Detection of culturable and nonculturable Legionella species from hot water systems of public buildings in Japan

Aims: To investigate the prevalence of culturable and nonculturable Legionella species in hot water systems of public buildings in Japan and assess the risk factors associated with Legionella contamination in hot water systems. Methods and Results: Legionella species were detected by conventional culture and molecular methods in 130 water samples collected from 40 buildings. A total of 26 (20·0%) water samples from 17 (42·5%) buildings were positive by culture, qualitative PCR or both methods: Legionella pneumophila and Leg. anisa were detected in four samples by a culture method, whereas 23 samples were positive by qualitative PCR, with the presence of various Legionella species confirmed by sequencing. Of these 23 samples, bacterial counts were quantifiable in 21 by real‐time PCR (from 1·7 × 10⁵ to 2·6 × 10¹¹ cells per litre). Phylogenetic analysis of amplified partial 16S rRNA gene showed close relations to various species of Legionella, including Leg. anisa and Leg. micdadei, all of which have been associated with respiratory diseases or increased antibody titres in human sera. Assessment of risk factors showed that turbidity, free chlorine concentration, iron concentration and heterotrophic plate count (HPC) were significantly associated with Legionella contamination (P < 0·05). Conclusions: Contamination of hot water systems of public buildings with culturable and nonculturable Legionella species may be a potential risk factor for Legionella infection in Japan. Adequate levels of chlorine, low levels of iron and HPC are important maintenance measures in the reduction of Legionella contamination in hot water systems. Significance and Impact of the Study: More than 40% of hot water systems in the Japanese public buildings examined were contaminated by not only culturable Leg. pneumophila and Leg. anisa but also by nonculturable pathogenic species. To our knowledge, this is the first report of both culturable and nonculturable Legionella contamination in hot water systems of public buildings in Japan.     

Distribution of Indigenous Bacterial Pathogens and Potential Pathogens Associated with Roof-Harvested Rainwater

The harvesting of rainwater is gaining acceptance among many governmental authorities in countries such as Australia, Germany, and South Africa, among others. However, conflicting reports on the microbial quality of harvested rainwater have been published. To monitor the presence of potential pathogenic bacteria during high-rainfall periods, rainwater from 29 rainwater tanks was sampled on four occasions (during June and August 2012) in a sustainable housing project in Kleinmond, South Africa. This resulted in the collection of 116 harvested rainwater samples in total throughout the sampling period. The identities of the dominant, indigenous, presumptive pathogenic isolates obtained from the rainwater samples throughout the sampling period were confirmed through universal 16S rRNA PCR, and the results revealed that Pseudomonas (19% of samples) was the dominant genus isolated, followed by Aeromonas (16%), Klebsiella (11%), and Enterobacter (9%). PCR assays employing genus-specific primers also confirmed the presence of Aeromonas spp. (16%), Klebsiella spp. (47%), Legionella spp. (73%), Pseudomonas spp. (13%), Salmonella spp. (6%), Shigella spp. (27%), and Yersinia spp. (28%) in the harvested rainwater samples. In addition, on one sampling occasion, Giardia spp. were detected in 25% of the eight tank water samples analyzed. This study highlights the diverse array of pathogenic bacteria that persist in harvested rainwater during high-rainfall periods. The consumption of untreated harvested rainwater could thus pose a potential significant health threat to consumers, especially children and immunocompromised individuals, and it is recommended that harvested rainwater be treated for safe usage as an alternative water source.     

A rapid detection method using flow cytometry to monitor the risk of Legionella in bath water

