Distinct difference of flaA genotypes of Legionella pneumophila between isolates from bath water and cooling tower water

To investigate the genetic difference of Legionella pneumophila in human‐made environments, we collected isolates of L. pneumophila from bath water (n = 167) and cooling tower water (n = 128) primarily in the Kanto region in 2001 and 2005. The environmental isolates were serogrouped and sequenced for a target region of flaA. A total of 14 types of flaA genotypes were found: 10 from cooling tower water and nine from bath water. The flaA genotypes of isolates from cooling tower water were quite different from those of bath water.     

Inhibition of Legionella pneumophila by Bacillus sp.

This study presents the potential of a biological control of Legionella pneumophila. It was verified to what extent the enrichment of various Bacillus species in water may decrease or prevent L. pneumophila from growing in water. During in vitro tests, B. subtilis BS104 was able to induce an average decrease in L. pneumophila numbers of 1.9 + 0.2 log units after 120 h. Furthermore, the spore and cell free filtrate of B. subtilis BS104 also decreased L. pneumophila by 2.6 + 0.4 log units after 120 h. An addition of Bacillus BS104 to a cooling tower test system with a water volume of 8 m³ resulted in a L. pneumophila level below 1000 CFU/L after 3 weeks, whereas the starting point was 5.3 × 10⁴ CFU/L. Further experiments also indicated that B. subtilis BS104 might inhibit the internal replication of L. pneumophila in Acanthamoeba castellanii. This paper demonstrates that certain strains of Bacillus have the potential of reducing the number of viable L. pneumophila in water, or at least prevent its increase. These results are the first indication that a biological abatement of L. pneumophila could be possible.     

Development of a new CARD-FISH protocol for quantification of Legionella pneumophila and its application in two hospital cooling towers

Aims: Open cooling towers are frequent sources of infections with Legionella pneumophila. The gold standard for the detection of Leg. pneumophila is based on cultivation lasting up to 10 days and detecting only culturable cells. Alternative fluorescence in situ hybridization (FISH) protocols have been proposed, but they result in faint fluorescence signals and lack specificity because of cross‐hybridization with other Legionella species. Our aim was thus to develop a new FISH protocol for rapid and specific detection of Leg. pneumophila in water samples. Methods and Results: A novel catalysed reporter deposition FISH (CARD‐FISH) protocol for the detection of Leg. pneumophila was developed, which significantly enhanced signal intensity as well as specificity of the probe through the use of a novel competitor probe. The developed protocol was compared with the culture method for monitoring the seasonal development of culturable and nonculturable Leg. pneumophila in two hospital cooling tower systems. Seasonal fluctuations of Leg. pneumophila concentrations detected via CARD‐FISH were related to the development of the total bacterial community in both cooling towers, with temperature and biocide as the main factors controlling this development. Conclusions: Our results clearly showed that the majority of the Leg. pneumophila cells were in a nonculturable state. Thus, detection of Leg. pneumophila with culture methods may underestimate the total numbers of Leg. pneumophila present. Significance and Impact of the Study: Rapid, sensitive and specific detection and quantification of Leg. pneumophila in water systems is prerequisite for reliable risk estimation. The new protocol significantly improves current methodology and can be used to monitor and screen for Leg. pneumophila concentrations in cooling towers or other water systems.     

Distribution and seasonality ofLegionella pneumophila in colling towers

The numbers of presumptiveLegionella pneumophila cells in waters and sediments of nine different cooling towers located on the same site in the northeastern United States were determined at approximately monthly intervals for 18 months. All systems received makeup water from the same source and received the same chemical treatments. PresumptiveL. pneumophila were found in both water and sediment samples from all systems on all sampling dates. An important result of this study was the finding that tower sediments represent large reservoirs ofL. pneumophila. The only correlation between levels of presumptiveL. pneumophila and any of the physical, chemical, or operating characteristics evaluated was with winter shutdown and drainage followed by a nonoperational period. These systems showed a definite seasonal response with the highest levels of presumptiveL. pneumophila found in the summer and fall. Systems operated year round showed relatively constant numbers ofL. pneumophila in both water and sediments.     

