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.     

Detection of Legionella species in sewage and ocean water by polymerase chain reaction, direct fluorescent-antibody, and plate culture methods.

Legionella spp. are ubiquitous in most environmental water sources; however, sewage treatment plants have not been examined as potential environmental reservoirs for these bacteria. This study used polymerase chain reaction, direct fluorescent-antibody staining, and culture methods to examine raw and treated sewage, ocean-receiving waters, and nearshore coastal environments for the presence of Legionella pneumophila and other Legionella spp. The study concluded that Legionella spp. are present in all phases of sewage treatment and that population numbers do not significantly decline through the treatment process. Ocean-receiving waters located 5 miles offshore, where the treated sewage is discharged, were found to contain Legionella spp., but ocean water between the discharge site and coastal bathing beaches was negative. This suggests that the Legionella spp. from the ocean discharge site were not reaching the nearshore beach waters. A flood control channel and river that entered the ocean were found to contain Legionella spp., and a nearby beach swimming area was also found to be positive, suggesting that land runoff from the flood control channel and river were the source of the Legionella spp. in the beach water samples that tested positive.     

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.     

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.     

Abundance and distribution of Legionellaceae in Puerto Rican waters

Waters in marine and freshwater areas of Puerto Rico were analyzed for the presence of Legionella spp. by direct fluorescent antibody assay with guinea pig confirmation. Several species, including L. bozemanii, L. dumoffii, L. gormanii, L. longbeachae, L. micdadei, and L. pneumophila, were widely distributed among all sites. Legionellaceae, including L. pneumophila, were found in high densities in water collected in the rain forest from epiphytes in trees 30 ft. (about 9.25 m) above the ground. Both interspecific and intersite variations were significant. L. pneumophila was the most abundant species at all sites, with average densities of 10(4) cells ml-1, very close to the range which is potentially pathogenic for humans. Densities of L. pneumophila were highest in sewage-contaminated coastal waters. These are the highest densities of Legionella spp. ever reported for marine habitats. Densities of L. pneumophila were positively correlated with concentrations of sulfates, phosphates, and pH. A survey of 88 fatal atypical pneumonia cases at a Puerto Rico hospital showed that 15% of the patients had L. pneumophila infections. This study establishes L. pneumophila as a relatively common cause of atypical pneumonia in Puerto Rico and suggests natural aquatic habitats as possible sources or reservoirs of pathogenic Legionella spp. in the tropics.     

Abundance and distribution of Legionellaceae in Puerto Rican waters.

Waters in marine and freshwater areas of Puerto Rico were analyzed for the presence of Legionella spp. by direct fluorescent antibody assay with guinea pig confirmation. Several species, including L. bozemanii, L. dumoffii, L. gormanii, L. longbeachae, L. micdadei, and L. pneumophila, were widely distributed among all sites. Legionellaceae, including L. pneumophila, were found in high densities in water collected in the rain forest from epiphytes in trees 30 ft. (about 9.25 m) above the ground. Both interspecific and intersite variations were significant. L. pneumophila was the most abundant species at all sites, with average densities of 10(4) cells ml-1, very close to the range which is potentially pathogenic for humans. Densities of L. pneumophila were highest in sewage-contaminated coastal waters. These are the highest densities of Legionella spp. ever reported for marine habitats. Densities of L. pneumophila were positively correlated with concentrations of sulfates, phosphates, and pH. A survey of 88 fatal atypical pneumonia cases at a Puerto Rico hospital showed that 15% of the patients had L. pneumophila infections. This study establishes L. pneumophila as a relatively common cause of atypical pneumonia in Puerto Rico and suggests natural aquatic habitats as possible sources or reservoirs of pathogenic Legionella spp. in the tropics.     

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.     

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.     

Isolation of amoebae and Pseudomonas and Legionella spp. from eyewash stations.

