Concentration and diversity of uncultured Legionella spp. in two unchlorinated drinking water supplies with different concentrations of natural organic matter

Two unchlorinated drinking water supplies were investigated to assess the potential of water treatment and distribution systems to support the growth of Legionella spp. The treatment plant for supply A distributed treated groundwater with a low concentration (<0.5 ppm of C) of natural organic matter (NOM), and the treatment plant for supply B distributed treated groundwater with a high NOM concentration (8 ppm of C). In both supplies, the water temperature ranged from about 10°C after treatment to 18°C during distribution. The concentrations of Legionella spp. in distributed water, analyzed with quantitative PCR (Q-PCR), averaged 2.9 (± 1.9) × 10(2) cells liter(-1) in supply A and 2.5 (± 1.6) × 10(3) cells liter(-1) in supply B. No Legionella was observed with the culture method. A total of 346 clones (96 operational taxonomical units [OTUs] with ≥97% sequence similarity) were retrieved from water and biofilms of supply A and 251 (43 OTUs) from supply B. The estimation of the average value of total species richness (Chao1) in supply A (153) was clearly higher than that for supply B (58). In each supply, about 77% of the sequences showed <97% similarity to described species. Sequences related to L. pneumophila were only incidentally observed. The Legionella populations of the two supplies are divided into two distinct clusters based on distances in the phylogenetic tree as fractions of the branch length. Thus, a large variety of mostly yet-undescribed Legionella spp. proliferates in unchlorinated water supplies at temperatures below 18°C. The lowest concentration and greatest diversity were observed in the supply with the low NOM concentration.     

Survival and multiplication of Legionella pneumophila in municipal drinking water systems

Studies were conducted to investigate the survival and multiplication of Legionella spp. in public drinking water supplies. An attempt was made, over a period of several years, to isolate legionellae from a municipal system. Sampling sites included the river water supply, treatment plant, finished water reservoir system, mains, and distribution taps. Despite the use of several isolation techniques, Legionella spp. could not be detected in any of the samples other than those collected from the river. It was hypothesized that this was due to the maintenance of a chlorine residual throughout the system. To investigate the potential for Legionella growth, additional water samples, collected from throughout the system, were dechlorinated, pasteurized, and inoculated with Legionella pneumophila. Subsequent growth indicated that many of these samples, especially those collected from areas affected by an accumulation of algal materials, exhibited a much greater ability to support Legionella multiplication than did river water prior to treatment. Chemical analyses were also performed on these samples. Correlation of chemical data and experimental growth results indicated that the chemical environment significantly affects the ability of the water to support multiplication, with turbidity, organic carbon, and certain metals being of particular importance. These studies indicate that the potential exists for Legionella growth within municipal systems and support the hypothesis that public water supplies may contaminate the plumbing systems of hospitals and other large buildings. The results also suggest that useful methods to control this contamination include adequate treatment plant filtration, maintenance of a chlorine residual throughout the treatment and distribution network, and effective covering of open reservoirs.     

Survival and multiplication of Legionella pneumophila in municipal drinking water systems.

Studies were conducted to investigate the survival and multiplication of Legionella spp. in public drinking water supplies. An attempt was made, over a period of several years, to isolate legionellae from a municipal system. Sampling sites included the river water supply, treatment plant, finished water reservoir system, mains, and distribution taps. Despite the use of several isolation techniques, Legionella spp. could not be detected in any of the samples other than those collected from the river. It was hypothesized that this was due to the maintenance of a chlorine residual throughout the system. To investigate the potential for Legionella growth, additional water samples, collected from throughout the system, were dechlorinated, pasteurized, and inoculated with Legionella pneumophila. Subsequent growth indicated that many of these samples, especially those collected from areas affected by an accumulation of algal materials, exhibited a much greater ability to support Legionella multiplication than did river water prior to treatment. Chemical analyses were also performed on these samples. Correlation of chemical data and experimental growth results indicated that the chemical environment significantly affects the ability of the water to support multiplication, with turbidity, organic carbon, and certain metals being of particular importance. These studies indicate that the potential exists for Legionella growth within municipal systems and support the hypothesis that public water supplies may contaminate the plumbing systems of hospitals and other large buildings. The results also suggest that useful methods to control this contamination include adequate treatment plant filtration, maintenance of a chlorine residual throughout the treatment and distribution network, and effective covering of open reservoirs.     

