Detection and quantification of Legionella pneumophila in water samples using competitive PCR

The presence of high levels of Legionella pneumophila in man-made aquatic systems correlates with the incidence of nosocomial Legionnaires' disease. This requires a rapid, reliable, and sensitive quantification of L. pneumophila concentrations in suspected water systems. In this research, a homologous competitor was developed and evaluated in a L. pneumophila competitive polymerase chain reaction (cPCR) to quantify this human pathogen in a quick, cost-effective, and reliable way. Accuracy of cPCR was evaluated by analyzing cooling tower and tap water samples spiked with known concentrations of L. pneumophila bacteria, in parallel with the standard culture method. Legionella pneumophila amounts detected and calculated from cPCR and culture correlated very well: r = 0.998, P = 0.002 for tap water and r = 0.990, P = 0.009 for cooling tower water. Nevertheless, for both kinds of water samples, mean numbers of L. pneumophila calculated from cPCR results were always higher than those obtained by culture. This study makes it clear that the rapid, sensitive, and cost-effective L. pneumophila cPCR is a promising alternative to the standard time-consuming culture method and expensive real-time PCR to enumerate L. pneumophila bacteria in environmental water samples.     

Introduction of a boost of Legionella pneumophila into a stagnant-water model by heat treatment

An environmentally representative stagnant-water model was developed to monitor the growth dynamics of Legionella pneumophila. This model was evaluated for three distinct water treatments: untreated tap water, heat-treated tap water, and heat-treated tap water supplemented with Pseudomonas putida, a known biofilm-forming bacterium. Bringing heat-treated tap water after subsequent cooling into contact with a densely formed untreated biofilm was found to promote the number of L. pneumophila by 4 log units within the biofilm, while the use of untreated water only sustained the L. pneumophila levels. Subsequent colonization of the water phase by L. pneumophila was noticed in the heat-treated stagnant-water models, with concentrations as high as 1 x 10(10) mip gene copies L(-1) stagnant water. Denaturing gradient gel electrophoresis in combination with clustering analysis of the prokaryotic community in the water phase and in the biofilm phase suggests that the different water treatments induced different communities. Moreover, boosts of L. pneumophila arising from heat treatment of water were accompanied by shifts to a more diverse eukaryotic community. Stimulated growth of L. pneumophila after heating of the water may explain the rapid recolonization of L. pneumophila in water systems. These results highlight the need for additional or alternative measures to heat treatment of water in order to prevent or abate potential outbreaks of L. pneumophila.     

Effectiveness of 1-bromo-3-chloro-5,5-dimethylhydantoin against Legionella pneumophila in a cooling tower.

Cooling towers are considered to be man-made amplifiers of Legionella spp. Thus, the proper maintenance and choice of biocides is important. The only biocidal measure that has thus far been shown to be effective in field tests is the judicious use of chlorination. Perturbation studies with 1-bromo-3-chloro-5, 5-dimethylhydantoin (Bromicide; Great Lakes Chemical Corp., West Lafayette, Ind.) (BCD) were conducted on an industrial cooling tower shown to contain Legionella pneumophila. At the concentrations recommended by the manufacturer, neither the density nor the activity of L. pneumophila was affected. At comcentrations greater than 2.0 ppm (2.0 micorgram/ml) free of residual, BCD was not effective in reducing L. pneumophila to source water concentrations, nor was it effective in reducing the 2-p-iodophenyl-3-p-nitrophenyl-5-phenyl tetrazolium chloride activity of the bacterium in situ. The data indicate that at concentrations up to 2.0 ppm, BCD is not effective in these tower studies.     