Comparison between standard culture and peptide nucleic acid 16S rRNA hybridization quantification to study the influence of physico-chemical parameters on Legionella pneumophila survival in drinking water biofilms

Legionella pneumophila is a waterborne pathogen that is mainly transmitted by the inhalation of contaminated aerosols. In this article, the influence of several physico-chemical parameters relating to the supply of potable water was studied using a L. pneumophila peptide nucleic acid (PNA) specific probe to quantify total L. pneumophila in addition to standard culture methods. A two-stage chemostat was used to form the heterotrophic biofilms, with biofilm generating vessels fed with naturally occurring L. pneumophila. The substratum was the commonly used potable water pipe material, uPVC. It proved impossible to recover cultivable L. pneumophila due to overgrowth by other microorganisms and/or the loss of cultivability of this pathogen. Nevertheless, results obtained for total L. pneumophila cells in biofilms using a specific PNA probe showed that for the two temperatures studied (15 and 20°C), there were no significant differences when shear stress was increased. However, when a source of carbon was added there was a significant increase in numbers at 20°C. A comparison of the two temperatures showed that at 15°C, the total cell numbers for L. pneumophila were generally higher compared with the total microbial flora, suggesting that lower temperatures support the inclusion of L. pneumophila in drinking water biofilms. The work reported in this article suggests that standard culture methods are not accurate for the evaluation of water quality in terms of L. pneumophila. This raises public health concerns since culture methods are still considered to be the gold standard for assessing the presence of this opportunistic pathogen in water.     

Efficacy of methylchloro/methylisothiazolone biocide againstLegionella pneumophila in cooling tower water

Summary Methylchloro/methylisothiazolone biocide was tested for efficacy in cooling tower water againstLegionella pneumophila (strains Flint 1 and 2) andL. gormanii. These analyses are the first reports on the efficacy of isothiazolones againstLegionella in actual cooling tower water; the results of data reported previously obtained from more arbitrary laboratory tests are discussed and compared. The biocide was effective in cooling tower water at the two pH levels tested (pH 8.0 and 6.7). The concentration of biocide required to eliminate theL. pneumophila depended on contact time and pH: 99% of the bacteria were killed after 6 and 3 h of contact by 1.07 and 3.13 ppm active ingredient, respectively, when the pH was 8.0: 99% of the bacteria were killed after 6 and 3 h of contact by 2.23 and 9.43 ppm active ingredient, respectively, when the pH was 6.7. 24 h of contact with 0.35 ppm active ingredient methylchloro/methylisothiazolone biocide reduced the concentration of viable bacterial cells by more than four orders of magnitude (>99.99%), regardless of pH.     

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.     

PCR methods for the rapid detection and identification of four pathogenic Legionella spp. and two Legionella pneumophila subspecies based on the gene amplification of gyrB

A total of 25 gyrB gene sequences from 20 Legionella pneumophila subsp. pneumophila strains and five L. pneumophila subsp. fraseri strains were obtained and analyzed, and a multiplex PCR for the simultaneous detection of Legionella bozemanae, Legionella longbeachae, Legionella micdadei and Legioenella pneumophila, and two single PCRs for the differentiation of L. pneumophila subsp. pneumophila and L. pneumophila subsp. fraseri were established. The multiplex PCR method was shown to be highly specific and reproducible when tested against 41 target strains and 17 strains of other bacteria species. The sensitivity of the multiplex PCR was also analyzed and was shown to detect levels as low as 1 ng of genomic DNA or 10 colony-forming units (CFUs) per milliliter in mock water samples. Sixty-three air conditioner condensed water samples from Shanghai City were examined, and the result was validated using 16S rRNA sequencing. The data reported here demonstrate that the multiplex PCR method described is efficient and convenient for the detection of Legionella species in water samples. Twenty L. pneumophila subsp. pneumophila strains and five L. pneumophila subsp. fraseri strains were used for the validation of the two L. pneumophila subspecies-specific PCR methods, and the results indicated that the two PCR methods were both highly specific and convenient for the identification of L. pneumophila at the subspecies level.     

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.     

