Legionella pneumophila in cooling towers: fluctuations in counts, determination of genetic variability by pulsed-field gel electrophoresis (PFGE), and persistence of PFGE patterns

The concentrations of Legionella pneumophila in cooling towers may vary considerably over short periods of time, producing significant fluctuations throughout the year. Despite genetic variability, in small geographical areas the same indistinguishable pulsed-field gel electrophoresis patterns may be shared among different cooling towers and persist over time.     

Genotypic variability and persistence of Legionella pneumophila PFGE patterns in 34 cooling towers from two different areas

Genotypic variability and clonal persistence are important concepts in molecular epidemiology as they facilitate the search for the source of sporadic cases or outbreaks of legionellosis. We studied the genotypic variability and persistence of Legionella pulsed-field gel electrophoresis (PFGE) patterns over time (period > 6 months) in 34 positive cooling towers from two different areas. In area A, radius of 70 km, 52 indistinguishable PFGE patterns were differentiated among the 27 cooling towers. In 13 cooling towers we observed ≥ 2 PFGE patterns. Each cooling tower had its own indistinguishable Legionella PFGE pattern which was not shared with any other cooling tower. In area B, radius of 1 km, 10 indistinguishable PFGE patterns were obtained from the seven cooling towers. In four, we observed ≥ 2 PFGE patterns. Three of these 10 indistinguishable PFGE patterns were shared by more than one cooling tower. In 27 of 34 cooling towers the same PFGE pattern was recovered after 6 months to up to 5 years of follow-up. The large genotypic diversity of Legionella observed in the cooling towers aids in the investigation of community outbreaks of Legionnaires' disease. However, shared patterns in small areas may confound the epidemiological investigation. The persistence of some PFGE patterns in cooling towers makes the recovery of the Legionella isolate causing the outbreak possible over time.     

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

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

Production of Respirable Vesicles Containing Live Legionella pneumophila Cells by Two Acanthamoeba spp.

Two Acanthamoeba species, fed at three temperatures, expelled vesicles containing living Legionella pneumophila cells. Vesicles ranged from 2.1 to 6.4 μm in diameter and theoretically could contain several hundred bacteria. Viable L. pneumophila cells were observed within vesicles which had been exposed to two cooling tower biocides for 24 h. Clusters of bacteria in vesicles were not dispersed by freeze-thawing and sonication. Such vesicles may be agents for the transmission of legionellosis associated with cooling towers, and the risk may be underestimated by plate count methods.     

The efficacy of biocides and other chemical additives in cooling water systems in the control of amoebae

Aims:  In vitro experiments were undertaken to evaluate biocide formulations commonly used in cooling water systems against protozoa previously isolated from cooling towers. The investigations evaluated the efficacy of these formulations against amoebic cysts and trophozoites.
Methods and Results:  Laboratory challenges against protozoa isolated from cooling towers using chlorine, bromine and isothiazolinone biocides showed that all were effective after 4 h. The presence of molybdate and organic phosphates resulted in longer kill times for bromine and isothiazolinones. All treatments resulted in no detectable viable protozoa after 4 h of exposure.
Conclusions:  The chemical disinfection of planktonic protozoa in cooling water systems is strongly influenced by the residence time of the formulation and less so by its active constituent. Bromine and isothiazolinone formulations may require higher dosage of concentrations than currently practiced if used in conjunction with molybdate- and phosphate-based scale/corrosion inhibitors.
Significance and Impact of the Study:  Cooling water systems are complex microbial ecosystems in which predator–prey relationships play a key role in the dissemination of Legionella . This study demonstrated that at recommended dosing concentrations, biocides had species-specific effects on environmental isolates of amoebae that may act as reservoirs for Legionella multiplication in cooling water systems.     

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.     

Isolation of Legionella pneumophila from Cooling Tower Water by Filtration

Methods are described for detection of Legionella pneumophila in cooling tower water or other water sources by direct fluorescent-antibody staining. A procedure for isolation of Legionella bacteria from water samples by guinea pig inoculation is described. Two different serogroups of L. pneumophila were isolated repeatedly from one of the cooling towers.     

