Cooling Tower Water Samples Research Articles

Detecting Legionella pneumophila in Cooling Tower Water Samples with a DNAzyme/Bead‐Based Fluorescence Assay

AbstractLegionella pneumophila is the causative agent behind the deadly waterborne disease Legionnaires’, which is commonly transmitted by the spread of contaminated droplets from cooling tower water samples. The lack of effective detection methods presents a challenge for L. pneumophila outbreak control. Previously, an RNA‐cleaving DNAzyme called LP1 was reported to specifically target L. pneumophila. In this study, LP1 was immobilized onto agarose beads via streptavidin‐biotin interaction to develop a bead‐based fluorescence assay for L. pneumophila detection. This bead‐based assay demonstrated excellent stability and functionality in various cooling tower water samples. To improve L. pneumophila monitoring in real‐world samples, a lysozyme treatment was used to enhance L. pneumophila recognition. The limit of detection of this DNAzyme‐based bead assay can reach 103 CFUs in cell‐spiked cooling tower water samples without cell culturing or signal amplification steps.

Detection of Diverse Sequence Types of Legionella pneumophila by Legiolert Enzymatic-Based Assay and the Development of a Long-Term Storage Protocol.

Legiolert is a rapid culture-based enzymatic method for the detection and quantification of Legionella pneumophila in potable and nonpotable water samples. We aimed to assess the ability of this assay to detect diverse sequence types and validated a simple method to preserve samples. We used this assay on 253 potable and 165 nonpotable cooling tower water samples from various buildings in Québec, Canada, and performed sequence-based typing on 96 isolates. Six sequence types were identified, including ST1, ST378, ST1427, ST2859, ST3054, and ST3069. Whole-genome sequencing revealed that ST2859 was a member of the L. pneumophila subspecies fraseri. Additional tests with pure isolates also found that subspecies Pascullei and Raphaeli could be detected via Legiolert. Eight storage methods, including the current recommendation to store Legiolert trays at 4°C, were evaluated for their ability to preserve viable cultures. Of those, storage of Legiolert culture with 10% glycerol at -80°C produced the best results, fully preserving culturable Legionella for at least 12.5 months. We incorporated these findings into a standard procedure for processing Legiolert packets. Overall, Legiolert captures a variety of common and new STs in addition to important L. pneumophila subspecies and can be easily stored, which allows the conservation of a population of isolates for later characterization. IMPORTANCE Legionnaires' disease is caused by the bacterium Legionella pneumophila, which can be found in a variety of water systems. When outbreaks of Legionnaires' disease occur, it is necessary to find the water systems transmitting the bacterium to humans. Access to historical isolates from water system samples is key for success in identifying sources but current regulations and isolation protocols mean very few isolates are obtained and stored long-term. We showed here that the Legiolert test could detect and produce isolates of a variety of L. pneumophila subspecies and types. In addition, the Legiolert test medium containing a representative population of isolates could be preserved for at least 12 months at -80°C with the addition of glycerol to the test medium. Therefore, we confirmed that the Legiolert method could be a useful tool for retrospective analysis of potential sources for an outbreak.

Assessment of flow cytometry for microbial water quality monitoring in cooling tower water and oxidizing biocide treatment efficiency

The control of Legionella proliferation in cooling tower water circuits requires regular monitoring of water contamination and effective disinfection procedures. In this study, flow cytometry was assessed to monitor water contamination and disinfection treatment efficiency on bacterial cells regarding nucleic acid injury (SYBR® Green II), cell integrity (SYBR® Green II and propidium iodide) and metabolism activity (ChemChrome V6). A total of 27 cooling tower water samples were analyzed in order to assess water contamination levels regarding viable populations: standard culture, ATP measurement and flow cytometry methods were compared. Flow cytometry and plate counts methods showed a significant correlation for changes in concentrations despite a 1 to 2-log difference regarding absolute quantification. Concerning intracellular activity, the use of two different flow cytometers (FACSCanto™ II and Accuri™ C6) showed no statistical difference while a difference was observed between flow cytometry and usual methods (culture and ATP measurement). The standard culture and flow cytometry methods were also compared for in vitro bacteria inactivation measurements in the presence of 3 different types of oxidizing biocides commonly used for cooling tower disinfection. Reductions observed ranged between 1 and 2 log depending on (1) the detection method, (2) the bacterial population origin and/or (3) the active biocide molecule used. In conclusion, flow cytometry represents an efficient, accurate and fast approach to monitor water contamination and biocide treatment efficiency in cooling towers.