Comparison of culture methods and an immunofluorescence assay for the detection ofLegionella pneumophila in domestic hot water devices

The objective of this study was to compare an indirect immunofluorescence assay with culture methods for the identification ofLegionella pneumophila serogroups 1 to 6 in hot water samples taken from domestic environments. Hot water samples were obtained from the water heater, the shower heads, and the most frequently used faucet of 211 private houses. Concentrated water samples were inoculated on buffered charcoal yeast extract agar (BCYE) and on a semi-selective culture medium (GPV). Colonies with a morphology similar to that ofLegionellaceae were subcultured on BCYE and on blood agar plates; those that grew on the former but not the latter were further characterized and identified by direct immunofluorescence techniques. The concentrated samples were also smeared on multiple-well microscope slides and tested by indirect immunofluorescence with monoclonal antibodies againstL. pneumophila, serogroups 1 to 6. Of the houses studied, 30% were found to contain culturableL. pneumophila in at least one water sample, whereas 63% were positive by indirect immunofluorescence. The sensitivity of this assay compared with culture varied from 16.7–21.1%, and its specificity was between 76.7% and 88.3% depending on the sample source (water heater, shower heads, or faucet). In the 38 houses with at least one positive sample found by both immunofluorescence and culture, total or partial agreement between serogroups identified by both techniques was only 34%. The results obtained in this study strongly suggest that indirect immunofluorescence is not an adequate alternative for the identification ofL. pneumophila in hot water systems.     

Automated dead-end ultrafiltration of large volume water samples to enable detection of low-level targets and reduce sample variability

Aims

A Portable Multi‐use Automated Concentration System (PMACS) concentrates micro‐organisms from large volumes of water through automated dead‐end ultrafiltration and backflushing. The ability to detect microbial targets from ground, surface and cooling tower waters collected using standard methods was compared with samples from the PMACS in this study.

Methods and Results

PMACS (100 l) and standard grab samples (100–500 ml) were collected from sites in Florida and South Carolina, USA. Samples were analysed for the presence of faecal indicator bacteria (FIB; ground and surface water) or Legionella pneumophila (Lp; cooling tower water). FIB were enumerated by growth on selective media following membrane filtration or in IDEXX defined substrate media. Lp cells were detected by direct fluorescence immunoassay using FITC‐labelled monoclonal antibodies targeting serogroups 1, 2, 4 and 6. FIB were found in PMACS samples from ground and surface waters when their concentrations were below detection limits in grab samples. The concentrations of Lp in cooling tower samples collected over 5 months were more consistent in PMACS samples than grab samples.

Conclusions

These data demonstrate that PMACS concentration is advantageous for water monitoring. FIB were detected in PMACS samples when their concentrations were below the detection limits of the standard methods used. PMACS processing provided more representative samples of cooling tower waters reducing sample variability during long‐term monitoring.

Significance and Impact of the Study

This study highlights the utility of PMACS processing for enhanced monitoring of water for low‐level microbial targets and for reducing sample variability in long‐term monitoring programmes.     

Rapid quantification of viable legionellae in water and biofilm using ethidium monoazide coupled with real‐time quantitative PCR

Aims: To optimize ethidium monoazide (EMA) coupled with real‐time quantitative PCR (qPCR) and to evaluate its environmental applicability on quantifying viable legionellae in water and biofilm of cooling towers and hot water systems. Methods and Results: EMA (0·9–45·5 μg ml^?1) and propidium monoazide (PMA, 0·9 and 2·3 μg ml^?1) combined with qPCR (i.e. EMA‐qPCR and PMA‐qPCR, respectively) were applied to unheated and heated (70°C for 30 min) Legionella pneumophila to quantify viable cells, which was also simultaneously determined by BacLight Bacterial Viability kit with epifluorogenic microscopic enumeration (BacLight‐EM). The effects of nontarget microflora and sample matrix on the performance of EMA‐qPCR were also evaluated. In comparison with BacLight‐EM results, qPCR with EMA at 2·3 μg ml^?1 was determined as the optimal EMA‐qPCR assay, which performed equally well as PMA‐qPCR for unheated Leg. pneumophila but better than PMA‐qPCR for heated Leg. pneumophila (P < 0·05). Moreover, qPCR with EMA at 2·3 μg ml^?1 accurately quantified viable Leg. pneumophila, Legionella anisa and Legionella‐like amoebal pathogens 6 (LLAP 6) without interferences by heated legionellae, unheated nonlegionellae cells and cooling tower water matrix (P > 0·05). As for water and biofilm samples collected from cooling towers and hot water systems, the viable legionellae counts determined by EMA‐qPCR were mostly greater than the culturable counts by culture assay but consistently lower than the total cell counts quantified by qPCR. Conclusions: The qPCR with EMA at 2·3 μg ml^?1 may accurately quantify viable legionellae (including fastidious LLAP 6) and Leg. pneumophila pretreated with superheating and is applicable for water and biofilm samples obtained from cooling towers and hot water systems. Significance and Impact of the Study: The EMA‐qPCR assay may be useful in environmental surveillance for viable legionellae and in evaluation of superheating efficacy against legionellae.     

