≥99.5% similarity to H. vermiformis. None of the other protozoa serving as hosts in in vitro studies were detected in the BBTs. In several tests with growth of L. pneumophila, the protozoa Diphylleia rotans, Echinamoeba thermarum, and Neoparamoeba sp. were identified as candidate hosts. In vitro studies are needed to confirm their role as hosts for L. pneumophila. Unidentified protozoa were implicated as hosts for uncultured Legionella spp. grown in BBT flasks at 15°C.Legionella pneumophila, the causative agent of Legionnaires'' disease, is a common inhabitant of natural freshwater environments and human-made water systems, including cooling towers, whirlpools, air-conditioning systems, and installations for warm tap water (14). In the aquatic environment L. pneumophila proliferates within certain free-living protozoa, which serve as its hosts (15, 30, 59). Environmental factors favoring the growth and survival of L. pneumophila in freshwater systems include a water temperature between 20°C and 45°C (41, 60) and the presence of biofilms and sediments on which the protozoan hosts can graze (30, 41, 56).Rowbotham (44) was the first to report the growth of L. pneumophila within free-living amoebae, which belonged to the genera Acanthamoeba and Naegleria. In vitro studies with cocultures have revealed that 14 species of amoebae, viz., Acanthamoeba spp. (1, 35, 44, 53), Balamuthia mandrillaris (47), Echinamoeba exundans (15), Hartmannella spp. (43), Naegleria spp. (38, 44, 53), and Vahlkampfia jugosa (43); the slime mold Dictyostelium discoideum (20, 48); and two species of the ciliate genus Tetrahymena (15, 26) can serve as hosts for L. pneumophila. Recently, it has been reported that L. pneumophila can also replicate within the intestinal tract of the microbiovorous nematode Caenorhabditis elegans (3).A number of the free-living protozoa mentioned above and others, e.g., Vannella spp. and Saccamoeba spp., have been observed in aquatic environments from which L. pneumophila was cultivated or in which it was detected with PCR (4, 42, 51, 52). However, it remains unknown which of these protozoa actually serve as hosts for L. pneumophila in the aquatic environment, including human-made water systems. Moreover, it cannot be excluded that free-living protozoa other than those tested in vitro can serve as hosts for L. pneumophila as well. Information is also lacking about protozoan hosts for Legionella anisa (13, 49), which is frequently present in water installations in temperate regions (11, 62). Furthermore, it is unknown which free-living protozoa serve as hosts for uncultured Legionella bacteria that can grow at temperatures of about 15°C (61; B. A. Wullings, G. Bakker, and D. van der Kooij, submitted for publication).L. pneumophila can proliferate in samples of surface water, effluent of wastewater treatment plants, potable water, and water from cooling towers incubated at 25°C, 35°C, or 37°C (28, 45, 56). Consequently, incubation of freshwater samples can be used to amplify protozoan hosts for L. pneumophila and other Legionella spp. In this study, different human-made water types were investigated using a biofilm batch test (BBT) system to (i) amplify and subsequently identify predominating, known, and yet-undescribed hosts for L. pneumophila and (ii) identify potential protozoan hosts for Legionella bacteria that can grow at 15°C.     

嗜肺军团菌生物膜形成及其影响因素

嗜肺军团菌在自然环境和人工供水系统中普遍存在,能够在阿米巴原虫和其他原生动物体内繁殖,所引起的军团菌病主要表现为严重的呼吸系统疾病。但在自然环境中,嗜肺军团菌的生存和繁殖受到多菌种生物膜形成和繁殖的影响,一些军团菌病的暴发与生物膜的存在相关。因此,阻止自然环境和人工水系统中生物膜的形成显然已成为降低水污染的有效策略之一。根据近年来的报道分别对影响嗜肺军团菌生物膜形成的生化因子和嗜肺军团菌的毒力因子,以及其他生物物种在嗜肺军团菌生物膜形成过程中所起的不同作用等进行综述。     

