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.     

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.     

Introduction of monochloramine into a municipal water system: impact on colonization of buildings by Legionella spp

Legionnaires' disease (LD) outbreaks are often traced to colonized potable water systems. We collected water samples from potable water systems of 96 buildings in Pinellas County, Florida, between January and April 2002, during a time when chlorine was the primary residual disinfectant, and from the same buildings between June and September 2002, immediately after monochloramine was introduced into the municipal water system. Samples were cultured for legionellae and amoebae using standard methods. We determined predictors of Legionella colonization of individual buildings and of individual sampling sites. During the chlorine phase, 19 (19.8%) buildings were colonized with legionellae in at least one sampling site. During the monochloramine phase, six (6.2%) buildings were colonized. In the chlorine phase, predictors of Legionella colonization included water source (source B compared to all others, adjusted odds ratio [aOR], 6.7; 95% confidence interval [CI], 2.0 to 23) and the presence of a system with continuously circulating hot water (aOR, 9.8; 95% CI, 1.9 to 51). In the monochloramine phase, there were no predictors of individual building colonization, although we observed a trend toward greater effectiveness of monochloramine in hotels and single-family homes than in county government buildings. The presence of amoebae predicted Legionella colonization at individual sampling sites in both phases (OR ranged from 15 to 46, depending on the phase and sampling site). The routine introduction of monochloramine into a municipal drinking water system appears to have reduced colonization by Legionella spp. in buildings served by the system. Monochloramine may hold promise as community-wide intervention for the prevention of LD.     

Isolation of Legionella species from drinking water

Three different species of Legionella were recovered from samples of water taken from chlorinated public water supplies where no coliform bacteria were simultaneously detected. Five of 856 samples yielded Legionella isolates. Three isolates were identified as Legionella pneumophila serogroup 1, the fourth was identified as Legionella dumoffii, and the fifth was identified as Legionella jordanis. Studies to determine the survival of L. pneumophila Flint 1 serogroup 1 in tap water at various temperatures and in tap water with added sodium hypochlorite were done. These organisms were found to survive for 299 days in tap water at 24 and 5 degrees C but not at 35 degrees C. A concentration of at least 0.2 mg of residual chlorine per ml was required to eliminate at least 90% of L. pneumophila and Escherichia coli inocula in 2 h.     

Isolation of Legionella species from drinking water.

Three different species of Legionella were recovered from samples of water taken from chlorinated public water supplies where no coliform bacteria were simultaneously detected. Five of 856 samples yielded Legionella isolates. Three isolates were identified as Legionella pneumophila serogroup 1, the fourth was identified as Legionella dumoffii, and the fifth was identified as Legionella jordanis. Studies to determine the survival of L. pneumophila Flint 1 serogroup 1 in tap water at various temperatures and in tap water with added sodium hypochlorite were done. These organisms were found to survive for 299 days in tap water at 24 and 5 degrees C but not at 35 degrees C. A concentration of at least 0.2 mg of residual chlorine per ml was required to eliminate at least 90% of L. pneumophila and Escherichia coli inocula in 2 h.     

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.     

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.     

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.     

Legionella contamination of dental-unit waters.

Water samples collected from 28 dental facilities in six U.S. states were examined for the presence of Legionella pneumophila and other Legionella spp. by the PCR-gene probe, fluorescent-antibody microscopic, and viable-plate-count detection methods. The PCR and fluorescent-antibody detection methods, which detect both viable and viable nonculturable Legionella spp., gave higher counts and rates of detection than the plate count method. By the PCR-gene probe detection method, Legionella spp. were detected in 68% of the dental-unit water samples and L. pneumophila was detected in 8%. Concentrations of Legionella spp. in dental-unit water reached 1,000 organisms per ml or more in 36% of the samples, and 19% of the samples were in the category of 10,000/ml or above. L. pneumophila, when present in dental-unit water, never reached concentrations of 1,000/ml or more. Microscopic examination with fluorescent-antibody staining indicated that the contamination was in the dental-unit water lines rather than in the handpieces. Legionella spp. were present in 61% of potable water samples collected for comparative analysis from domestic and institutional faucets and drinking fountains; this percentage was not significantly different from the rate of detection of Legionella spp. in dental-unit water. However, in only 4% of the potable water samples did Legionella spp. reach concentrations of 1,000 organisms per ml, and none was in the 10,000 organisms-per-ml category, and so health-threatening levels of Legionella spp. in potable water were significantly lower than in dental-unit water. L. pneumophila was found in 2% of the potable water samples, but only at the lowest detectable level.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relationships between free-living protozoa, cultivable Legionella spp., and water quality characteristics in three drinking water supplies in the Caribbean