Legionella species are the causative agents of human legionellosis, and bathing facilities have been identified as the sources of infection in several outbreaks in Japan. Researchers in Japan have recently reported evidence of significant associations between bacterial counts and the occurrence of Legionella in bathing facilities and in a hot tub model. A convenient and quantitative bacterial enumeration method is therefore required as an indicator of Legionella contamination or disinfection to replace existing methods such as time-consuming Legionella culture and expensive Legionella-DNA amplification. In this study, we developed a rapid detection method (RDM) to monitor the risk of Legionella using an automated microbial analyzing device based on flow cytometry techniques to measure the total number of bacteria in water samples within two minutes, by detecting typical patterns of scattered light and fluorescence. We first compared the results of our RDM with plate counting results for five filtered hot spring water samples spiked with three species of bacteria, including Legionella. Inactivation of these samples by chlorine was also assessed by the RDM, a live/dead bacterial fluorescence assay and plate counting. Using the RDM, the lower limit of quantitative bacterial counts in the spiked samples was determined as 3.0 × 10³ (3.48 log) counts mL^− 1. We then used a laboratory model of a hot tub and found that the RDM could monitor the growth curve of naturally occurring heterotrophic bacteria with 1 and 2 days' delayed growth of amoeba and Legionella, respectively, and could also determine the killing curve of these bacteria by chlorination. Finally, samples with ≥ 3.48 or < 3.48 log total bacterial counts mL^− 1 were tested using the RDM from 149 different hot tubs, and were found to be significantly associated with the positive or negative detection of Legionella with 95% sensitivity and 84% specificity. These findings indicated that the RDM can be used for Legionella control at bathing facilities, especially those where the effectiveness of chlorine is reduced by the presence of Fe²⁺, Mn²⁺, NH₄⁺, skin debris, and/or biofilms in the water.     

Examination of microbial contaminants of emergency showers and eyewash stations

To evaluate the effects of regular flushing, water from fifty emergency eyewash and shower stations was cultured for the presence of potentially pathogenic protozoa, heterotrophic bacteria, and Legionella species. This study also provided the opportunity to evaluate a commercially available molecular assay for the direct detection of Legionella sp in environmental samples. The Perkin Elmer Legionella EnviroAmp polymerase chain reaction (PCR) kit and culture on buffered charcoal yeast extract agar were used to detect Legionella species in water samples. Chemical and physical parameters of station water measured included: pH, hardness, alkalinity, turbidity, conductivity, total chlorine and assimilable organic carbon. Protozoal isolates were identified by classical identification methods, and isolates from the stations were identified as Hartmannella sp, Vexillifera sp, Vahlkampfia sp, Acanthamoeba sp, and Vanella sp. Heterotrophic plate counts ranged from 10² to 10⁶ CFU ml⁻¹ and acridine orange total counts ranged from 10³ to 10⁶ cells ml⁻¹ after regular flushing. PCR and gene probe analysis showed that 89% of the stations (eyewash and shower) were positive for Legionella species by PCR, while 6% of the samples were culture positive. These results indicate that routine flushing alone is not sufficient to control microbial contamination and disinfection must also be included in a routine maintenance program. In addition, regular maintenance, disinfection, and monitoring of emergency eyewash and shower stations is important in preventing potential secondary microbial infections by either direct inoculation or aerosol transmission. Received 02 September 1997/ Accepted in revised form 29 November 1997     

Comparison of Detection Methods for Legionella Species in Environmental Water by Colony Isolation,Fluorescent Antibody Staining,and Polymerase Chain Reaction

Three detection methods for Legionella species in water samples from cooling towers and a river were examined. Direct counting of bacteria stained with fluorescent antibody (FA) for L. pneumophila (serogroups 1 to 6) could detect the cell of 10⁴ to 10⁶ cell/100 ml in all 14 samples, while colony counting method detected 10 to 10³ CFU/100 ml only in 8 samples from cooling towers. Polymerase chain reaction (PCR) assay with primers to amplify 16S ribosomal DNA sequence of most Legionella species (LEG primer) detected legionellae in 13 samples, while species-specific primers for L. pneumophila detected the DNAs from 3 samples. In laboratory examination, LEG primers could amplify DNAs of 29 species of genus Legionella with high sensitivity, even from 1 cell of L. pneumophila GIFU 9134. The PCR assay with LEG primers was specific and sensitive methods to be satisfied the survey of legionellae. Thus, PCR assay is a suitable method to detect and monitor Legionella species in an environment.     