Necrotrophic Growth of Legionella pneumophila

This study examined whether Legionella pneumophila is able to thrive on heat-killed microbial cells (necrotrophy) present in biofilms or heat-treated water systems. Quantification by means of plate counting, real-time PCR, and flow cytometry demonstrated necrotrophic growth of L. pneumophila in water after 96 h, when at least 100 dead cells are available to one L. pneumophila cell. Compared to the starting concentration of L. pneumophila, the maximum observed necrotrophic growth was 1.89 log units for real-time PCR and 1.49 log units for plate counting. The average growth was 1.57 ± 0.32 log units (n = 5) for real-time PCR and 1.14 ± 0.35 log units (n = 5) for plate counting. Viability staining and flow cytometry showed that the fraction of living cells in the L. pneumophila population rose from the initial 54% to 82% after 96 h. Growth was measured on heat-killed Pseudomonas putida, Escherichia coli, Acanthamoeba castellanii, Saccharomyces boulardii, and a biofilm sample. Gram-positive organisms did not result in significant growth of L. pneumophila, probably due to their robust cell wall structure. Although necrotrophy showed lower growth yields compared to replication within protozoan hosts, these findings indicate that it may be of major importance in the environmental persistence of L. pneumophila. Techniques aimed at the elimination of protozoa or biofilm from water systems will not necessarily result in a subsequent removal of L. pneumophila unless the formation of dead microbial cells is minimized.     

Effect of Bacterial Interference on Biofilm Development by Legionella pneumophila

In the ecology of Legionella pneumophila a crucial role may be played by its relationship with the natural flora; thus we investigated the interactions between Legionella and other aquatic bacteria, particularly within biofilms. Among 80 aquatic bacteria screened for the production of bacteriocin-like substances (BLSs), 66.2% of them were active against L. pneumophila. The possible effect of some of these aquatic bacteria on the development and stability of L. pneumophila biofilms was studied. Pseudomonas fluorescens, the best BLS producer, showed the greatest negative effect on biofilm formation and strongly enhanced the detachment of Legionella. Pseudomonas aeruginosa, Burkholderia cepacia, Pseudomonas putida, Aeromonas hydrophila, and Stenotrophomonas maltophilia, although producing BLSs at different levels, were less active in the biofilm experiments. Acinetobacter lwoffii did not produce any antagonistic compound and was the only one able to strongly enhance L. pneumophila biofilm. Our results highlight that BLS production may contribute to determining the fate of L. pneumophila within ecological niches. The interactions observed in this study are important features of L. pneumophila ecology, which knowledge may lead to more effective measures to control the persistance of the germ in the environment.     

Distribution of Sequence-Based Types of Legionella pneumophila Serogroup 1 Strains Isolated from Cooling Towers,Hot Springs,and Potable Water Systems in China

Legionella pneumophila serogroup 1 causes Legionnaires'' disease. Water systems contaminated with Legionella are the implicated sources of Legionnaires'' disease. This study analyzed L. pneumophila serogroup 1 strains in China using sequence-based typing. Strains were isolated from cooling towers (n = 96), hot springs (n = 42), and potable water systems (n = 26). Isolates from cooling towers, hot springs, and potable water systems were divided into 25 sequence types (STs; index of discrimination [IOD], 0.711), 19 STs (IOD, 0.934), and 3 STs (IOD, 0.151), respectively. The genetic variation among the potable water isolates was lower than that among cooling tower and hot spring isolates. ST1 was the predominant type, accounting for 49.4% of analyzed strains (n = 81), followed by ST154. With the exception of two strains, all potable water isolates (92.3%) belonged to ST1. In contrast, 53.1% (51/96) and only 14.3% (6/42) of cooling tower and hot spring, respectively, isolates belonged to ST1. There were differences in the distributions of clone groups among the water sources. The comparisons among L. pneumophila strains isolated in China, Japan, and South Korea revealed that similar clones (ST1 complex and ST154 complex) exist in these countries. In conclusion, in China, STs had several unique allelic profiles, and ST1 was the most prevalent sequence type of environmental L. pneumophila serogroup 1 isolates, similar to its prevalence in Japan and South Korea.     