Prevalence and Molecular Characteristics of Waterborne Pathogen Legionella in Industrial Cooling Tower Environments.

Cooling towers are a source of Legionnaires’ disease. It is important from a public health perspective to survey industrial cooling towers for the presence of Legionella. Prospective surveillance of the extent of Legionella pollution was conducted at factories in Shijiazhuang, China between March 2011 and September 2012. Overall, 35.7% of 255 industrial cooling tower water samples showed Legionella-positive, and their concentrations ranged from 100 Colony-Forming Units (CFU)/liter to 88,000 CFU/liter, with an average concentration of 9100 CFU/liter. A total of 121 isolates were obtained. All isolates were L. pneumophila, and the isolated serogroups included serogroups 1 (68 isolates, 56.2%), 6 (25, 20.7%), 5 (12, 9.9%), 8 (8, 6.6%), 3 (6, 5.0%) and 9 (2, 1.6%). All 121 isolates were analyzed by pulsed-field gel electrophoresis (PFGE) and 64 different patterns were obtained. All 121 isolates were analyzed sequence-based typing (SBT), a full 7-allele profile was obtained from 117 isolates. One hundred and seventeen isolates were divided into 49 sequence types. Two virulence genes, lvh and rtxA, are analyzed by polymerase chain reaction (PCR). 92.6% (112/121) and 98.3% (119/121) isolates carried lvh and rtxA respectively and 90.9% (110/121) of tested isolates carried both genes. Our results demonstrated high prevalence and genetic polymorphism of L. pneumophila in industrial cooling tower environments in Shijiazhang, China, and the SBT and virulence gene PCR results suggested that the isolates were pathogenic. Improved control and prevention strategies are urgently needed.

Molecular characterization of viable Legionella spp. in cooling tower water samples by combined use of ethidium monoazide and PCR.

Viable Legionella spp. in environmental water samples were characterized phylogenetically by a clone library analysis combining the use of ethidium monoazide and quantitative PCR. To examine the diversity of Legionella spp., six cooling tower water samples and three bath water samples were collected and analyzed. A total of 617 clones were analyzed for their 16S rRNA gene sequences and classified into 99 operational taxonomic units (OTUs). The majority of OTUs were not clustered with currently described Legionella spp., suggesting the wide diversity of not-yet-cultured Legionella groups harbored in cooling tower water environments.

Detection of Legionella species in environmental water by the quantitative PCR method in combination with ethidium monoazide treatment.

We detected Legionella species in 111 bath water samples and 95 cooling tower water samples by using a combination of conventional plate culture, quantitative polymerase chain reaction (qPCR) and qPCR combined with ethidium monoazide treatment (EMA-qPCR) methods. In the case of bath water samples, Legionella spp. were detected in 30 samples by plate culture, in 85 samples by qPCR, and in 49 samples by EMA-qPCR. Of 81 samples determined to be Legionella-negative by plate culture, 56 and 23 samples were positive by qPCR and EMA-qPCR, respectively. Therefore, EMA treatment decreased the number of Legionella-positive bath water samples detected by qPCR. In contrast, EMA treatment had no effect on cooling tower water samples. We therefore expect that EMA-qPCR is a useful method for the rapid detection of viable Legionella spp. from bath water samples.