Adaptation of amoebae to cooling tower biocides

Adaptation of amoebae to four cooling tower Biocides, which included a thiocarbamate compound, tributyltin neodecanoate mixed with quaternary ammonium compounds (TBT/QAC), another QAC alone, and an isothiazolin derivative, was studied. Previously we found that amoebae isolated from waters of cooling towers were more resistant to cooling tower biocides than amoebae from other habitats. Acanthamoeba hatchetti and Cochliopodium bilimbosum, obtained from American Type Culture Collection and used in the previous studies, were tested to determine whether they could adapt to cooling tower Biocides. A. hatchetti was preexposed to subinhibitory concentrations of the four Biocides for 72h, after which they were tested for their resistance to the same and other biocides. C. bilimbosum was exposed to only two biocides, as exposure to the other two was lethal after 72 h. Preexposure to the subinhibitory concentrations of the Biocides increased the resistance of the amoebae, as indicated by a significant increase in the minimum inhibitory concentration (up to 30-fold). In addition, cross-resistance was also observed, i.e., exposure to one biocide caused resistance to other biocides. These results show that amoebae can adapt to biocides in a short time. The phenomenon of cross-resistance indicates that regularly alternating biocides, as is done to control microbial growth in cooling towers, may not be effective in keeping amoeba populations in check. On the contrary, exposure to one biocide may boost the amoebae's resistance to a second biocide before the second biocide is used in the cooling tower. Since amoebae may harbor Legionella, or alone cause human diseases, these results may be important in designing effective strategies for controlling pathogens in cooling towers. Correspondence to: S.G. Berk.     

Integrated real-time PCR for detection and monitoring of Legionella pneumophila in water systems

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

Despersion and survival ofLegionella in air

Summary From the time of the report of the Philadelphia outbreak in 1976, the airborne contamination of patients through cooling towers had been suspected although never truly corroborated. Since then, contamination through different air conditioning systems or cooling towers has often been implicated in both epidemics and endemics as well as in sporadic cases. Whatever the conditions, contaminated water present in the different air conditioning systems remains the main source of infection. From the contaminated sites an aerosol occurs. The infectivity of the aerosol is linked to the size of the droplets (d≥μm). A close surveillance is mandatory and preventive measures have to be tanken.     

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

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

Disinfection of bacterial biofilms in pilot-scale cooling tower systems

The impact of continuous chlorination and periodic glutaraldehyde treatment on planktonic and biofilm microbial communities was evaluated in pilot-scale cooling towers operated continuously for 3 months. The system was operated at a flow rate of 10,080 l day^?1. Experiments were performed with a well-defined microbial consortium containing three heterotrophic bacteria: Pseudomonas aeruginosa, Klebsiella pneumoniae and Flavobacterium sp. The persistence of each species was monitored in the recirculating cooling water loop and in biofilms on steel and PVC coupons in the cooling tower basin. The observed bacterial colonization in cooling towers did not follow trends in growth rates observed under batch conditions and, instead, reflected differences in the ability of each organism to remain attached and form biofilms under the high-through flow conditions in cooling towers. Flavobacterium was the dominant organism in the community, while P. aeruginosa and K. pneumoniae did not attach well to either PVC or steel coupons in cooling towers and were not able to persist in biofilms. As a result, the much greater ability of Flavobacterium to adhere to surfaces protected it from disinfection, whereas P. aeruginosa and K. pneumoniae were subject to rapid disinfection in the planktonic state.     

Legionnaires' disease outbreaks and cooling towers with amplifiedLegionella concentrations

This study was designed to investigate the possible association of high colony counts of legionellae from cooling towers and evaporative condensers with Legionnaires' disease outbreaks. We obtained legionellae counts from samples of cooling towers and evaporative condensers that were the likely sources of two different Legionnaires' disease outbreaks and compared these counts with those from cooling towers that were not associated with reports of human disease. Among 675 potential control cooling tower that were samples from 258 facilities, 136 facilities had one or more cooling towers that met our criteria for inclusion into the study. Samples taken from buildings where an outbreak had occurred had much higherLegionella counts than did samples from other buildings. Colony counts from the two outbreak-associated facilities were significantly higher than colony counts from other facilities [Wilcoxon Rank Sum Test (Exact), p<0.01]. The results of the study suggest that, among cooling towers that test positive for the presence of legionellae, higher colony counts are associated with higher risk of Legionnaires' disease.     