Integrated Real-Time PCR for Detection and Monitoring of Legionella pneumophila in Water Systems

We evaluated a ready-to-use real-time quantitative Legionella pneumophila PCR assay system by testing 136 hot-water-system samples collected from 55 sites as well as 49 cooling tower samples collected from 20 different sites, in parallel with the standard culture method. The PCR assay was reproducible and suitable for routine quantification of L. pneumophila. An acceptable correlation between PCR and culture results was obtained for sanitary hot-water samples but not for cooling tower samples. We also monitored the same L. pneumophila-contaminated cooling tower for 13 months by analyzing 104 serial samples. The culture and PCR results were extremely variable over time, but the curves were similar. The differences between the PCR and culture results did not change over time and were not affected by regular biocide treatment. This ready-to-use PCR assay for L. pneumophila quantification could permit more timely disinfection of cooling towers.     

Growth of Legionella pneumophila in continuous culture

A method was developed to grow Legionella pneumophila in continuous culture. A chemostat was used to simulate nutrient-limited, submaximal growth in the natural environmental and to provide a precisely controlled growth regimen. Cultures grew under forced aeration under conditions yielding up to 38% saturation of dissolved oxygen; supplemental CO2 (5%) at the same gas flow rates as ambient air had no effect on culture growth. Pleomorphism was observed during growth under all conditions. Pigment was produced only at D less than 0.03 h-1. Catalase was produced at higher growth rates but not at higher temperatures. The pathogenicity was unaffected by altering either the growth rate or the growth temperature.     

Rapid Detection and Enumeration of Legionella pneumophila in Hot Water Systems by Solid-Phase Cytometry

A new method for the rapid and sensitive detection of Legionella pneumophila in hot water systems has been developed. The method is based on an IF assay combined with detection by solid-phase cytometry. This method allowed the enumeration of L. pneumophila serogroup 1 and L. pneumophila serogroups 2 to 6, 8 to 10, and 12 to 15 in tap water samples within 3 to 4 h. The sensitivity of the method was between 10 and 100 bacteria per liter and was principally limited by the filtration capacity of membranes. The specificity of the antibody was evaluated against 15 non-Legionella strains, and no cross-reactivity was observed. When the method was applied to natural waters, direct counts of L. pneumophila were compared with the number of CFU obtained by the standard culture method. Direct counts were always higher than culturable counts, and the ratio between the two methods ranged from 1.4 to 325. Solid-phase cytometry offers a fast and sensitive alternative to the culture method for L. pneumophila screening in hot water systems.     

Influence of aquatic microorganisms on Legionella pneumophila survival

The ability of aquatic bacteria Pseudomonas fluorescens SSD (Ps-D) and Pseudomonas putida SSC (Ps-C) to support the persistence of Legionella pneumophila (Lp-1) in an artificial water microcosm was investigated for 42 day, at two different incubation temperatures. At 4 degrees C, individually suspended Lp-1 was no longer detectable just after 24 hours, while in co-cultures with Pseudomonas, Lp1 showed a better survival capability. At 30 degrees C, Lp-1 alone displayed high survival rates over the entire period of observation. When Lp-1 was inoculated with Ps-C and Ps-D, its count showed a marked decrease, followed by a gradual and costant decline.     

Growth of Legionella pneumophila in continuous culture.

A method was developed to grow Legionella pneumophila in continuous culture. A chemostat was used to simulate nutrient-limited, submaximal growth in the natural environmental and to provide a precisely controlled growth regimen. Cultures grew under forced aeration under conditions yielding up to 38% saturation of dissolved oxygen; supplemental CO2 (5%) at the same gas flow rates as ambient air had no effect on culture growth. Pleomorphism was observed during growth under all conditions. Pigment was produced only at D less than 0.03 h-1. Catalase was produced at higher growth rates but not at higher temperatures. The pathogenicity was unaffected by altering either the growth rate or the growth temperature.     

Multiplication of Legionella pneumophila in Unsterilized Tap Water

[This corrects the article on p. 1333 in vol. 43.].     