Effectiveness of 1-bromo-3-chloro-5,5-dimethylhydantoin against Legionella pneumophila in a cooling tower

Cooling towers are considered to be man-made amplifiers of Legionella spp. Thus, the proper maintenance and choice of biocides is important. The only biocidal measure that has thus far been shown to be effective in field tests is the judicious use of chlorination. Perturbation studies with 1-bromo-3-chloro-5, 5-dimethylhydantoin (Bromicide; Great Lakes Chemical Corp., West Lafayette, Ind.) (BCD) were conducted on an industrial cooling tower shown to contain Legionella pneumophila. At the concentrations recommended by the manufacturer, neither the density nor the activity of L. pneumophila was affected. At comcentrations greater than 2.0 ppm (2.0 micorgram/ml) free of residual, BCD was not effective in reducing L. pneumophila to source water concentrations, nor was it effective in reducing the 2-p-iodophenyl-3-p-nitrophenyl-5-phenyl tetrazolium chloride activity of the bacterium in situ. The data indicate that at concentrations up to 2.0 ppm, BCD is not effective in these tower studies.     

Isolation of Legionella pneumophila from water systems: methods and preliminary results.

A preliminary survey of water systems in hospitals and hotels showed that Legionella pneumophila may be found in water storage and distribution systems as well as in the recirculating cooling water of air-conditioning plants. Altogether 42 isolates of L pneumophila were made from 31 establishments, six of which were associated with cases of legionnaires'' disease but in 25 of which there was no known association with disease. In the six establishments implicated epidemiologically as the source of legionnaires'' disease, these organisms were found in each of their water-distribution systems and also in the cooling water from each of the three with cooling towers. In establishments not associated with cases, water from three out of nine cooling towers, four out of 24 taps or showers, and one out of 15 storage tanks was found to contain legionellae. The organisms were isolated by guinea-pig inoculation and subsequent culture of their peritoneal fluid, liver, and spleen. Finding L pneumophila in water systems in the absence of cases of legionnaires'' disease should not at present be an indication for attempts at eradication.     

Effects of metals on Legionella pneumophila growth in drinking water plumbing systems.

An investigation of the chemical environment and growth of Legionella pneumophila in plumbing systems was conducted to gain a better understanding of its ecology in this habitat. Water samples were collected from hospital and institutional hot-water tanks known to have supported L. pneumophila and were analyzed for 23 chemical parameters. The chemical environment of these tanks was found to vary extensively, with the concentrations of certain metals reaching relatively high levels due to corrosion. The effect of various chemical conditions on L. pneumophila growth was then examined by observing its multiplication in the chemically analyzed hot-water tank samples after sterilization and reinoculation with L. pneumophila. L. pneumophila and associated microbiota used in these experiments were obtained from a hot-water tank. These stains were maintained in tap water and had never been passaged on agar. The results of the growth studies indicate that although elevated concentrations of a number of metals are toxic, lower levels of certain metals such as iron, zinc, and potassium enhance growth of naturally occurring L. pneumophila. Parallel observations on accompanying non-Legionellaceae bacteria failed to show the same relationship. These findings suggest that metal plumbing components and associated corrosion products are important factors in the survival and growth of L. pneumophila in plumbing systems and may also be important in related habitats such as cooling towers and air-conditioning systems.     

Ecological studies of Legionella species. I. Viable counts of Legionella pneumophila in cooling tower water

The occurrence and viable counts of Legionella pneumophila in acid-treated water samples of 62 cooling towers on the main island of Japan were determined by inoculating them onto plates of Wadowsky-Yee-Okuda (WYO) agar medium. WYO plate cultures of 39 (63%) of the samples yielded L. pneumophila with viable counts ranging from 10 to 10(4) colony-forming units per 100 ml. Of the L. pneumophila isolates, 157 were serologically identified as serogroup 1, and the remaining 21 were agglutinated by serogroup 3 (2 strains) and serogroup 6 (19 strains) antisera. In each culture-positive water sample, the pH and the number of other bacteria were found not be statistically significantly correlated with the viable counts of L. pneumophila. However, a higher rate of recovery of L. pneumophila was obtained with the water samples with a smaller number of other bacteria. Practical use of commercially available antialgal or antimicrobial agents was found not to be significantly effective for controlling the occurrence and growth of L. pneumophila in cooling tower water.     