Thionine‐Bromate as a New Reaction System for Kinetic Spectrophotometric Determination of Hydrazine in Cooling Tower Water Samples

A simple, selective, and inexpensive kinetic method was developed for the determination of hydrazine based on its inhibitory effect on the thionine‐bromate system in sulfuric acid media. The reaction was monitored spectrophotometrically at 601 nm by a fixed time method. The effect of different parameters such as concentration of reactants, ionic strength, temperature, and time on the rate of reaction was investigated, and the optimum conditions were obtained. Under optimum conditions, the calibration curve was linear in the concentration range from 0.8–23.0 μg mL−1 of hydrazine, and the detection limit of the method was 0.22 μg mL−1. The relative standard deviation for five replicate determinations of 1.0 μg mL−1 of hydrazine was 0.74%. The potential of interfering effect of foreign species on the hydrazine determination was studied. The proposed method was successfully applied for the determination of hydrazine in different water samples.

Simultaneous detection of Legionella species and Legionella pneumophila by duplex PCR (dPCR) assay in cooling tower water samples from Jakarta, Indonesia

Aim: Since culture method is time-consuming and has low sensitivity, we developed a duplex PCR (dPCR) assay for the detection of Legionella sp. and L. pneumophila in cooling tower samples. We used culture method as a gold standard. Methods: Optimization of dPCR method was performed to obtain an assay with high sensitivity and specifi city. The optimized method was used to detect Legionella sp. dan L. pneumophila in 9 samples obtained from 9 buildings in Jakarta. For culture method, the bacteria were grown or isolated on selective growth factor supplemented-buffered charcoal yeast extract (BCYE) media. Results: Of 9 samples tested by dPCR assay, 6 were positive for Legionella species,1 was positive for L. pneumophila, and 2 showed negative results. For the same samples, no Legionella sp. was detected by the culture method. Conclusion: dPCR assay was much more sensitive than the culture method and was potentially used as a rapid, specifi c and sensitive test for routine detection of Legionella sp. dan for L. pneumophila in water samples. (Med J Indones 2010; 19:223-7)

Hospital-acquired legionellosis originating from a cooling tower during a period of thermal inversion

A case of hospital-acquired legionellosis occurred in a 75-year-old male patient who underwent surgery due to malignant melanoma. Legionellosis was proven by culture of Legionella pneumophila serogroup 1 from bronchoalveolar lavage (BAL) fluid. Being a chronic smoker the patient used to visit the sickroom balcony that was located about 90 m to the west of a hospital cooling tower. Routine cooling tower water samples drawn during the presumed incubation period revealed 1.0x10(4) CFU/100 ml (L. pneumophila serogroup 1). One of three isolates from the cooling tower water matched the patient's isolate by monoclonal antibody (mab)- and genotyping (sequence-based typing). Horizontal transport of cooling tower aerosols probably was favoured by meteorological conditions with thermal inversion. The case report stresses the importance of routine maintenance and microbiological control of hospital cooling towers.

Enhanced Antifungal Effect of the Selective Medium for the Detection of Legionella Species by a Combination of Cycloheximide, Amphotericin B and Thiabendazole

The accurate detection of Legionella from environmental water samples using conventional plate culture methods is often made difficult by the overgrowth of non-target microorganisms on the selective agar plates. Acid pretreatment is a very effective pretreatment to decrease the overgrowth of heterotrophic bacteria. However, acid pretreatment would not be expected to eliminate molds. We evaluated the effects of four kinds of antifungal agents, individually and in combination, on the growth of Legionella strains and molds. Consequently, it was demonstrated that the combination of cycloheximide, amphotericin B and thiabendazole was very effective in eliminating molds on agar plates, and resulted in the improved detection of Legionella. Examination of 214 cooling tower water samples using the enhanced antifungal selective medium (CAT alpha) instead of GVPC alpha selective medium demonstrated a decrease in contamination by molds from 13.6% to 1.9% without affecting the growth of Legionella.