Planktonic Replication Is Essential for Biofilm Formation by Legionella pneumophila in a Complex Medium under Static and Dynamic Flow Conditions

Legionella pneumophila persists for a long time in aquatic habitats, where the bacteria associate with biofilms and replicate within protozoan predators. While L. pneumophila serves as a paradigm for intracellular growth within protozoa, it is less clear whether the bacteria form or replicate within biofilms in the absence of protozoa. In this study, we analyzed surface adherence of and biofilm formation by L. pneumophila in a rich medium that supported axenic replication. Biofilm formation by the virulent L. pneumophila strain JR32 and by clinical and environmental isolates was analyzed by confocal microscopy and crystal violet staining. Strain JR32 formed biofilms on glass surfaces and upright polystyrene wells, as well as on pins of “inverse” microtiter plates, indicating that biofilm formation was not simply due to sedimentation of the bacteria. Biofilm formation by an L. pneumophila fliA mutant lacking the alternative sigma factor σ²⁸ was reduced, which demonstrated that bacterial factors are required. Accumulation of biomass coincided with an increase in the optical density at 600 nm and ceased when the bacteria reached the stationary growth phase. L. pneumophila neither grew nor formed biofilms in the inverse system if the medium was exchanged twice a day. However, after addition of Acanthamoeba castellanii, the bacteria proliferated and adhered to surfaces. Sessile (surface-attached) and planktonic (free-swimming) L. pneumophila expressed β-galactosidase activity to similar extents, and therefore, the observed lack of proliferation of surface-attached bacteria was not due to impaired protein synthesis or metabolic activity. Cocultivation of green fluorescent protein (GFP)- and DsRed-labeled L. pneumophila led to randomly interspersed cells on the substratum and in aggregates, and no sizeable patches of clonally growing bacteria were observed. Our findings indicate that biofilm formation by L. pneumophila in a rich medium is due to growth of planktonic bacteria rather than to growth of sessile bacteria. In agreement with this conclusion, GFP-labeled L. pneumophila initially adhered in a continuous-flow chamber system but detached over time; the detachment correlated with the flow rate, and there was no accumulation of biomass. Under these conditions, L. pneumophila persisted in biofilms formed by Empedobacter breve or Microbacterium sp. but not in biofilms formed by Klebsiella pneumoniae or other environmental bacteria, suggesting that specific interactions between the bacteria modulate adherence.     

Necessity and Effect of Combating Legionella pneumophila in Municipal Shower Systems

The objective was to obtain research-based, holistic knowledge about necessity and effect of practiced measures against L. pneumophila in municipal shower systems in Stavanger, Norway. The effects of hot water treatment and membrane-filtering were investigated and compared to no intervention at all. The studies were done under real-world conditions. Additionally, a surveillance pilot study of municipal showers in Stavanger was performed. The validity of high total plate count (TPC) as an indication of L. pneumophila was evaluated. A simplified method, named “dripping method”, for detection and quantification of L. pneumophila was developed. The sensitivity of the dripping method is 5 colony-forming units of L. pneumophila/ml. The transference of L. pneumophila from shower water to aerosols was studied. Interviews and observational studies among the stakeholders were done in order to identify patterns of communication and behavior in a Legionella risk perspective. No substantial effects of the measures against L. pneumophila were demonstrated, except for a distally placed membrane filter. No significant positive correlation between TPC and L. pneumophila concentrations were found. L. pneumophila serogroup 2–14 was demonstrated in 21% of the 29 buildings tested in the surveillance pilot. Relatively few cells of L. pneumophila were transferred from shower water to aerosols. Anxiety appeared as the major driving force in the risk governance of Legionella. In conclusion, the risk of acquiring Legionnaires'' disease from municipal shower systems is evaluated as low and uncertain. By eliminating ineffective approaches, targeted Legionella risk governance can be practiced. Risk management by surveillance is evaluated as appropriate.     