Protocol for sampling environmental sites for legionellae

A protocol for sampling environmental sites was developed and used to identify possible sources of Legionella species in support of epidemiologic investigations at two hospitals. In hospital A, legionellae were isolated from 43 of 106 (40%) different sites. Three separate Legionella pneumophila serotypes and a previously unrecognized species were present in different combinations in the positive samples. Two of five cooling towers contained the same L. pneumophila serogroup 1 monoclonal type (1,2,4,5) as was isolated from patients. The same monoclonal type was also isolated from make-up water for the two cooling towers, a hot water tank, water separators in four main air compressor systems for respiratory therapy, and cold and hot water faucets. In hospital B, 13 of 37 (38%) sample sites contained legionellae, all of which were L. pneumophila serogroup 1. The monoclonal type matching isolates from patients (1,2,4,5) was found at the highest concentration in a hot water tank, but it was also present at four other sample sites. Since legionellae not related to disease may be found in many of the sites sampled, an epidemiologic association with the probable source should be established before intervention methods, such as disinfection, are undertaken.     

Protocol for sampling environmental sites for legionellae.

A protocol for sampling environmental sites was developed and used to identify possible sources of Legionella species in support of epidemiologic investigations at two hospitals. In hospital A, legionellae were isolated from 43 of 106 (40%) different sites. Three separate Legionella pneumophila serotypes and a previously unrecognized species were present in different combinations in the positive samples. Two of five cooling towers contained the same L. pneumophila serogroup 1 monoclonal type (1,2,4,5) as was isolated from patients. The same monoclonal type was also isolated from make-up water for the two cooling towers, a hot water tank, water separators in four main air compressor systems for respiratory therapy, and cold and hot water faucets. In hospital B, 13 of 37 (38%) sample sites contained legionellae, all of which were L. pneumophila serogroup 1. The monoclonal type matching isolates from patients (1,2,4,5) was found at the highest concentration in a hot water tank, but it was also present at four other sample sites. Since legionellae not related to disease may be found in many of the sites sampled, an epidemiologic association with the probable source should be established before intervention methods, such as disinfection, are undertaken.     

Detection of Legionella pneumophila in water samples by species-specific real-time and nested PCR assays

AIMS: Legionella pneumophila is a contaminant of man-made water systems, including potable water, cooling towers, water systems of large buildings, etc. It is the most common causative agent of legionellosis, a respiratory infection, which may give rise to restricted outbreaks. To survey environmental water samples from hospitals and private habitations in Bologna, we developed a species-specific nested and a TaqMan real-time PCR for the detection of L. pneumophila. We compared the two assays and both to cultural isolation. METHODS AND RESULTS: The targeted gene was macrophage infectivity potentiator (mip), conserved in L. pneumophila, and divergent in other legionellae. One assay was based on a nested PCR and the other on a TaqMan real-time PCR protocol. Their sensitivities were 14 % or 5% higher than that of cultural isolation respectively. The detection limits were 1-2 genome equivalents per 50-microl reaction. Specificity was assessed using DNA from nine target and 20 nontarget organisms. CONCLUSIONS: When applied to water samples, both assays detected L. pneumophila at 80% or higher frequency. SIGNIFICANCE AND IMPACT OF THE STUDY: The species-specific molecular diagnosis of L. pneumophila by means of nested PCR does not require a specific instrumentation, exhibits a high sensitivity, and is advantageous over the cultural isolation and real-time PCR detection. It allows to quickly monitor water samples for the risk assessment of environmental contaminations.     

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.     

The impact of electrochemical disinfection on Escherichia coli and Legionella pneumophila in tap water

In order to reduce the risks of Legionnaires' disease, caused by the bacterium Legionella pneumophila, disinfection of tap water systems contaminated with this bacterium is a necessity. This study investigates if electrochemical disinfection is able to eliminate such contamination. Hereto, water spiked with bacteria (10(4)CFU Escherichia coli or L. pneumophila/ml) was passed through an electrolysis cell (direct effect) or bacteria were added to tap water after passage through such disinfection unit (residual effect). The spiked tap water was completely disinfected, during passage through the electrolysis cell, even when only a residual free oxidant concentration of 0.07 mg/l is left (L. pneumophila). The residual effect leads to a complete eradication of cultivable E. coli, if after reaction time at least a free oxidant concentration of 0.08 mg/l is still present. Similar conditions reduce substantially L. pneumophila, but a complete killing is not realised.     