Effects of Disinfection on Legionella spp., Eukarya, and Biofilms in a Hot Water System

Legionella species are frequently detected in hot water systems, attached to the surface as a biofilm. In this work, the dynamics of Legionella spp. and diverse bacteria and eukarya associated together in the biofilm, coming from a pilot scale 1 system simulating a real hot water system, were investigated throughout 6 months after two successive heat shock treatments followed by three successive chemical treatments. Community structure was assessed by a fingerprint technique, single-strand conformation polymorphism (SSCP). In addition, the diversity and dynamics of Legionella and eukarya were investigated by small-subunit (SSU) ribosomal cloning and sequencing. Our results showed that pathogenic Legionella species remained after the heat shock and chemical treatments (Legionella pneumophila and Legionella anisa, respectively). The biofilm was not removed, and the bacterial community structure was transitorily affected by the treatments. Moreover, several amoebae had been detected in the biofilm before treatments (Thecamoebae sp., Vannella sp., and Hartmanella vermiformis) and after the first heat shock treatment, but only H. vermiformis remained. However, another protozoan affiliated with Alveolata, which is known as a host cell for Legionella, dominated the eukaryal species after the second heat shock and chemical treatment tests. Therefore, effective Legionella disinfection may be dependent on the elimination of these important microbial components. We suggest that eradicating Legionella in hot water networks requires better study of bacterial and eukaryal species associated with Legionella in biofilms.     

Legionella pneumophila associated with the protozoan Hartmannella vermiformis in a model multi-species biofilm has reduced susceptibility to disinfectants

Legionella pneumophila will infect biofilm-associated protozoa, and in this way might be protected from disinfectants in potable water systems. A base biofilm containing Pseudomonas aeruginosa, Klebsiella pneumoniae, and Flavobacterium spp. was grown on steel coupons in potable water prior to the addition of L. pneumophila and the protozoan H. vermiformis. After 7 d, coupons were removed and treated with 0.5 mgl(-1) free residual chlorine (FRC) or 0.5 mgl(-1) monochloramine (MCA) for 15, 60, or 180 min or 24 h. In a second experiment, only L. pneumophila and the base biofilm organisms were present but with an identical treatment protocol. Treatment of L. pneumophila for 180 min in a system without H. vermiformis resulted in log reductions of 2.07 and 2.11 for FRC and MCA, respectively. When H. vermiformis was present, however, the treatment resulted in log reductions of 0.67 and 0.81 for FRC and MCA, respectively. A similar pattern was observed for 15 and 60 min contact times. These results indicate that L. pneumophila was less susceptible to MCA or FRC when associated with biofilm-associated H. vermiformis in a model potable water biofilm.     

Multiplication of Legionella spp. in tap water containing Hartmannella vermiformis