Forty eyewash units were sampled for protozoa, bacteria, and fungi. Total heterotrophic bacterial counts on nutrient agar and R2A agar (Difco Laboratories, Detroit, Mich.) ranged from 0 to 10(5) CFU/ml, with Pseudomonas spp. being the most frequently isolated. Total counts of 10(4) and 10(8) cells per ml were obtained with the acridine orange staining procedure. All samples were examined for Legionella spp. by direct fluorescent-antibody staining and by culturing on buffered charcoal-yeast extract agar containing alpha-ketoglutarate and glycine and supplemented with cycloheximide, vancomycin, and polymyxin B. DNA-DNA hybridization was used to confirm identification of the Legionella isolates. Legionellae were detected in 35 of 40 (87.5%) samples by direct fluorescent-antibody staining, with 3 samples yielding both Legionella spp. and amoebae. Amoebae identified as Hartmannella, Vahlkampfia, Acanthamoeba, and Cochliopodium spp. were detected in 19 of 40 (47:5%) samples. Sabouraud dextrose agar was used to obtain a crude estimate of viable fungal populations, pH, hardness, and ammonia, alkalinity, chlorine, copper, and iron contents were recorded for all water samples collected from eyewash stations; 33% of the samples had greater than or equal to 10 mg of CO2 per liter. It is concluded that eyewash stations not regularly flushed and/or cleaned and used to flush traumatized eye tissue may be a source of infection and can contaminate laboratory environments via aerosol transmission.     

Longitudinal evaluation of the efficacy of heat treatment procedures against Legionella spp. in hospital water systems by using a flow cytometric assay

Legionella spp. are frequently isolated in hospital water systems. Heat shock (30 min at 70°C) is recommended by the World Health Organization to control its multiplication. The aim of the study was to evaluate retrospectively the efficacy of heat treatments by using a flow cytometry assay (FCA) able to identify viable but nonculturable (VBNC) cells. The study included Legionella strains (L. pneumophila [3 clusters] and L. anisa [1 cluster]) isolated from four hot water circuits of different hospital buildings in Saint-Etienne, France, during a 20-year prospective surveillance. The strains recovered from the different circuits were not epidemiologically related, but the strains isolated within a same circuit over time exhibited an identical genotypic profile. After an in vitro treatment of 30 min at 70°C, the mean percentage of viable cells and VBNC cells varied from 4.6% to 71.7%. The in vitro differences in heat sensitivity were in agreement with the observed efficacy of preventive and corrective heating measures used to control water contamination. These results suggest that Legionella strains can become heat resistant after heating treatments for a long time and that flow cytometry could be helpful to check the efficacy of heat treatments on Legionella spp. and to optimize the decontamination processes applied to water systems for the control of Legionella proliferation.     

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.     

Occurrence of Giardia and Cryptosporidium spp. in surface water supplies.

Giardia and Cryptosporidium levels were determined by using a combined immunofluorescence test for source waters of 66 surface water treatment plants in 14 states and 1 Canadian province. The results showed that cysts and oocysts were widely dispersed in the aquatic environment. Giardia spp. were detected in 81% of the raw water samples. Cryptosporidium spp. were found in 87% of the raw water locations. Overall, Giardia or Cryptosporidium spp. were detected in 97% of the raw water samples. Higher cyst and oocyst densities were associated with source waters receiving industrial or sewage effluents. Significant correlations were found between Giardia and Cryptosporidium densities and raw water quality parameters such as turbidity and total and fecal coliform levels. Statistical modeling suggests that cyst and oocyst densities could be predicted on the basis of watershed and water quality characteristics. The occurrence of high levels of Giardia cysts in raw water samples may require water utilities to apply treatment beyond that outlined in the Surface Water Treatment Rule of the U.S. Environmental Protection Agency.     

Occurrence of Giardia and Cryptosporidium spp. in surface water supplies.

Giardia and Cryptosporidium levels were determined by using a combined immunofluorescence test for source waters of 66 surface water treatment plants in 14 states and 1 Canadian province. The results showed that cysts and oocysts were widely dispersed in the aquatic environment. Giardia spp. were detected in 81% of the raw water samples. Cryptosporidium spp. were found in 87% of the raw water locations. Overall, Giardia or Cryptosporidium spp. were detected in 97% of the raw water samples. Higher cyst and oocyst densities were associated with source waters receiving industrial or sewage effluents. Significant correlations were found between Giardia and Cryptosporidium densities and raw water quality parameters such as turbidity and total and fecal coliform levels. Statistical modeling suggests that cyst and oocyst densities could be predicted on the basis of watershed and water quality characteristics. The occurrence of high levels of Giardia cysts in raw water samples may require water utilities to apply treatment beyond that outlined in the Surface Water Treatment Rule of the U.S. Environmental Protection Agency.     

Distribution of Legionella longbeachae serogroup 1 and other legionellae in potting soils in Australia.