Simulating an invasion: unsealed water storage (rainwater tanks) and urban block design facilitate the spread of the dengue fever mosquito,Aedes aegypti,in Brisbane,Australia

Aedes aegypti (Linnaeus) was once highly prevalent across eastern Australia, resulting in epidemics of dengue fever. Drought conditions have led to a rapid rise in semi-permanent, urban water storage containers called rainwater tanks known to be critical larval habitat for the species. The presence of these larval habitats has increased the risk of establishment of highly urbanised, invasive mosquito vectors such as Ae. aegypti. Here we use a spatially explicit network model to examine the role that unsealed rainwater tanks may play in population connectivity of an Ae. aegypti invasion in suburbs of Brisbane, a major Australian city. We characterise movement between rainwater tanks as a diffusion-like process, limited by a maximum distance of movement, average life expectancy, and a probability that Ae. aegypti will cross wide open spaces such as roads. The simulation model was run against a number of scenarios that examined population spread through the rainwater tank network based on non-compliance rates of tanks (unsealed or sealed) and road grids. We show that Ae. aegypti tank infestation and population spread was greatest in areas of high tank density and road lengths were shortest e.g. cul-de-sacs. Rainwater tank non-compliance rates of over 30% show increased connectivity when compared to less than 10%, suggesting rainwater tanks non-compliance should be maintained under this level to minimize the spread of an invading Ae. aegypti population. These results presented as risk maps of Ae. aegypti spread across Brisbane, can assist health and government authorities on where to optimally target rainwater tank surveillance and educational activities.

     

Inhibitory effects of silver ions on Legionella pneumophila grown on agar, intracellular in Acanthamoeba castellanii and in artificial biofilms

Aims: We undertook a series of experiments to investigate the susceptibility of Legionella pneumophila grown under extracellular and intracellular conditions and other water‐related bacteria to silver ions. Methods and Results: In this study, the antimicrobial effect of silver ions to intra‐ and extra‐cellular grown Legionella bacteria was investigated. The minimal inhibitory concentration (MIC) after 24 h exposure, leading to a 5 log reduction, was c. 64 μg l^?1 AgNO₃ for extracellular grown Legionella and other tested Gram‐positive and Gram‐negative bacteria. In contrast, the MIC for intracellularly grown Legionella was up to 4096 μg l^?1 AgNO₃ after 24 h. Furthermore, the heterotrophic bacteria grown within a biofilm model were killed at a concentration of 4–16 μg l^?1 AgNO₃. In contrast, biofilm‐associated Legionella were less sensitive (MIC 128–512 μg l^?1 AgNO₃). Conclusion: Intracellularly and biofilm‐grown legionellae are less sensitive against silver compared with agar‐grown bacteria. Significance and Impact of the Study: The reduced sensitivity of Legionella grown in amoebae might explain why the effect of silver decontamination requires an extended exposure in field trials.     

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     

Inhibition of growth ofLegionella species by heterotrophic plate count bacteria isolated from chlorinated drinking water

The ability of heterotrophic plate count bacterial strains isolated from chlorinated drinking water on low-nutrient media to inhibit the growth ofLegionella species was examined. Between 16% and 32% of these strains were able to inhibit the growth ofLegionella species when tested on buffered charcoal yeast extract agar. The exact proportion of inhibiting strains varied with the individualLegionella species. Two strains that inhibited the growth of severalLegionella species could also stimulate the growth of the same species when both the test strain and theLegionella species were grown on buffered charcoal yeast extract agar that lacked the essential amino acidl-cysteine.     

Diverse Legionella‐Like Bacteria Associated with Testate Amoebae of the Genus Arcella (Arcellinida: Amoebozoa)

Diverse species of Legionella and Legionella‐like amoebal pathogens (LLAPs) have been identified as intracellular bacteria in many amoeboid protists. There are, however, other amoeboid groups such as testate amoeba for which we know little about their potential to host such bacteria. In this study, we assessed the occurrence and diversity of Legionella spp. in cultures and environmental isolates of freshwater arcellinid testate amoebae species, Arcella hemispherica, Arcella intermedia, and Arcella vulgaris, via 16S rRNA gene sequence analyses and fluorescent in situ hybridization (FISH). Analysis of the 16S rRNA gene sequences indicated that A. hemispherica, A. intermedia, and A. vulgaris host Legionella‐like bacteria with 94–98% identity to other Legionella spp. based on NCBI BLAST search. Phylogenetic analysis placed Legionella‐like Arcella‐associated bacteria (LLAB) in three different clusters within a tree containing all other members of Legionella and LLAPs. The intracellular localization of the Legionella within Arcella hosts was confirmed using FISH with a Legionella‐specific probe. This study demonstrates that the host range of Legionella and Legionella‐like bacteria in the Amoebozoa extends beyond members of “naked” amoebae species, with members of the testate amoebae potentially serving an ecological role in the dispersal, protection, and replication of Legionella spp. in natural environments.     