環境水由来レジオネラ属菌の検出における有機酸緩衝液前処理の検討

Anti-microbial effect of the pretreatment with various organic acid buffer solutions against co-existing microorganisms which were isolated from cooling-tower water samples along with Legionella spp. was examined. Among several buffer solutions, a 0.1 M potassium citrate-citric acid buffer solution (hereafter, citrate buffer solution, pH 2.2) hardly affected the recovery of Legionella spp., but effectively inhibited the growth of co-existing microorganisms. To evaluate the buffer action of these buffer solutions, pHs of 9 cooling-tower water samples were evaluated after addition of an equal volume of each buffer solution. When a citrate buffer solution. pH 2.2 was combined to a 200-fold concentrated solution of each cooling-tower water sample, the pH of the combined solution became 2.5 to 2.7 and maintained a stably low pH value than that (pH 3.0 to 7.4) obtained after mixture of a 0.2 M HCl-KCl buffer solution (hereafter, HCl buffer solution, pH 2.2), suggesting strong buffer action of the citrate buffer solution, pH 2.2 in the combined solutions. Furthermore, when cooling-tower water samples were pretreated with a citrate buffer solution, pH 2.2, the recovery of Legionella spp. was successful in 7 out of 9 cooling-tower water samples, suggesting 3 times higher recovery rate than that obtained by addition of a HCl buffer solution, pH 2.2 (3 out of 9 cooling-tower water samples).

環境水由来レジオネラ属菌の選択培地による効率的検出法の検討

To examine the optimal pH range for growth on media, growth of Legionella spp. on its selective media, BCYE alpha, WYO alpha and MWY agar media, in a pH range of 6.0-8.0 (at 0.5 intervals) was compared. The growth of two strains of L. pneumophila and one strain of L. micdadei on a WYO alpha agar supplemented with some selecting antimicrobial agents was markedly inhibited at all pH range except 6.0 and 7.0, suggesting a narrow optimal pH range for growth of these species compared to the BCYE alpha without selecting antimicrobioal agents. Vancomycin (VCM) added to the selective agar suppressed the growth of some Legionella spp. depending on the concentration. However, the extent of suppression was different among species and/or strains of Legionella spp. The selectivity for species other than Legionella spp. was also affected similarly by VCM concentration added to their media, suggesting that it is important to use proper amounts of the selecting antimicrobial agent depending on the species and/or strains of Legionella spp. or the other species in water samples. Amphothericin B (AMPH-B) added to a selective medium, MWY agar, in the concentration of 80 micrograms/ml hardly affected the growth of Legionella spp. examined, but effectively inhibited the growth of fungal strains identified as Aspergillus sp., Trichoderma sp., Scolecobasidium sp. and Mucor sp. which were isolated from cooling-tower water samples together with Legionella spp. Furthermore, the growth of a combination culture of one each of the 4 strains of isolated fungi and one each of the 3 strains of Legionella spp. was examined at various concentration of AMPH-B. Addition of AMPH-B to the selective medium at the concentration of 80 micrograms/ml suppressed the growth of spreading fungi, permitting the growth of Legionella spp. to allow efficient detection of the species.

Sampling and detection of Legionella pneumophila aerosols generated from an industrial cooling tower

