Presence Of Legionella Research Articles

Biodiversity of amoebae and amoeba-associated bacteria in water treatment plants

In this study, we enlarged our previous investigation focusing on the biodiversity of chlamydiae and amoebae in a drinking water treatment plant, by the inclusion of two additional plants and by searching also for the presence of legionellae and mycobacteria. Autochthonous amoebae were recovered onto non-nutritive agar, identified by 18S rRNA gene sequencing, and screened for the presence of bacterial endosymbionts. Bacteria were also searched for by Acanthamoeba co-culture. From a total of 125 samples, we recovered 38 amoebae, among which six harboured endosymbionts (three chlamydiae and three legionellae). In addition, we recovered by amoebal co-culture 11 chlamydiae, 36 legionellae (no L. pneumophila), and 24 mycobacteria (all rapid-growers). Two plants presented a similar percentage of samples positive for chlamydiae (11%), mycobacteria (20%) and amoebae (27%), whereas in the third plant the number of recovered bacteria was almost twice higher. Each plant exhibited a relatively high specific microbiota. Amoebae were mainly represented by various Naegleria species, Acanthamoeba species and Hartmannella vermiformis. Parachlamydiaceae were the most abundant chlamydiae (8 strains in total), and in this study we recovered a new genus-level strain, along with new chlamydiae previously reported. Similarly, about 66% of the recovered legionellae and 47% of the isolated mycobacteria could represent new species. Our work highlighted a high species diversity among legionellae and mycobacteria, dominated by putative new species, and it confirmed the presence of chlamydiae in these artificial water systems.

Evaluation of the usefulness of a new direct immunofluorescence assay (ScanVIT-Legionella™) for monitoring hospital water systems contaminated withLegionellaspp.

To compare the efficiency of the ScanVIT-Legionella test (Vermicon, Munich, Germany) vs a conventional culture method for the quantification of Legionella spp. in hospital water samples in daily hospital practice. The detection of Legionella spp. takes place on a cultivated filter brought into contact with dye-marked gene probes. The results are analysed under fluorescence microscopy. Bacteria that light up green belong to the genus Legionella; those that light up both green and red belong to the species Legionella pneumophila. Our results showed that the ScanVIT test has a sensitivity of 90%; agreement between the two methods was 82%. In the 48 samples that tested positive with both methods, the Legionella concentration detected by the culture method was consistently higher. A statistically significant difference between the results obtained with the two test methods emerged at the Wilcoxon test (P < 0.001). The ScanVIT test may be recommended for investigating the presence of Legionella by qualitative testing. Given the simplicity of colony identification by fluorescence, the ScanVIT test can be used in laboratories where staffs are not experienced in identifying typical colonies of Legionella.

Resistance of Legionella pneumophila serotype 1 biofilms to chlorine-based disinfection

The presence of Legionella spp. in potable water systems is a major concern to municipal water providers and consumers alike. Despite the inclusion of chlorine in potable supplies and frequent chlorination cycles, the bacterium is a recalcitrant human pathogen capable of causing incidents of Legionnaires' disease, Pontiac fever and community-acquired pneumonia in humans. Using two materials routinely employed for the delivery of potable water as a substratum, copper and stainless steel, the development of Legionella pneumophila biofilms and their response to chlorination was monitored over a three-day and a three-month period, respectively. Preliminary in vitro studies using broth and sterile tap water as culture media indicated that the bacterium was capable of surviving in low numbers for 28 days in the presence of chlorine. Subsequently, biofilms were grown for three days, one month and two months, respectively, on stainless steel and copper sections, which are widely used for the conveyance of potable water. Immediately after exposure to 50mg/L chlorine for 1h, the biofilms yielded no recoverable colonies, but colonies did reappear in low numbers over the following days. Despite chlorination at 50mg/L for 1h, both one- and two-month-old L. pneumophila biofilms were able to survive this treatment and to continue to grow, ultimately exceeding 1x10(6)cfu per disc. This research provides an insight into the resistance afforded to L. pneumophila against high levels of chlorine by the formation of biofilms and has implications for the delivery of potable water.