A model was developed to study the multiplication of various Legionella spp. in tap water containing Hartmannella vermiformis. Tap water cultures prepared with the following components were suitable for the multiplication studies: Legionella spp., 10(3) CFU/ml; H. vermiformis, 10(4.4) cysts per ml; and killed Pseudomonas paucimobilis, 10(9) cells per ml. Cocultures were incubated at 37 degrees C for at least 1 week. The following legionellae multiplied in tap water cocultures in each replicate experiment: L. bozemanii (WIGA strain), L. dumoffii (NY-23 and TX-KL strains), L. micdadei (two environmental strains), and L. pneumophila (six environmental strains and one clinical isolate). Growth yield values for these strains were 0.6 to 3.5 log CFU/ml. Legionellae which did not multiply in replicate cocultures included L. anisa (one strain), L. bozemanii (MI-15 strain), L. micdadei (a clinical isolate), L. longbeachae, (one strain), and L. pneumophila (Philadelphia 1 strain). L. gormanii and an environmental isolate of L. pneumophila multiplied in only one of three experiments. None of the legionellae multiplied in tap water containing only killed P. paucimobilis. The mean growth yield (+/- standard deviation) of H. vermiformis in the cocultures was 1.2 +/- 0.1 log units/ml. H. vermiformis supports multiplication of only particular strains of legionellae, some of which are from diverse origins.     

Multiplication of Legionella spp. in tap water containing Hartmannella vermiformis.

A model was developed to study the multiplication of various Legionella spp. in tap water containing Hartmannella vermiformis. Tap water cultures prepared with the following components were suitable for the multiplication studies: Legionella spp., 10(3) CFU/ml; H. vermiformis, 10(4.4) cysts per ml; and killed Pseudomonas paucimobilis, 10(9) cells per ml. Cocultures were incubated at 37 degrees C for at least 1 week. The following legionellae multiplied in tap water cocultures in each replicate experiment: L. bozemanii (WIGA strain), L. dumoffii (NY-23 and TX-KL strains), L. micdadei (two environmental strains), and L. pneumophila (six environmental strains and one clinical isolate). Growth yield values for these strains were 0.6 to 3.5 log CFU/ml. Legionellae which did not multiply in replicate cocultures included L. anisa (one strain), L. bozemanii (MI-15 strain), L. micdadei (a clinical isolate), L. longbeachae, (one strain), and L. pneumophila (Philadelphia 1 strain). L. gormanii and an environmental isolate of L. pneumophila multiplied in only one of three experiments. None of the legionellae multiplied in tap water containing only killed P. paucimobilis. The mean growth yield (+/- standard deviation) of H. vermiformis in the cocultures was 1.2 +/- 0.1 log units/ml. H. vermiformis supports multiplication of only particular strains of legionellae, some of which are from diverse origins.     

Occurrence and genetic diversity of uncultured Legionella spp. in drinking water treated at temperatures below 15 degrees C

Representatives of the genus Legionella were detected by use of a real-time PCR method in all water samples collected directly after treatment from 16 surface water (SW) supplies prior to postdisinfection and from 81 groundwater (GW) supplies. Legionella concentrations ranged from 1.1 x 10(3) to 7.8 x 10(5) cells liter(-1) and were significantly higher in SW treated with multiple barriers at 4 degrees C than in GW treated at 9 to 12 degrees C with aeration and filtration but without chemical disinfection. No Legionellae (<50 CFU liter(-1)) were detected in treated water by the culture method. Legionella was also observed in untreated SW and in untreated aerobic and anaerobic GW. Filtration processes in SW and GW treatment had little effect or increased the Legionella concentration, but ozonation in SW treatment caused about 1-log-unit reduction. A phylogenetic analysis of 16S rRNA gene sequences of 202 clones, obtained from a selection of samples, showed a high similarity (>91%) with Legionella sequences in the GenBank database. A total of 40 (33%) of the 16S rRNA gene sequences obtained from treated water were identified as described Legionella species and types, including L. bozemanii, L. worsleiensis, Legionella-like amoebal pathogen types, L. quateirensis, L. waltersii, and L. pneumophila. 16S rRNA gene sequences with a similarity of below 97% from described species were positioned all over the phylogenetic tree of Legionella. Hence, a large diversity of yet-uncultured Legionellae are common members of the microbial communities in SW and GW treated at water temperatures of below 15 degrees C.     