The study whose results are presented here aimed at identifying free-living protozoa (FLP) and conditions favoring the growth of these organisms and cultivable Legionella spp. in drinking water supplies in a tropical region. Treated and distributed water (±30°C) of the water supplies of three Caribbean islands were sampled and investigated with molecular techniques, based on the 18S rRNA gene. The protozoan host Hartmannella vermiformis and cultivable Legionella pneumophila were observed in all three supplies. Operational taxonomic units (OTUs) with the highest similarity to the potential or candidate hosts Acanthamoeba spp., Echinamoeba exundans, E. thermarum, and an Neoparamoeba sp. were detected as well. In total, 59 OTUs of FLP were identified. The estimated protozoan richness did not differ significantly between the three supplies. In supply CA-1, the concentration of H. vermiformis correlated with the concentration of Legionella spp. and clones related to Amoebozoa predominated (82%) in the protozoan community. These observations, the low turbidity (<0.2 nephelometric turbidity units [NTU]), and the varying ATP concentrations (1 to 12 ng liter(-1)) suggest that biofilms promoted protozoan growth in this supply. Ciliophora represented 25% of the protozoan OTUs in supply CA-2 with elevated ATP concentrations (maximum, 55 ng liter(-1)) correlating with turbidity (maximum, 62 NTU) caused by corroding iron pipes. Cercozoan types represented 70% of the protozoan clones in supply CA-3 with ATP concentrations of <1 ng liter(-1) and turbidity of <0.5 NTU in most samples of distributed water. The absence of H. vermiformis in most samples from supply CA-3 suggests that growth of this protozoan is limited at ATP concentrations of <1 ng liter(-1).     

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.     

An alkaline approach to treating cooling towers for control of Legionella pneumophila

Earlier field and laboratory studies have shown that Legionella species survive and multiply in the pH range 5.5 to 9.2. Additionally, the technical feasibility of operating cooling towers at elevated alkalinities and pH has previously been documented by published guidelines. The guidelines indicate that these conditions facilitate corrosion control and favor chlorine persistence which enhances the effectiveness of continuous chlorination in biofouling control. This information suggests that control of Legionella species in cooling towers can be accomplished by operating the towers under alkaline conditions. To test this possibility, we collected water samples over a period of months from a hospital cooling tower. The samples were analyzed for a variety of chemical parameters. Subsamples were pasteurized and inoculated with non-agar-passaged Legionella pneumophila which had been maintained in tap water. Correlation of subsequent Legionella growth with corresponding pH and alkalinity values revealed statistically significant inverse associations. These data support the hypothesis that operating cooling towers outside of the optimal conditions for Legionella growth (e.g., at elevated alkalinities and a pH greater than 9) may be a useful approach to controlling growth in this habitat.     

An alkaline approach to treating cooling towers for control of Legionella pneumophila.

Earlier field and laboratory studies have shown that Legionella species survive and multiply in the pH range 5.5 to 9.2. Additionally, the technical feasibility of operating cooling towers at elevated alkalinities and pH has previously been documented by published guidelines. The guidelines indicate that these conditions facilitate corrosion control and favor chlorine persistence which enhances the effectiveness of continuous chlorination in biofouling control. This information suggests that control of Legionella species in cooling towers can be accomplished by operating the towers under alkaline conditions. To test this possibility, we collected water samples over a period of months from a hospital cooling tower. The samples were analyzed for a variety of chemical parameters. Subsamples were pasteurized and inoculated with non-agar-passaged Legionella pneumophila which had been maintained in tap water. Correlation of subsequent Legionella growth with corresponding pH and alkalinity values revealed statistically significant inverse associations. These data support the hypothesis that operating cooling towers outside of the optimal conditions for Legionella growth (e.g., at elevated alkalinities and a pH greater than 9) may be a useful approach to controlling growth in this habitat.     

Poor efficacy of residual chlorine disinfectant in drinking water to inactivate waterborne pathogens in distribution systems.

To evaluate the inactivating power of residual chlorine in a distribution system, test microorganisms (Escherichia coli, Clostridium perfringens, bacteriophage phi-X 170, and poliovirus type 1) were added to drinking water samples obtained from two water treatment plants and their distribution system. Except for Escherichia coli, microorganisms remained relatively unaffected in water from the distribution systems tested. When sewage was added to the water samples, indigenous thermotolerant coliforms were inactivated only when water was obtained from sites very close to the treatment plant and containing a high residual chlorine concentration. Clostridium perfringens was barely inactivated, suggesting that the most resistant pathogens such as Giardia lamblia, Cryptosporidium parvum, and human enteric viruses would not be inactivated. Our results suggest that the maintenance of a free residual concentration in a distribution system does not provide a significant inactivation of pathogens, could even mask events of contamination of the distribution, and thus would provide only a false sense of safety with little active protection of public health. Recent epidemiological studies that have suggested a significant waterborne level of endemic gastrointestinal illness could then be explained by undetected intrusions in the distribution system, intrusions resulting in the infection of a small number of individuals without eliciting an outbreak situation.     