Fecal indicators and zoonotic pathogens in household drinking water taps fed from rainwater tanks in Southeast Queensland, Australia

In this study, the microbiological quality of household tap water samples fed from rainwater tanks was assessed by monitoring the numbers of Escherichia coli bacteria and enterococci from 24 households in Southeast Queensland (SEQ), Australia. Quantitative PCR (qPCR) was also used for the quantitative detection of zoonotic pathogens in water samples from rainwater tanks and connected household taps. The numbers of zoonotic pathogens were also estimated in fecal samples from possums and various species of birds by using qPCR, as possums and birds are considered to be the potential sources of fecal contamination in roof-harvested rainwater (RHRW). Among the 24 households, 63% of rainwater tank and 58% of connected household tap water (CHTW) samples contained E. coli and exceeded Australian drinking water guidelines of <1 CFU E. coli per 100 ml water. Similarly, 92% of rainwater tanks and 83% of CHTW samples also contained enterococci. In all, 21%, 4%, and 13% of rainwater tank samples contained Campylobacter spp., Salmonella spp., and Giardia lamblia, respectively. Similarly, 21% of rainwater tank and 13% of CHTW samples contained Campylobacter spp. and G. lamblia, respectively. The number of E. coli (P = 0.78), Enterococcus (P = 0.64), Campylobacter (P = 0.44), and G. lamblia (P = 0.50) cells in rainwater tanks did not differ significantly from the numbers observed in the CHTW samples. Among the 40 possum fecal samples tested, Campylobacter spp., Cryptosporidium parvum, and G. lamblia were detected in 60%, 13%, and 30% of samples, respectively. Among the 38 bird fecal samples tested, Campylobacter spp., Salmonella spp., C. parvum, and G. lamblia were detected in 24%, 11%, 5%, and 13% of the samples, respectively. Household tap water samples fed from rainwater tanks tested in the study appeared to be highly variable. Regular cleaning of roofs and gutters, along with pruning of overhanging tree branches, might also prove effective in reducing animal fecal contamination of rainwater tanks.     

Intracellular multiplication ofLegionella pneumophila in amoebae isolated from hospital hot water tanks

We studied the ability ofLegionella to multiply in potable water samples obtained from investigations of nosocomial legionellosis. AutochthonousLegionella multiplied in three of 14 hospital water samples after incubation at 35°C and 42°C. All three samples were from hot water tanks. Multiplication did not occur when a selected sample was filtered through a 0.45-m membrane and reinoculated with indigenousLegionella. We isolated bothLegionella pneumophila and one or more species of free-living amoebae, primarity members of theHartmannellidae, from each of these hot water tank samples. Amoebae from a total of six hot water tank samples were used for cocultivation studies withL. pneumophila. All amoebae supported multiplication ofLegionella in coculture at 35°C. Four of six isolates of amoebae supported multiplication oflegionella at 42°C, while none supported multiplication at 45°C. Gimenez staining and electron microscopy showed thatLegionella multiplied intracellularly in amoebae. Control of these amoebae in potable water may prevent colonization and multiplication ofLegionella in domestic hot water systems.     

Growth of Iron-Oxidizing Bacteria Gallionella ferruginea and Leptothrix cholodnii in Oligotrophic Environments: Ca,Mg, and C as Limiting Factors and G. ferruginea Necromass as C-Source

Abstract

Iron-oxidizing bacteria (FeOB) can successively populate low-nutrient aquatic environments and adapt to a broad concentration range of alkaline earth metals, with optimum conditions widely differing from one species to another. For the most abundant known FeOB genera Gallionella and Leptothrix, there is a lack of reports on substrate affinity for calcium and magnesium and necromass assimilability. Single nutrient and combined affinity for Ca and Mg of a wild Gallionella ferruginea isolate and a Leptothrix cholodnii strain as well as growth of heterotrophic L. cholodnii on necromass of autotrophic G. ferruginea were determined by cell density measurements. G. ferruginea responds with Monod-shaped preferences and thus favors waters rich in Ca and Mg. Maximum growth occurred at Ca concentrations five times above those of commonly used modified mineral Wolfe’s medium. L. cholodnii showed a Monod-shaped preference in the low concentration range and an inhibitory response to increasing hardness: concentrations >2?mM Ca or >0.6?mM Mg allow only 50 or 75%, respectively, of maximum specific cell densities. Considering the concentration range with a Monod-shaped response (for L. cholodnii only lower concentration range), both FeOB show a type I independent colimitation for Ca and Mg with lower requirements of Mg than Ca. On a C-limited medium containing G. ferruginea necromass as the only C-source, L. cholodnii cell counts were higher by two orders of magnitude compared to pyruvate medium. Thus, the necromass may serve as a primary C-source for heterotrophic FeOB and other heterotrophic bacteria with technical and economical relevance.     