Legionella incidence and density in potable drinking water supplies

The incidence and density of Legionella spp. in raw water, water at various stages of treatment, and in potable distribution water were determined by direct immunofluorescence. The number of cells reacting with Legionella-specific fluorescent antibody conjugates in raw waters ranged from about 10(4) to 10(5) cells/liter, whereas the concentrations of fluorescent antibody-positive cells in the distribution waters were generally 10- to 100-fold lower than in the raw source waters. No viable or virulent Legionella strains were isolated from either the source or distribution waters. However, Legionella spp. are infrequently isolated from water at temperatures below 15 degrees C as was the case in the system surveyed in this study.     

Legionella incidence and density in potable drinking water supplies.

The incidence and density of Legionella spp. in raw water, water at various stages of treatment, and in potable distribution water were determined by direct immunofluorescence. The number of cells reacting with Legionella-specific fluorescent antibody conjugates in raw waters ranged from about 10(4) to 10(5) cells/liter, whereas the concentrations of fluorescent antibody-positive cells in the distribution waters were generally 10- to 100-fold lower than in the raw source waters. No viable or virulent Legionella strains were isolated from either the source or distribution waters. However, Legionella spp. are infrequently isolated from water at temperatures below 15 degrees C as was the case in the system surveyed in this study.     

Quantitative detection of the free-living amoeba Hartmannella vermiformis in surface water by using real-time PCR

A real-time PCR-based method targeting the 18S rRNA gene was developed for the quantitative detection of Hartmannella vermiformis, a free-living amoeba which is a potential host for Legionella pneumophila in warm water systems and cooling towers. The detection specificity was validated using genomic DNA of the closely related amoeba Hartmannella abertawensis as a negative control and sequence analysis of amplified products from environmental samples. Real-time PCR detection of serially diluted DNA extracted from H. vermiformis was linear for microscopic cell counts between 1.14 x 10(-1) and 1.14 x 10(4) cells per PCR. The genome of H. vermiformis harbors multiple copies of the 18S rRNA gene, and an average number (with standard error) of 1,330 +/- 127 copies per cell was derived from real-time PCR calibration curves for cell suspensions and plasmid DNA. No significant differences were observed between the 18S rRNA gene copy numbers for trophozoites and cysts of strain ATCC 50237 or between the copy numbers for this strain and strain KWR-1. The developed method was applied to water samples (200 ml) collected from a variety of lakes and rivers serving as sources for drinking water production in The Netherlands. Detectable populations were found in 21 of the 28 samples, with concentrations ranging from 5 to 75 cells/liter. A high degree of similarity (> or =98%) was observed between sequences of clones originating from the different surface waters and between these clones and the reference strains. Hence, H. vermiformis, which is highly similar to strains serving as hosts for L. pneumophila, is a common component of the microbial community in fresh surface water.     

The incidence of Bdellovibrio spp. in man-made water systems: coexistence with legionellas

Bdellovibrios have been isolated from surface waters but there are no reports of its occurrence in mains water supplies. One hundred and thirty five water samples from 81 sources were examined for the presence of Bdellovibrio bacteriovorus and Legionella spp. Bdellovibrios were isolated by a double-layer agar technique with two strains of Legionella pneumophila serogroup 1 as the host organisms. Bdellovibrio spp. were isolated from 57.8% and Legionella spp. from 9.5% of the samples. The two species occurred together in 4.4% of samples. The incidence of Bdellovibrio spp. and its occurrence with legionellas in man-made water systems is discussed.     

The role of biofilms and protozoa in Legionella pathogenesis: implications for drinking water

Current models to study Legionella pathogenesis include the use of primary macrophages and monocyte cell lines, various free-living protozoan species and murine models of pneumonia. However, there are very few studies of Legionella spp. pathogenesis aimed at associating the role of biofilm colonization and parasitization of biofilm microbiota and release of virulent bacterial cell/vacuoles in drinking water distribution systems. Moreover, the implications of these environmental niches for drinking water exposure to pathogenic legionellae are poorly understood. This review summarizes the known mechanisms of Legionella spp. proliferation within Acanthamoeba and mammalian cells and advocates the use of the amoeba model to study Legionella pathogenicity because of their close association with Legionella spp. in the aquatic environment. The putative role of biofilms and amoebae in the proliferation, development and dissemination of potentially pathogenic Legionella spp. is also discussed. Elucidating the mechanisms of Legionella pathogenicity development in our drinking water systems will aid in elimination strategies and procedural designs for drinking water systems and in controlling exposure to Legionella spp. and similar pathogens.     