Microbial Characterization of Water and Biofilms in Drinking Water Distribution Systems at Sport Facilities

The present study investigated the in situ microbial indicators' occurrence in water and biofilm in drinking water distribution systems of sport facilities such as gyms. The presence of Legionella, such as a potential pathogen, was also verified. Water and biofilm were contemporaneously collected and microbiologically analyzed. Few colonies of coliforms were detected in biofilm but not in the corresponding water samples. Conversely, some colonies of heterotrophs were counted at 22 degrees C in both the 80% biofilm samples and the 53% water samples. Legionella was detected in 29% of the hot water systems, and, in particular, L. pneumophila in 21% and L. non pneumophila in 9% of the samples. Higher counts were found in biofilm samples. As it was recommended for swimming pools, it is to be hoped that hygiene risk assessment criteria and safety plans could be also developed for gyms to reduce potential hazards to health for all attendants.

Biofilm Formation on Copper and Galvanized Steel Surfaces in a Cooling-Water System

Cooling water systems provide ideal aquatic environment for the microorganism multiplication. Copper and galvanized steel are frequently used in the construction of cooling towers because of their well-known antifouling property. The current study would examine the changes in total aerobic mesophilic heterotrophic bacteria (TAMHB) counts, the presence of Legionella and free-living amoeba on copper and galvanized steel surfaces and the presence of biocide in a cooling water system. In this study biofilm was confirmed by SEM on copper and galvanized coupons in experiments. Statistical analysis demostrated that heterotrophic bacteria counts were significantly (P<0.01 ) higher on the surface of galvanized steel coupons than on the surface of copper coupons. Our results showed that the isolates were characterized as members of genus Pseudomonas or were closely related to this genus. Free-living amoeba were detected on the surface of copper coupons and galvanized steel coupons whereas Legionella pneumophila was not established by culture method.

Legionella species colonization of water distribution systems, pools and air conditioning systems in cruise ships and ferries.

BackgroundLegionnaires' disease continues to be a public health concern in passenger ships. This study was scheduled in order to investigate Legionella spp. colonization of water distribution systems (WDS), recreational pools, and air-conditioning systems on board ferries and cruise ships in an attempt to identify risk factors for Legionella spp. colonization associated with ship water systems and water characteristics.MethodsWater systems of 21 ferries and 10 cruise ships including WDS, air conditioning systems and pools were investigated for the presence of Legionella spp.ResultsThe 133 samples collected from the 10 cruise ships WDS, air conditioning systems and pools were negative for Legionella spp. Of the 21 ferries WDS examined, 14 (66.7%) were legionellae-positive. A total of 276 samples were collected from WDS and air conditioning systems. Legionella spp. was isolated from 37.8% of the hot water samples and 17.5% of the cold water samples. Of the total 96 positive isolates, 87 (90.6%) were L. pneumophila. Legionella spp. colonization was positively associated with ship age. The temperature of the hot water samples was negatively associated with colonization of L. pneumophila serogroup (sg) 1 and that of L. pneumophila sg 2 to 14. Increases in pH ≥7.8 and total plate count ≥400 CFU/L, correlated positively with the counts of L. pneumophila sg 2 to 14 and Legionella spp. respectively. Free chlorine of ≥0.2 mg/L inhibited colonization of Legionella spp.ConclusionWDS of ferries can be heavily colonized by Legionella spp. and may present a risk of Legionnaires' disease for passengers and crew members. Guidelines and advising of Legionnaires' disease prevention regarding ferries are needed, in particular for operators and crew members.