<Emphasis Type="Italic">Legionella</Emphasis>, Protozoa,and Biofilms: Interactions Within Complex Microbial Systems

Currently, the investigation of Legionella ecology falls into two distinct areas of research activity: (1) that Legionella multiply within water sources by parasitizing amoebic or ciliate hosts or (2) that Legionella grows extracellularly within biofilms. Less focus has been given to the overlaps that may occur between these two areas or the likelihood that Legionella employs multiple survival strategies to persist in water sources. It is likely that Legionella interacts with protozoa, bacteria, algae, fungi, etc., and biofilm components in a more complex fashion than multiplication or death due to the presence or absence of single components of these complex microbial systems. This paper addresses gaps that exist in the understanding of Legionella ecology and serves to pinpoint areas of future research. To assume that only one other class of organism is important to Legionella ecology may limit our understanding of how this bacterium proliferates in heated water sources and also limit our strategies for its control in the built environment.     

Application of EMA-qPCR as a complementary tool for the detection and monitoring of <Emphasis Type="Italic">Legionella</Emphasis> in different water systems

Legionella are prevalent in human-made water systems and cause legionellosis in humans. Conventional culturing and polymerase chain reaction (PCR) techniques are not sufficiently accurate for the quantitative analysis of live Legionella bacteria in water samples because of the presence of viable but nonculturable cells and dead cells. Here, we report a rapid detection method for viable Legionella that combines ethidium monoazide (EMA) with quantitative real-time PCR (qPCR) and apply this method to detect Legionella in a large number of water samples from different sources. Results yielded that samples treated with 5 μg/ml EMA for 10 min and subsequently exposed to light irradiation for 5 min were optimal for detecting Legionella. EMA treatment before qPCR could block the signal from approximately 4 log₁₀ of dead cells. When investigating environmental water samples, the percent-positive rate obtained by EMA-qPCR was significantly higher than conventional PCR and culture methods, and slightly lower than qPCR. The bacterial count of Legionella determined by EMA-qPCR were mostly greater than those determined by culture assays and lower than those determined by qPCR. Acceptable correlations were found between the EMA-qPCR and qPCR results for cooling towers, piped water and hot spring water samples (r = 0.849, P < 0.001) and also found between the EMA-qPCR and culture results for hot spring water samples (r = 0.698, P < 0.001). The results indicate that EMA-qPCR could be used as a complementary tool for the detection and monitoring of Legionella in water systems, especially in hot spring water samples.     

Cross-reactivity of the IDEXX Legiolert method with other Gram-negative bacteria and waterborne pathogens leads to false-positive assay results

Legionella species are the causative agent of Legionnaires’ disease, a potentially fatal bacterial pneumonia. New regulations and standards have prioritized the development of water safety plans to minimize the growth and spread of Legionella species in buildings. To determine the presence and type of Legionella in a water system, microbiological culturing is the gold standard method. However, recently new methodologies have been developed that claim to be sensitive and specific for Legionella at the genus or L. pneumophila at the species level. Published and anecdotal reports suggest that one of these newer culture-based, enzyme-substrate methods, the IDEXX Legiolert test, may exhibit false positivity with other microbes common to water sources. We experimentally evaluated the IDEXX Legiolert method using these other waterborne bacteria including Elizabethkingia meningoseptica, Pseudomonas aeruginosa, Proteus mirabilis and Serratia marcescens at real-world environmental concentrations. We saw false-positive results for the Legiolert test with several of these organisms, at sample concentrations as low as 60 CFU per ml. False-positive Legionella results can trigger costly remediation and water-use restrictions, that may be implemented while waiting for additional, confirmatory microbiological testing that could, in this case, yield no L. pneumophila.     