Cooling tower water has frequently been cited as a source of infection in outbreaks of Legionnaires' disease. However, there have been few reports on the presence of legionellae in aerosols from cooling towers. This paper describes our use of an impinger or a six-stage microbial impactor for detecting legionellae in air around a cooling tower contaminated with L. pneumophila (1.2±0.3×105 CFU/100 ml). Phosphate-buffered saline, Page's saline, 2% yeast extract solution and buffered yeast extract (BYE) broth were tested to evaluate their collection efficiency. These solutions were compared in laboratory experiments using an aerosol of L. pneumophila serogroup (SG) 1. Because BYE broth was the most efficient and storable collecting fluid among them, it was used for outdoor air sampling. In the outdoor air sampling, aerosolized L. pneumophila SG 6 was detected in the air around the cooling tower by the impinger (0.09 CFU/l. air). No legionellae were detected by the impactor with Legionella-selective agar plates (WYOα) because the plates were overgrown with fungi. Repetitive element PCR (rep–PCR) and arbitrarily primed PCR (AP–PCR) were employed to assess the epidemiological relationship among Legionella isolates from the air sample and the cooling tower water samples. L. pneumophila SG 6 isolated from the aerosols produced rep–PCR and AP–PCR fingerprints identical to those of L. pneumophila SG 6 strains from the cooling tower water, suggesting that the bacterium was aerosolized from the cooling tower.

Sampling and detection of <italic>Legionella pneumophila</italic> aerosols generated from an industrial cooling tower

Cooling tower water has frequently been cited as a source of infection in outbreaks of Legionnaires' disease. However, there have been few reports on the presence of legionellae in aerosols from cooling towers. This paper describes our use of an impinger or a six-stage microbial impactor for detecting legionellae in air around a cooling tower contaminated with L. pneumophila (1.2+/-0.3x10(5) CFU/100 ml). Phosphate-buffered saline, Page's saline, 2% yeast extract solution and buffered yeast extract (BYE) broth were tested to evaluate their collection efficiency. These solutions were compared in laboratory experiments using an aerosol of L. pneumophila serogroup (SG) 1. Because BYE broth was the most efficient and storable collecting fluid among them, it was used for outdoor air sampling. In the outdoor air sampling, aerosolized L. pneumophila SG 6 was detected in the air around the cooling tower by the impinger (0.09 CFU/l. air). No legionellae were detected by the impactor with Legionella-selective agar plates (WYOalpha) because the plates were overgrown with fungi. Repetitive element PCR (rep-PCR) and arbitrarily primed PCR (AP-PCR) were employed to assess the epidemiological relationship among Legionella isolates from the air sample and the cooling tower water samples. L. pneumophila SG 6 isolated from the aerosols produced rep-PCR and AP-PCR fingerprints identical to those of L. pneumophila SG 6 strains from the cooling tower water, suggesting that the bacterium was aerosolized from the cooling tower.

Detection of Legionella spp. in cooling tower water by the polymerase chain reaction method

The presence of Legionella spp. in cooling tower water was investigated by using the polymerase chain reaction. Total Legionella spp. detection was performed with 20-mer 5S rRNA complementary DNA sequence primers, and specific Legionella pneumophila detection was performed with 20-mer and then 21-mer macrophage infectivity potentiator gene sequence primers. Of 27 cooling tower water samples, 25 were positive for Legionella spp., and 14 of these contained L. pneumophila.

Legionella moravica sp. Nov. and Legionella brunensis sp. Nov. Isolated from cooling-tower water

Two Legionella-like organisms were isolated from cooling-tower water samples in Czechoslovakia. They were presumptively identified as legionellae by their growth on buffered charcoal-yeast extract agar (BCYE) containing L-cysteine and their absence of growth on BCYE without L-cysteine. Both strains contained predominantly branch-chained cellular fatty acids and were therefore definitively placed in the genus Legionella. They were serologically distinct from other described Legionella species and were shown by DNA studies to constitute two new Legionella species, Legionella moravica (type strain 316-36; ATCC 43877) and Legionella brunensis (type strain 441-1; ATCC 43878). Two Legionella-like organisms were isolated from cooling-tower water samples in Czechoslovakia. They were presumptively identified as legionellae by their growth on buffered charcoal-yeast extract agar (BCYE) containing L-cysteine and their absence of growth on BCYE without L-cysteine. Both strains contained predominantly branch-chained cellular fatty acids and were therefore definitively placed in the genus Legionella. They were serologically distinct from other described Legionella species and were shown by DNA studies to constitute two new Legionella species, Legionella moravica (type strain 316-36; ATCC 43877) and Legionella brunensis (type strain 441-1; ATCC 43878).