Enhancement of Legionella pneumophila culture isolation from microenvironments by macrophage infectivity potentiator (mip) gene-specific nested polymerase chain reaction

The combination of a Legionella pneumophila culture isolation technique and macrophage infectivity potentiator (mip) gene-specific nested polymerase chain reaction (PCR) is pivotal for effective routine use in an environmental water system laboratory. Detection of Legionella organisms in 169 environmental samples was performed by using modified buffered charcoal yeast extract (MBCYE) agar for conventional culture. Nested PCR specific for L. pneumophila was performed using boiled genomic DNA extracts from filtered and Chelex 100-treated water samples, or by using silica-gel membrane spin column-eluted DNA from concentrated pond, canal and river samples. Overall, the nested PCR was twelvefold more sensitive than the culture method. The target amplicons (471 basepairs) of all 4 biochemically characterized L. pneumophila isolates were sequenced. They had homology at the DNA and protein levels to 3' proximity of the mip-coding gene of L. pneumophila deposited in genome databases. EcoRI- or KpnI-digested PCR fragments with expected sizes were also confirmed in all 52 PCR-positive samples that were isolated from cooling towers and condenser drains. Viable but nonculturable L. pneumophila might have been present in 48 PCR-positive samples. This study demonstrates that detection of the genetically stable mip gene by nested PCR with a modified process of water sample preparation can be rapidly and effectively used to enhance isolation of the L. pneumophila taxon from microenvironments.     

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.     

Total and viable Legionella pneumophila cells in hot and natural waters as measured by immunofluorescence-based assays and solid-phase cytometry

A new method was developed for the rapid and sensitive detection of viable Legionella pneumophila. The method combines specific immunofluorescence (IF) staining using monoclonal antibodies with a bacterial viability marker (ChemChrome V6 cellular esterase activity marker) by means of solid-phase cytometry (SPC). IF methods were applied to the detection and enumeration of both the total and viable L. pneumophila cells in water samples. The sensitivity of the IF methods coupled to SPC was 34 cells liter(-1), and the reproducibility was good, with the coefficient of variation generally falling below 30%. IF methods were applied to the enumeration of total and viable L. pneumophila cells in 46 domestic hot water samples as well as in cooling tower water and natural water samples, such as thermal spring water and freshwater samples. Comparison with standard plate counts showed that (i) the total direct counts were always higher than the plate counts and (ii) the viable counts were higher than or close to the plate counts. With domestic hot waters, when the IF assay was combined with the viability test, SPC detected up to 3.4 × 10(3) viable but nonculturable L. pneumophila cells per liter. These direct IF methods could be a powerful tool for high-frequency monitoring of domestic hot waters or for investigating the occurrence of viable L. pneumophila in both man-made water systems and environmental water samples.     

Application of molecular genetic methods during Legionnaires' disease outbreak in town Verkhnyaya Pyshma

The aim of the study was to perform molecular genetic analysis based on multi-locus sequence typing in order to identify source of Legionnaires' disease outbreak in town Verkhnyaya Pyshma in July 2007 and genetic profile of the causative agent. Sequence-based typing protocol recommended by European Working Group on Legionella infection (EWGLI) was used. It was not possible to obtain satisfactory results of Fla gene sequencing for all samples. Obtained allelic profiles of other genes were typical for L. pneumophila. Allelic profiles of L. pneumophila isolated from patients were identical and matched with L. pneumophila DNA detected in water from hot water supply of domestic building, but differed from cooling tower's isolates and isolates from showerhead in apartment of one patient. Identity of 5 genes of L. pneumophila isolated from autopsy samples and from hot water of central hot water supply of domestic building confirms aspiration route of infection through hot water contaminated by the microorganism. L. pneumophila detected in water from cooling tower, showerhead in apartment of one patient, and from drainage canal of hot water supply station belonged to other allelic variants and, therefore, are not related with the outbreak.     