LAMP-based method for a rapid identification of Legionella spp. and Legionella pneumophila

Legionella pneumophila is accounted for more than 80% of Legionella infection. However it is difficult to discriminate between the L. pneumophila and non-L. pneumophila species rapidly. In order to detect the Legionella spp. and distinguish L. pneumophila from Legionella spp., a real-time loop-mediated isothermal amplification (LAMP) platform that targets a specific sequence of the 16S rRNA gene was developed. LS-LAMP amplifies the fragment of the 16S rRNA gene to detect all species of Legionella genus. A specific sequence appears at the 16S rRNA gene of L. pneumophila, while non-L. pneumophila strains have a variable sequence in this site, which can be recognized by the primer of LP-LAMP. In the present study, 61 reference strains were used for the method verification. We found that the specificity was 100% for both LS-LAMP and LP-LAMP, and the sensitivity of LAMP assay for L. pneumophila detection was between 52 and 5.2 copies per reaction. In the environmental water samples detection, a total of 107 water samples were identified by the method. The culture and serological test were used as reference methods. The specificity of LS-LAMP and LP-LAMP for the samples detection were 91.59% (98/107) and 93.33% (56/60), respectively. The sensitivity of LS-LAMP and LP-LAMP were 100% (51/51) and 100% (18/18). The results suggest that real-time LAMP, as a new assay, provides a specific and sensitive method for rapid detection and differentiation of Legionella spp. and L. pneumophila and should be utilized to test environmental water samples for increased rates of detection.     

A Comparison of Legionella pneumophila Occurrence in Hot Water Tanks and Instantaneous Devices in Domestic, Nosocomial, and Community Environments

The aim of this study was to compare the occurrence of L. pneumophila in hot water samples from hot water tanks and instantaneous devices. Tanks and devices were all operated by heat exchangers employed in the town's district heating system. Thirty-six out of 171 (21%) hot water samples tested positive for L. pneumophila isolation, with 14.6% belonging to serogroup 1 and 6.4% to serogroups 2–14. The proportion of L. pneumophila detected in hot water reservoirs (30%) was higher than that observed in hot water instantaneous devices (6.2%). Differences in L. pneumophila isolation reflected different temperatures registered at the faucet: ≤50°C for hot water from reservoir devices, and >60°C for hot water from instantaneous devices. These data emphasize the need to control temperature in hot water distribution devices, thus inhibiting the formation of biofilm and L. pneumophila colonization. Received: 4 April 2000 / Accepted: 31 May 2000     

Growth ofLegionella pneumophila in two-membered cultures with green algae and cyanobacteria

Environmental and clinical isolates ofLegionella pneumophila were grown in minimal-salts media (no organic compounds added) in associated with various green algae and cyanobacteria. Growth was observed to level off after a period of hours to days with no subsequent significant loss in the numbers of viableL. pneumophila even several days after growth had ceased. Transfer to new algal or cyanobacterial cultures resulted in a new burst of growty by theL. pneumophila.     

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.     

Growth phase-dependent surface properties of Legionella pneumophila and their role in adhesion to stainless steel coated QCM-D sensors

Legionella pneumophila cell surface hydrophobicity and charge are important determinants of their mobility and persistence in engineered water systems (EWS). These surface properties may differ depending on the growth phase of L. pneumophila resulting in variable adhesion and persistence within EWS. We describe the growth-dependent variations in L. pneumophila cell surface hydrophobicity and surface charge using the microbial adhesion to hydrocarbon assay and microelectrophoresis, respectively, and their role in cell adhesion to stainless steel using a quartz crystal microbalance with dissipation (QCM-D) monitoring instrument. We observed a steady increase in L. pneumophila hydrophobicity during their lifecycle in culture media. Cell surfaces of stationary phase L. pneumophila were significantly more hydrophobic than their lag and midexponential counterparts. No significant changes in L. pneumophila cell surface charge were noted. Morphology of L. pneumophila remained relatively constant throughout their lifecycle. In the QCM-D study, lag and exponential phase L. pneumophila weakly adhered to stainless steel surfaces resulting in viscoelastic layers. In contrast, stationary phase bacteria were tightly and irreversibly bound to the surfaces, forming rigid layers. Our results suggest that the stationary phase of L. pneumophila would highly favour their adhesion to plumbing surfaces and persistence in EWS.     