Survival of protozoa in cooling tower biocides

Protozoa from cooling towers may serve as hosts for legionellae, but such protozoa have not been examined with respect to effects of cooling tower biocides. In this study, two ciliate species,Tetrahymena sp andColpoda sp, and two amoebae species,Vannella miroides andAcanthamoeba hatchetti, were isolated from a cooling tower and tested for survival in the presence of four cooling tower biocides. The protozoa were exposed for 24 h to a thiocarbamate compound, an isothiazolone compound, quaternary ammonium compounds (QAC), and tributyltin neodecanoate with quarternary ammonium compounds (TBT/QAC). After exposure, cells were examined for viability. The highest concentration of each biocide in which cells could survive was compared to the manufacturers' recommended maintenance dosage (MRMD) of the biocides.Tetrahymena andColpoda survived concentrations within the range of the MRMD of thiocarbamate and QAC.Vannella andAcanthamoeba survived concentrations within the MRMD of thiocarbamate, isothiazolone, and QAC.Colpoda cysts andAcanthamoebae cysts remained viable after exposure to concentrations much greater than the MRMD of thiocarbamate, isothiazolone, and QAC. None of the protozoa in any stage could survive the MRMD of TBT/QAC. These results show that protozoa indigenous to cooling towers may survive the recommended concentration of certain biocides, and this information may be important in devising procedures for eradicating hosts for legionellae.     

Predicted survival of the bay anchovy (Anchoa mitchilli) in the heated effluent of a power plant on Galveston Bay,Texas

Synopsis The bay anchovy (Anchoa mitchilli), collected from the intake canal of the P.H. Robinson Generating Station, Bacliff, Texas, were tested for 180 min at various constant temperatures during June 1974 through September 1975 to determine if they could survive passage through the discharge canal system.Three-h survival temperatures were significantly lower, throughout the year, than the temperatures in the discharge canal both afferent and efferent to the cooling towers. This indicates that the bay anchovy entrained and entrapped from the intake canal and exposed to the heated effluent could not survive in the cooling water system. However, the cooling towers should aid survival of the fish in the canal below the towers.     

Routine Sampling and the Control of Legionella spp. inCooling Tower Water Systems

Cooling water samples from 31 cooling tower systems were cultured for Legionella over a 16-week summer period. The selected systems were known to be colonized by Legionella. Mean Legionella counts and standard deviations were calculated and time series correlograms prepared for each system. The standard deviations of Legionella counts in all the systems were very large, indicating great variability in the systems over the time period. Time series analyses demonstrated that in the majority of cases there was no significant relationship between the Legionella counts in the cooling tower at time of collection and the culture result once it was available. In the majority of systems (25/28), culture results from Legionella samples taken from the same systems 2 weeks apart were not statistically related. The data suggest that determinations of health risks from cooling towers cannot be reliably based upon single or infrequent Legionella tests. Received: 19 January 2000 / Accepted: 9 May 2000     

Disinfection of bacterial biofilms in pilot-scale cooling tower systems

The impact of continuous chlorination and periodic glutaraldehyde treatment on planktonic and biofilm microbial communities was evaluated in pilot-scale cooling towers operated continuously for 3 months. The system was operated at a flow rate of 10,080 l day(-1). Experiments were performed with a well-defined microbial consortium containing three heterotrophic bacteria: Pseudomonas aeruginosa, Klebsiella pneumoniae and Flavobacterium sp. The persistence of each species was monitored in the recirculating cooling water loop and in biofilms on steel and PVC coupons in the cooling tower basin. The observed bacterial colonization in cooling towers did not follow trends in growth rates observed under batch conditions and, instead, reflected differences in the ability of each organism to remain attached and form biofilms under the high-through flow conditions in cooling towers. Flavobacterium was the dominant organism in the community, while P. aeruginosa and K. pneumoniae did not attach well to either PVC or steel coupons in cooling towers and were not able to persist in biofilms. As a result, the much greater ability of Flavobacterium to adhere to surfaces protected it from disinfection, whereas P. aeruginosa and K. pneumoniae were subject to rapid disinfection in the planktonic state.     

Failure of Legionella pneumophila sensitivities to predict culture results from disinfectant-treated air-conditioning cooling towers.

The disinfection of cooling towers based on manufacturers' treatment protocols, as employed in units installed at various public gathering places in Dallas, Tex. (hotels, municipal auditorium), and at the city health department, was evaluated for effectiveness in controlling Legionella pneumophila and compared with previous laboratory studies. In specimens collected in September and December, 1978, L. pneumophila was isolated from 2 of 4 specimens from untreated cooling towers, 2 of 4 specimens from towers treated with agents deemed ineffective in earlier laboratory tests, 6 of 11 specimens from towers treated with putatively effective agents, and 0 of 4 specimens from towers treated with an agent unknown efficacy. These results suggest the need for further studies to identify biocidal agents effective in eliminating L. pneumophila from air-conditioning cooling towers.