Microbial Flora of Pond-Reared Brown Shrimp (Penaeus aztecus)

Agar plate counts and microbial types are reported for brown shrimp reared in 2-acre natural marshland and in 0.5-acre artificial ponds during June to October 1970. Bacterial counts of pond-reared shrimp ranged from 5 × 10⁴ to 5.5 × 10⁶ per g. At final harvest in October, bacterial counts ranged from 2 × 10⁵ to 5.5 × 10⁶ per g. In marsh ponds, bacterial counts of shrimp and pond water were lowest in August when both water temperature and salinity were high. Coryneform bacteria and to a lesser extent Vibrio were the predominant isolates from fresh pond shrimp. Shrimp stored at 3 to 5 C for 7 days were acceptable as judged by appearance and odor. Between 7 and 14 days of refrigerated storage, bacterial counts increased sharply and about 50% of the samples became unacceptable. Refrigerated storage of pond shrimp caused increases in coryneform bacteria and micrococci and decreases in Vibrio, Flavobacterium, Moraxella, and Bacillus species. Pseudomonas species were not significant in fresh or stored pond shrimp. The microbial flora of pond water usually was dominated by coryneform bacteria, Flavobacterium, Moraxella, and Bacillus species.     

Prevalence of Virulence Genes Associated with Pathogenic Escherichia coli Strains Isolated from Domestically Harvested Rainwater during Low- and High-Rainfall Periods

The possible health risks associated with the consumption of harvested rainwater remains one of the major obstacles hampering its large-scale implementation in water limited countries such as South Africa. Rainwater tank samples collected on eight occasions during the low- and high-rainfall periods (March to August 2012) in Kleinmond, South Africa, were monitored for the presence of virulence genes associated with Escherichia coli. The identity of presumptive E. coli isolates in rainwater samples collected from 10 domestic rainwater harvesting (DRWH) tanks throughout the sampling period was confirmed through universal 16S rRNA PCR with subsequent sequencing and phylogenetic analysis. Species-specific primers were also used to routinely screen for the virulent genes, aggR, stx, eae, and ipaH found in enteroaggregative E. coli (EAEC), enterohemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), and enteroinvasive E. coli, respectively, in the rainwater samples. Of the 92 E. coli strains isolated from the rainwater using culture based techniques, 6% were presumptively positively identified as E. coli O157:H7 using 16S rRNA. Furthermore, virulent pathogenic E. coli genes were detected in 3% (EPEC and EHEC) and 16% (EAEC) of the 80 rainwater samples collected during the sampling period from the 10 DRWH tanks. This study thus contributes valuable information to the limited data available regarding the ongoing prevalence of virulent pathotypes of E. coli in harvested rainwater during a longitudinal study in a high-population-density, periurban setting.     

Application of multilocus sequence analysis (MLSA) for accurate identification of Legionella spp. Isolated from municipal fountains in Chengdu, China, based on 16S rRNA, mip, and rpoB genes

Legionellosis (Legionnaires’ disease; LD) is a form of severe pneumonia caused by species of Legionella bacteria. Because inhalation of Legionella-contaminated aerosol is considered the major infection route, routine assessments of potential infection sources such as hot water systems, air-conditioner cooling water, and municipal fountains are of great importance. In this study, we utilized in vitro culture and multilocus sequence analysis (MLSA) targeting 16S rRNA, mip, rpoB, and mip-rpoB concatenation to isolate and identify Legionella spp. from 5 municipal fountains in Chengdu City, Sichuan Province, China. Our results demonstrated that 16S rRNA was useful for initial identification, as it could recognize isolates robustly at the genus level, while the genes mip, rpoB, and mip-rpoB concatenation could confidently discriminate Legionella species. Notably, the three subspecies of L. pneumophila could be distinguished by the analysis based on rpoB. The serotyping result of strain CD-1 was consistent with genetic analysis based on the concatenation of mip and rpoB. Despite regular maintenance and sanitizing methods, 4 of the 5 municipal fountains investigated in this study were positive for Legionella contamination. Thus, regularly scheduled monitoring of municipal fountains is urgently needed as well as vigilant disinfection. Although the application of MLSA for inspection of potential sites of infection in public areas is not standard procedure, further investigations may prove its usefulness.