Legionella moravica sp. Nov. and Legionella brunensis sp. Nov. Isolated from cooling-tower water

Two Legionella-like organisms were isolated from cooling-tower water samples in Czechoslovakia. They were presumptively identified as legionellae by their growth on buffered charcoal-yeast extract agar (BCYE) containing L-cysteine and their absence of growth on BCYE without L-cysteine. Both strains contained predominantly branch-chained cellular fatty acids and were therefore definitively placed in the genus Legionella. They were serologically distinct from other described Legionella species and were shown by DNA studies to constitute two new Legionella species, Legionella moravica (type strain 316-36; ATCC 43877) and Legionella brunensis (type strain 441-1; ATCC 43878).

Nosocomial outbreak of Legionnaires' disease: molecular epidemiology and disease control measures.

Molecular laboratory techniques were used to study the epidemiology of an outbreak of nosocomial Legionnaires' disease. All patient isolates were Legionella pneumophila serogroup 1 and showed identical plasmid profiles and reactions with serogroup-specific monoclonal antibodies. L pneumophila was also cultured from four of five cooling tower water samples; however, the isolate from only one tower was serogroup 1 of the same subtype as patient isolates. Since the cases were temporally clustered and epidemiologically associated with exposure to cooling tower aerosols, the single cooling tower implicated by molecular analysis was the most likely source of the outbreak. Chlorination of cooling tower ponds has eradicated the epidemic strain. Since potable water also harbored the infecting organism and was the probable source for cooling tower contamination, decontamination of the hospital water system was also undertaken. Superchlorination of hot water holding tanks to 17 ppm on a weekly basis has effectively eradicated L pneumophila from the potable water system and appears to be a reasonable, simple, and relatively inexpensive alternative to previously described methods of control.

Ecological distribution of Legionella species in Japan and pathogenicity of environmental isolates

Distribution of Legionella species over Japan was investigated.Five hundred ml of various environmental sources (343 samples of cooling tower water, 25 samples of water of puddy field. 29 samples of river, 3 samples of lake, 1 sample of fountain, 2 samples of puddle, 4 samples of well water, 6 samples of hemodialysate, 5 samples of antiseptic solution for washing hands at operating room) were cultured for isolation of Legionella spp.Isolations of the organism were applied to intraperitoneal injection to guinea pigs and direct inoculation to semiselective medium (B-CYE agar with antibiotics) after low pH treatment.One hundred sixty-six strains of Legionella spp. were isolated from 126 (36.7%) out of 343 cooling tower water samples.The origin of positive samples ranged from Hokkaido (Northern Japan) to Okinawa (Southern Japan).L. pneumophila, serogroup 1 was the most frequent isolates (66.3%); next in frequency of occurrence were Legionella-like organisms (16.3%), L. pneumophila serogroup 4 (7.2%), L. bozemanii (7.2%), L. pneumophila serogroup 6 (1.8%) and serogroup 3 (1.2%).L. pneumophila serogroup 2, serogroup 5, L. micdadei, L. dumoffii, L. gormanii, L. longbeachae and L. jordanis were not isolated from cooling tower water samples in this study.Five out of 28 soil samples were positive culture of Legionella spp. with L. pneumophila serogroup 1 (5 strains), serogroup 4 (2 strains) and serogroup 5 (1 strain).One strain of L. pneumophila serogroup 1 was isolated from one of 2 samples of puddle in construction sites. No positive culture was obtained from remaining materials.These results indicated that many types of Legionella species were widespread in all over the country.Environmental isolates possessed to have almost same endotoxin-like activity as clinical isolates by limulus test. However, there was some variations in virulence to mice among environmental isolates.