Temperature-Regulated Formation of Mycelial Mat-Like Biofilms by Legionella pneumophila

Fifty strains representing 38 species of the genus Legionella were examined for biofilm formation on glass, polystyrene, and polypropylene surfaces in static cultures at 25°C, 37°C, and 42°C. Strains of Legionella pneumophila, the most common causative agent of Legionnaires' disease, were found to have the highest ability to form biofilms among the test strains. The quantity, rate of formation, and adherence stability of L. pneumophila biofilms showed considerable dependence on both temperature and surface material. Glass and polystyrene surfaces gave between two- to sevenfold-higher yields of biofilms at 37°C or 42°C than at 25°C; conversely, polypropylene surface had between 2 to 16 times higher yields at 25°C than at 37°C or 42°C. On glass surfaces, the biofilms were formed faster but attached less stably at 37°C or 42°C than at 25°C. Both scanning electron microscopy and confocal laser scanning microscopy revealed that biofilms formed at 37°C or 42°C were mycelial mat like and were composed of filamentous cells, while at 25°C, cells were rod shaped. Planktonic cells outside of biofilms or in shaken liquid cultures were rod shaped. Notably, the filamentous cells were found to be multinucleate and lacking septa, but a recA null mutant of L. pneumophila was unaffected in its temperature-regulated filamentation within biofilms. Our data also showed that filamentous cells were able to rapidly give rise to a large number of short rods in a fresh liquid culture at 37°C. The possibility of this biofilm to represent a novel strategy by L. pneumophila to compete for proliferation among the environmental microbiota is discussed.     

Microbial Biofilm Formation and Contamination of Dental-Unit Water Systems in General Dental Practice

Dental-unit water systems (DUWS) harbor bacterial biofilms, which may serve as a haven for pathogens. The aim of this study was to investigate the microbial load of water from DUWS in general dental practices and the biofouling of DUWS tubing. Water and tube samples were taken from 55 dental surgeries in southwestern England. Contamination was determined by viable counts on environmentally selective, clinically selective, and pathogen-selective media, and biofouling was determined by using microscopic and image analysis techniques. Microbial loading ranged from 500 to 10⁵ CFU · ml⁻¹; in 95% of DUWS water samples, it exceeded European Union drinking water guidelines and in 83% it exceeded American Dental Association DUWS standards. Among visible bacteria, 68% were viable by BacLight staining, but only 5% of this “viable by BacLight” fraction produced colonies on agar plates. Legionella pneumophila, Mycobacterium spp., Candida spp., and Pseudomonas spp. were detected in one, five, two, and nine different surgeries, respectively. Presumptive oral streptococci and Fusobacterium spp. were detected in four and one surgeries, respectively, suggesting back siphonage and failure of antiretraction devices. Hepatitis B virus was never detected. Decontamination strategies (5 of 55 surgeries) significantly reduced biofilm coverage but significantly increased microbial numbers in the water phase (in both cases, P < 0.05). Microbial loads were not significantly different in DUWS fed with soft, hard, deionized, or distilled water or in different DUWS (main, tank, or bottle fed). Microbiologically, no DUWS can be considered “cleaner” than others. DUWS deliver water to patients with microbial levels exceeding those considered safe for drinking water.     

Rapid Method for Enumeration of Viable Legionella pneumophila and Other Legionella spp. in Water

A sensitive and specific method has been developed to enumerate viable L. pneumophila and other Legionella spp. in water by epifluorescence microscopy in a short period of time (a few hours). This method allows the quantification of L. pneumophila or other Legionella spp. as well as the discrimination between viable and nonviable Legionella. It simultaneously combines the specific detection of Legionella cells using antibodies and a bacterial viability marker (ChemChrome V6), the enumeration being achieved by epifluorescence microscopy. The performance of this immunological double-staining (IDS) method was investigated in 38 natural filterable water samples from different aquatic sources, and the viable Legionella counts were compared with those obtained by the standard culture method. The recovery rate of the IDS method is similar to, or higher than, that of the conventional culture method. Under our experimental conditions, the limit of detection of the IDS method was <176 Legionella cells per liter. The examination of several samples in duplicates for the presence of L. pneumophila and other Legionella spp. indicated that the IDS method exhibits an excellent intralaboratory reproducibility, better than that of the standard culture method. This immunological approach allows rapid measurements in emergency situations, such as monitoring the efficacy of disinfection shock treatments. Although its field of application is as yet limited to filterable waters, the double-staining method may be an interesting alternative (not equivalent) to the conventional standard culture methods for enumerating viable Legionella when rapid detection is required.