Effect of point-of-use disinfection, flocculation and combined flocculation-disinfection on drinking water quality in western Kenya

AIMS: Point-of-use drinking water disinfection with sodium hypochlorite has been shown to improve water quality and reduce diarrhoeal disease. However, the chlorine demand of highly turbid water may render sodium hypochlorite less effective. METHODS AND RESULTS: We evaluated a novel combined flocculant-disinfectant point-of-use water treatment product and compared its effect on drinking water quality with existing technologies in western Kenya. In water from 30 sources, combined flocculant-disinfectant reduced Escherichia coli concentrations to <1 CFU100 ml(-1) for 29 (97%) and reduced turbidity to <5 nephelometric turbidity units (NTU) for 26 (87%). By contrast, water from 30 sources treated with sodium hypochlorite reduced E. coli concentrations to <1 CFU 100 ml(-1) for 25 (83%) and turbidity to <5 NTU for 5 (17%). CONCLUSIONS: For source waters over a range of turbidities in western Kenya, combined flocculant-disinfectant product effectively reduces turbidity to <5 NTU and reduces E. coli concentrations to <1 CFU 100 ml(-1). SIGNIFICANCE AND IMPACT OF THE STUDY: The novel flocculant-disinfectant product may be acceptable to consumers and may be effective in reducing diarrhoeal disease in settings where source water is highly turbid.     

Microbial ecology of drinking water distribution systems

The supply of clean drinking water is a major, and relatively recent, public health milestone. Control of microbial growth in drinking water distribution systems, often achieved through the addition of disinfectants, is essential to limiting waterborne illness, particularly in immunocompromised subpopulations. Recent inquiries into the microbial ecology of distribution systems have found that pathogen resistance to chlorination is affected by microbial community diversity and interspecies relationships. Research indicates that multispecies biofilms are generally more resistant to disinfection than single-species biofilms. Other recent findings are the increased survival of the bacterial pathogen Legionella pneumophila when present inside its protozoan host Hartmannella vermiformis and the depletion of chloramine disinfectant residuals by nitrifying bacteria, leading to increased overall microbial growth. Interactions such as these are unaccounted for in current disinfection models. An understanding of the microbial ecology of distribution systems is necessary to design innovative and effective control strategies that will ensure safe and high-quality 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.     

Legionella contamination in hot water of Italian hotels

A cross-sectional multicenter survey of Italian hotels was conducted to investigate Legionella spp. contamination of hot water. Chemical parameters (hardness, free chlorine concentration, and trace element concentrations), water systems, and building characteristics were evaluated to study risk factors for colonization. The hot water systems of Italian hotels were strongly colonized by Legionella; 75% of the buildings examined and 60% of the water samples were contaminated, mainly at levels of > or =10(3) CFU liter(-1), and Legionella pneumophila was the most frequently isolated species (87%). L. pneumophila serogroup 1 was isolated from 45.8% of the contaminated sites and from 32.5% of the hotels examined. When a multivariate logistic model was used, only hotel age was associated with contamination, but the risk factors differed depending on the contaminating species and serogroup. Soft water with higher chlorine levels and higher temperatures were associated with L. pneumophila serogroup 1 colonization, whereas the opposite was observed for serogroups 2 to 14. In conclusion, Italian hotels, particularly those located in old buildings, represent a major source of risk for Legionnaires' disease due to the high frequency of Legionella contamination, high germ concentration, and major L. pneumophila serogroup 1 colonization. The possible role of chlorine in favoring the survival of Legionella species is discussed.     