Susceptibility of Legionella pneumophila to chlorine in tap water

A study was conducted to compare the susceptibility of legionellae and coliforms to disinfection by chlorine. The chlorine residuals used were similar to concentrations that might be found in the distribution systems of large public potable water supplies. The effects of various chlorine concentrations, temperatures, and pH levels were considered. A number of different Legionella strains, both environmental and clinical, were tested. The results indicate that legionellae are much more resistant to chlorine than are coliform bacteria. At 21 degrees C, pH 7.6, and 0.1 mg of free chlorine residual per liter, a 99% kill of L. pneumophila was achieved within 40 min, compared with less than 1 min for Escherichia coli. The observed resistance is enhanced as conditions for disinfection become less optimal. The required contact time for the removal of L. pneumophilia was twice as long at 4 degrees C than it was at 21 degrees C. These data suggest that legionellae can survive low levels of chlorine for relatively long periods of time.     

Susceptibility of Legionella pneumophila to chlorine in tap water.

A study was conducted to compare the susceptibility of legionellae and coliforms to disinfection by chlorine. The chlorine residuals used were similar to concentrations that might be found in the distribution systems of large public potable water supplies. The effects of various chlorine concentrations, temperatures, and pH levels were considered. A number of different Legionella strains, both environmental and clinical, were tested. The results indicate that legionellae are much more resistant to chlorine than are coliform bacteria. At 21 degrees C, pH 7.6, and 0.1 mg of free chlorine residual per liter, a 99% kill of L. pneumophila was achieved within 40 min, compared with less than 1 min for Escherichia coli. The observed resistance is enhanced as conditions for disinfection become less optimal. The required contact time for the removal of L. pneumophilia was twice as long at 4 degrees C than it was at 21 degrees C. These data suggest that legionellae can survive low levels of chlorine for relatively long periods of time.     

Legionella spp. in Puerto Rico cooling towers.

Water samples from air conditioning cooling towers receiving different treatment protocols on five large municipal buildings in San Juan, P.R., were assayed for various Legionella spp. and serogroups by using direct immunofluorescence. Several water quality parameters were also measured for each sample. Guinea pigs were inoculated with water samples to confirm pathogenicity and recover viable organisms. Legionella pneumophila serogroups 1 to 6, L. bozemanii, L. micdadei, L. dumoffii, and L. gormanii were observed in at least one of the cooling towers. L. pneumophila was the most abundant species; its density reached 10(5) cells per ml, which is within the range that is considered potentially pathogenic to humans. A significantly higher density of L. pneumophila was observed in the cooling tower water that was not being treated with biocides. Percent respiration (INT) and total cell activity (acridine orange direct count) were inversely correlated with bacterial density. This study demonstrates that Legionella spp. are present in tropical air-conditioning cooling systems and that, without continuous biocide treatment, they may reach densities that present a health risk.     

Legionella spp. in Puerto Rico cooling towers

Water samples from air conditioning cooling towers receiving different treatment protocols on five large municipal buildings in San Juan, P.R., were assayed for various Legionella spp. and serogroups by using direct immunofluorescence. Several water quality parameters were also measured for each sample. Guinea pigs were inoculated with water samples to confirm pathogenicity and recover viable organisms. Legionella pneumophila serogroups 1 to 6, L. bozemanii, L. micdadei, L. dumoffii, and L. gormanii were observed in at least one of the cooling towers. L. pneumophila was the most abundant species; its density reached 10(5) cells per ml, which is within the range that is considered potentially pathogenic to humans. A significantly higher density of L. pneumophila was observed in the cooling tower water that was not being treated with biocides. Percent respiration (INT) and total cell activity (acridine orange direct count) were inversely correlated with bacterial density. This study demonstrates that Legionella spp. are present in tropical air-conditioning cooling systems and that, without continuous biocide treatment, they may reach densities that present a health risk.     

Aerosolization of mycobacteria and legionellae during dental treatment: low exposure despite dental unit contamination

Dental unit waterlines (DUWL) support growth of a dense microbial population that includes pathogens and hypersensitivity-inducing bacteria, such as Legionella spp. and non-tuberculous mycobacteria (NTM). Dynamic dental instruments connected to DUWL generate aerosols in the work environment, which could allow waterborne pathogens to be aerosolized. The use of the real-time quantitative polymerase chain reaction (qPCR) provides a more accurate estimation of exposure levels compared with the traditional culture approach. Bioaerosol sampling was performed 13 times in an isolated dental treatment room according to a standardized protocol that included four dental prophylaxis treatments. Inhalable dust samples were taken at the breathing zone of both the hygienist and patient and outside the treatment room (control). Total bacteria as well as Legionella spp. and NTM were quantified by qPCR in bioaerosol and DUWL water samples. Dental staff and patients are exposed to bacteria generated during dental treatments (up to 4.3 E + 05 bacteria per m(3) of air). Because DUWL water studied was weakly contaminated by Legionella spp. and NTM, their aerosolization during dental treatment was not significant. As a result, infectious and sensitization risks associated with legionellae and NTM should be minimal.     

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.