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.     

Development and evaluation of a Taqman duplex real-time PCR quantification method for reliable enumeration of Legionella pneumophila in water samples

This study describes the development and evaluation of a specific Legionella pneumophila Taqman duplex real-time PCR (qPCR) for fast and reliable quantification of this human pathogen in suspected man-made water systems. The qPCR assay was 100% specific for all L. pneumophila serogroups 1-15 with a sensitivity of 60 genome units/l and an amplification efficiency of 98%. Amplification inhibitors were detected via an exogenous internal positive control, which was amplified simultaneously with L. pneumophila DNA using its own primer and probe set. Mean recovery rates of the qPCR assay for tap water and cooling circuit water, spiked with a known number L. pneumophila bacteria, were 93.0% and 56.3%, respectively. Additionally, by using the Ultraclean Soil DNA isolation kit, we were able to remove amplification inhibitors ubiquitously present in cooling water. The practical value of our qPCR assay was evaluated through analysis of 30 water samples from showers, taps, eyewash stations, fire sprinklers and recirculation loops with qPCR and traditional culture. In conclusion, the described L. pneumophila Taqman duplex real-time assay proved to be specific, sensitive and reproducible. This makes it a promising method complementing the current time-consuming culture standard method.     

Role of stagnation and obstruction of water flow in isolation of Legionella pneumophila from hospital plumbing.

The stagnation of water in two of four hospital hot-water storage tanks found to contain Legionella pneumophila was reduced by keeping the two tanks continually on-line for 1 year. L. pneumophila colony counts in these two tanks fell quickly to low levels, whereas the organisms persisted in the two tanks that were not in use. L. pneumophila continued to be isolated from 50 to 100% of the hospital showerheads which were sampled during this period. We also examined aerators and other hospital faucet fixtures which obstruct water flow. L. pneumophila was isolated from 22 of 30 faucet aerators and 2 of 16 vacuum breakers but not from 26 nonobstructed faucets or 6 backflow preventers. Over a 7-month period, after nine faucet aerators were sterilized, 10 of 60 surveillance cultures revealed L. pneumophila, despite the inability to isolate the organism from the potable-water tanks in use. These data suggest that prevention of stagnation in hot-water tanks may be effective in reducing L. pneumophila concentrations in potable-water systems serving high-risk populations. We have also shown that faucet aerators, by providing a surface for L. pneumophila to colonize, can become secondary reservoirs for the organism in hospital plumbing.     

Role of stagnation and obstruction of water flow in isolation of Legionella pneumophila from hospital plumbing

The stagnation of water in two of four hospital hot-water storage tanks found to contain Legionella pneumophila was reduced by keeping the two tanks continually on-line for 1 year. L. pneumophila colony counts in these two tanks fell quickly to low levels, whereas the organisms persisted in the two tanks that were not in use. L. pneumophila continued to be isolated from 50 to 100% of the hospital showerheads which were sampled during this period. We also examined aerators and other hospital faucet fixtures which obstruct water flow. L. pneumophila was isolated from 22 of 30 faucet aerators and 2 of 16 vacuum breakers but not from 26 nonobstructed faucets or 6 backflow preventers. Over a 7-month period, after nine faucet aerators were sterilized, 10 of 60 surveillance cultures revealed L. pneumophila, despite the inability to isolate the organism from the potable-water tanks in use. These data suggest that prevention of stagnation in hot-water tanks may be effective in reducing L. pneumophila concentrations in potable-water systems serving high-risk populations. We have also shown that faucet aerators, by providing a surface for L. pneumophila to colonize, can become secondary reservoirs for the organism in hospital plumbing.