Legionella longbeachae serogroup 1 and other Legionella spp. were isolated from 73% of 45 potting soils made in Australia by 13 manufacturers but were not detected in 19 potting soils made in Greece, Switzerland, and the United Kingdom examined between March 1989 and May 1990. Several Legionella species were isolated from a small number of samples of uncomposted pine sawdusts, but it is not known whether sawdust was the source of some of the legionellae found in potting soils. Legionella spp. persisted for periods ranging from 3 to 10 months in a potting soil held at temperatures between -20 and 35 degrees C. Isolates of L. longbeachae serogroup 1 from soil did not grow at 43 degrees C, a temperature which was also lethal for this species in soil. Most Legionella spp. isolated from potting and natural soils belonged to one distinct group according to analysis of ubiquinones and were serologically related to several known species in this group. A small number of potting soils contained L. pneumophila and L. micdadei.     

Distribution of Legionella longbeachae serogroup 1 and other legionellae in potting soils in Australia

Legionella longbeachae serogroup 1 and other Legionella spp. were isolated from 73% of 45 potting soils made in Australia by 13 manufacturers but were not detected in 19 potting soils made in Greece, Switzerland, and the United Kingdom examined between March 1989 and May 1990. Several Legionella species were isolated from a small number of samples of uncomposted pine sawdusts, but it is not known whether sawdust was the source of some of the legionellae found in potting soils. Legionella spp. persisted for periods ranging from 3 to 10 months in a potting soil held at temperatures between -20 and 35 degrees C. Isolates of L. longbeachae serogroup 1 from soil did not grow at 43 degrees C, a temperature which was also lethal for this species in soil. Most Legionella spp. isolated from potting and natural soils belonged to one distinct group according to analysis of ubiquinones and were serologically related to several known species in this group. A small number of potting soils contained L. pneumophila and L. micdadei.     

Biofilms as major sources of Legionella spp. in hydrothermal areas and their dispersion into stream water

Abstract Water and biofilms from two hydrothermal areas in central Portugal, and one hydrothermal area in New Mexico, USA, were examined for Legionella spp. In general, Legionella spp were isolated in higher numbers from biofilms than from water, although one biofilm with a temperature of 50°C, did not yield isolates of these organisms. In one area L. pneumophila serogroup (sg) 3 constituted the major population in the thermal discharge by the stream and the biofilm below it; however, L. pneumophila sg 1 was predominant in the sediments of the stream bed with minor thermal springs below the main discharge and in the water downstream. No Legionellae were isolated from water upstream of the hydrothermal area indicating that the thermal area was the source of the organisms in the stream water. In the other two hydrothermal areas, L. pneumophila sg 1 constituted the major population isolated, whereas L. pneumophila sg 3 was absent or isolated in low numbers. Isolates of L. micdadei were also recovered from one hydrothermal area, while ‘ L. londoniensis ’ was isolated from another.     

Isolation and characterization of Legionella spp. and Pseudomonas spp. from greenhouse misting systems

AIMS: Greenhouse misting systems used for watering plants produce fine aerosols. They are a possible cause for bacterial infections. This study investigates the colonization of greenhouse misting systems with Legionella spp. and Pseudomonas spp. and evaluates a possible health hazard. METHODS AND RESULTS: Between June and September 2003, a total of 80 water samples were collected in 20 different greenhouse systems in Germany, each tested on two different occasions. Each time, water was drawn at a central tap and at the outlet of spray nozzles. Sampled greenhouses were used to cultivate various plants and trees for commercial, recreational or scientific reasons, some of them in tropical conditions. Legionella spp. were detected in 10% of the systems (two systems), but only in low numbers. On the contrary, Pseudomonas spp. were recovered from 70% of the greenhouse watering systems (14 systems), occasionally at counts greater than 10,000 CFU per 100 ml. A random amplified polymorphic DNA polymerase chain reaction typing method was used to demonstrate that each colonized greenhouse had one or several individual strains of Legionella and Pseudomonas that could not be detected in any other system. CONCLUSIONS: This study demonstrates that aerosolizing greenhouse watering systems may be contaminated with Legionella or Pseudomonas which under certain circumstances could become a potential source of infection for workers and visitors. SIGNIFICANCE AND IMPACT OF THE STUDY: The study results indicate that greenhouse misting systems should be included in Legionella and Pseudomonas monitoring and control programs.     

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.     

Application of flow cytometry to detection and characterization of Legionella spp.

Flow cytometry, using fluorescein-bound specific antibodies and propidium iodide, was shown to be effective in detecting Legionella spp. in cooling tower waters. The procedure was quicker and less labor intensive than fluorescent microscopy. The use of these procedures also identified qualitative differences, perhaps related to infectivity, in Legionella populations.