The incidence of Bdellovibrio spp. in man-made water systems: coexistence with legionellas

Bdellovibrios have been isolated from surface waters but there are no reports of its occurrence in mains water supplies. One hundred and thirty five water samples from 81 sources were examined for the presence of Bdellovibrio bacteriovorus and Legion-ella spp. Bdellovibrics were isolated by a double-layer agar technique with two strains of Legionella pneumophila serogroup 1 as the host organisms. Bdellovibrio spp. were isolated from 57·8% and Legionella spp. from 9·5% of the samples. The two species occurred together in 4·4% of samples. The incidence of Bdellovibrio spp. and its occurrence with legionellas in man-made water systems is discussed.     

Reduction of Legionella spp. in Water and in Soil by a Citrus Plant Extract Vapor

Legionnaires'' disease is a severe form of pneumonia caused by Legionella spp., organisms often isolated from environmental sources, including soil and water. Legionella spp. are capable of replicating intracellularly within free-living protozoa, and once this has occurred, Legionella is particularly resistant to disinfectants. Citrus essential oil (EO) vapors are effective antimicrobials against a range of microorganisms, with reductions of 5 log cells ml⁻¹ on a variety of surfaces. The aim of this investigation was to assess the efficacy of a citrus EO vapor against Legionella spp. in water and in soil systems. Reductions of viable cells of Legionella pneumophila, Legionella longbeachae, Legionella bozemanii, and an intra-amoebal culture of Legionella pneumophila (water system only) were assessed in soil and in water after exposure to a citrus EO vapor at concentrations ranging from 3.75 mg/liter air to 15g/liter air. Antimicrobial efficacy via different delivery systems (passive and active sintering of the vapor) was determined in water, and gas chromatography-mass spectrometry (GC-MS) analysis of the antimicrobial components (linalool, citral, and β-pinene) was conducted. There was up to a 5-log cells ml⁻¹ reduction in Legionella spp. in soil after exposure to the citrus EO vapors (15 mg/liter air). The most susceptible strain in water was L. pneumophila, with a 4-log cells ml⁻¹ reduction after 24 h via sintering (15 g/liter air). Sintering the vapor through water increased the presence of the antimicrobial components, with a 61% increase of linalool. Therefore, the appropriate method of delivery of an antimicrobial citrus EO vapor may go some way in controlling Legionella spp. from environmental sources.     

Detection of Legionella species in reclaimed water and air with the EnviroAmp Legionella PCR kit and direct fluorescent antibody staining.

Reclaimed water is an important resource for areas with inadequate water supplies. However, there have been few studies on the variety of microorganisms found in this type of water, since typically reclaimed water is examined only for the presence of coliform bacteria. Many microorganisms, including the legionellae, are known to be more resistant to chlorine than are coliform bacteria. Previously, we detected > 10(3) Legionella cells per ml in primary and secondary sewage effluents and observed no significant reduction in population numbers throughout the treatment process. In this study, we detected Legionella spp. in chlorinated effluent by using an EnviroAmp Legionella PCR kit and direct fluorescent antibody (DFA) staining. However, we were not able to isolate Legionella spp. from either natural or seeded reclaimed water samples. This suggests that the Legionella spp. detected by the PCR and DFA methods may be injured or viable but nonculturable after exposure to the high residual chlorine levels typically found in this type of water source. The numbers of coliform bacteria were low (< 2 cells per 100 ml) in most reclaimed water samples and were not correlated with the presence or absence of Legionella spp. We also collected air samples from above a secondary aeration basin and analyzed them by using the PCR, DFA, and plate culture methods. Legionella spp. were detected in the air obtained from above the secondary basin with all three methods. We concluded that the PCR was superior to the culture and DFA methods for detecting Legionella spp. in environmental water samples.