Molecular Determination of Infection Source of a Sporadic Legionella Pneumonia Case Associated with a Hot Spring Bath

To determine the infection source of a sporadic Legionella pneumonia case associated with a hot spring bath, we used five molecular methods, including repetitive element polymerase chain reaction (rep-PCR), arbitrarily primed PCR (AP-PCR), ribotyping, restriction endonuclease analysis (REA), and macrorestriction endonuclease analysis (MREA) by pulsed-field gel electrophoresis. L. pneumophila serogroup (SG) 3 strain EY 3702, isolated from an intratracheal specimen of a 71-year-old Japanese female who developed pneumonia after nearly drowning in a hot spring spa bath, produced rep-PCR and AP-PCR fingerprints identical to those of L. pneumophila SG 3 strains EY 3768 and EY 3769 isolated from the bath water. Four epidemiologically unrelated L. pneumophila SG 3 strains showed different rep-PCR or AP-PCR fingerprints from those of the three EY strains (EY 3702, 3768, and 3769). The three EY strains were also genotypically indistinguishable by ribotyping with EcoRI and PstI, by REA with EcoBI or HindIII, and by MREA with NotI. Based on these results, we identified the bath water of the hot spring spa as the source of infection of this patient, even though the viable number of the organisms in the bath water was low (3 CFU/100 ml) when determined 27 days after her nearly drowning.     

Quantitative Detection of Legionella pneumophila in Water Samples by Immunomagnetic Purification and Real-Time PCR Amplification of the dotA Gene

A new real-time PCR assay was developed and validated in combination with an immunomagnetic separation system for the quantitative determination of Legionella pneumophila in water samples. Primers that amplify simultaneously an 80-bp fragment of the dotA gene from L. pneumophila and a recombinant fragment including a specific sequence of the gyrB gene from Aeromonas hydrophila, added as an internal positive control, were used. The specificity, limit of detection, limit of quantification, repetitivity, reproducibility, and accuracy of the method were calculated, and the values obtained confirmed the applicability of the method for the quantitative detection of L. pneumophila. Moreover, the efficiency of immunomagnetic separation in the recovery of L. pneumophila from different kinds of water was evaluated. The recovery rates decreased as the water contamination increased (ranging from 59.9% for distilled water to 36% for cooling tower water), and the reproducibility also decreased in parallel to water complexity. The feasibility of the method was evaluated by cell culture and real-time PCR analysis of 60 samples in parallel. All the samples found to be positive by cell culture were also positive by real-time PCR, while only eight samples were found to be positive only by PCR. Finally, the correlation of both methods showed that the number of cells calculated by PCR was 20-fold higher than the culture values. In conclusion, the real-time PCR method combined with immunomagnetic separation provides a sensitive, specific, and accurate method for the rapid quantification of L. pneumophila in water samples. However, the recovery efficiency of immunomagnetic separation should be considered in complex samples.     

Phylogenetic Study of <Emphasis Type="Italic">Legionella</Emphasis> Species in Pristine and Polluted Aquatic Samples from a Tropical Atlantic Forest Ecosystem

Legionella species are ubiquitous bacteria in aquatic environments. To examine the effect of anthropogenic impacts and physicochemical characteristics on the Legionellaceae population, we collected water from two sites in the Itanhaém River system in the Atlantic Forest of Brazil. One sample was collected from an upstream pristine region, the other from a downstream estuarine region moderately affected by untreated domestic sewage. Cultures on a selective medium failed to isolate Legionella species. Culture-independent methods showed that water from the estuarine aquatic habitat contained DNA sequences homologous to the 16S ribosomal DNA gene of Legionella pneumophila and non-pneumophila species. In pristine water, only two sequences related to L. pneumophila were detected. The results suggest that salinity and anthropogenic factors, such as wastewater discharge, favor a diversity of Legionella species, whereas pristine freshwater selects for Legionella pneumophila.