Occurrence and Diversity of Legionellaceae in Polar Lakes of the Antarctic Peninsula

Legionellaceae is a family of gram-negative, mesophilic, and facultative intracellular parasitic bacteria that inhabits freshwater environments. In this article, the Legionella population of water samples from the North and South Lake, located close to the Brazilian Scientific Station on King George Island, Keller Peninsula, Antarctica has been characterized. Culture onto selective medium and a independent-culture method were applied to the samples. In our attempt to isolate Legionella species from Antarctic lakes, we were able to obtain one L. pneumophila colony by an amoebic coculture procedure followed by plate culture onto a selective medium. In addition, results obtained from phylogenetic inference showed the presence of noncharacterized specimens of Legionella spp. These findings indicated the presence of legionellae in Antarctica and suggest that these bacteria can adapt to extreme conditions and open new possibilities for understanding the survival strategies of mesophilic Legionellaceae living in Antarctic environments. Furthermore, the isolation of these symbiotic bacteria in Antarctic lakes will allow future studies on cold-resistant mechanisms of legionellae in polar environments.

Isolation of Legionella species/serogroups from water cooling systems compared with potable water systems in Spanish healthcare facilities

Surveillance of Legionella spp. in hospital water systems was performed in forty-four inpatient healthcare facilities in Spain during 2005-2006. A total of 2,341 samples were collected: 470 from cooling systems (cooling towers) and 1,871 from potable water systems. The latter included 211 from cold-water tanks and 260 from hot-water tanks, totalling 471 from central water reservoirs 136 from showers, 1,172 from unfiltered taps and 92 from filtered taps, totalling 1,400 from peripheral points. Temperature, chlorine levels and the presence of Legionella spp. were determined. In all, 373 (15.9%) samples yielded Legionella spp. Significantly higher isolation rates were obtained from cooling towers (23.8%) versus cold- and hot-water tanks (approximately 4.7%), due to the significantly higher number of samples positive for serogroup 1 (19.4 vs 0.9-3.5%). In potable water systems, no differences were found between central water tanks and showers, but significant differences in isolation rates between central water tanks and unfiltered taps were observed (4.7 vs 19.6%) due to differences in non-serogroup 1 L. pneumophila. Filters significantly decreased isolation rates of these serotypes (11 vs 0%). Some seasonal differences were noted, with higher isolation rates in summer for legionella serogroup 1 in cooling systems and for L. pneumophila serogroups 2-14 in potable water systems. In regression models, higher temperatures were associated with colonisation in cooling systems, while lower chlorine levels were associated with colonisation in potable water systems.

Detection of Legionella spp. and some of their amoeba hosts in floating biofilms from anthropogenic and natural aquatic environments

Floating biofilms develop at the water–air interface and harbor numerous microorganisms, some of which are human pathogens like Legionella pneumophila. The presence of Legionella spp. and especially L. pneumophila in such biofilms was investigated. In parallel, the occurrence of Naegleria spp., Acanthamoeba spp., Willaertia spp., Vahlkampfia spp. and Hartmanella spp. was determined and it was examined whether Acanthamoeba spp. isolates were naturally infected with L. pneumophila bacteria. Eight anthropogenic and 37 natural aquatic environments were sampled between June and August 2005. Both Legionella spp. and L. pneumophila were present in 100% of the floating biofilms of the anthropogenic aquatic systems. Eighty-one percent of all natural floating biofilm samples were positive for Legionella spp. and 70% of these samples were positive for L. pneumophila. Legionella concentrations were in the range of 10 1–10 2 cells/cm 2. Naegleria spp. and Acanthamoeba spp., two well-known L. pneumophila amoeba hosts, were present in 50–92% and 67–72% of floating biofilm samples, respectively. Acanthamoeba spp. isolates appeared to be naturally infected with L. pneumophila bacteria as proved by fluorescent in situ hybridization.

Environmental Survey of Legionella pneumophila in Hot Springs in Taiwan

Acquisition of sporadic community-acquired legionnaires' disease has been linked to hot springs and whirlpool baths. Outbreaks of hot spring-associated legionnaires' disease were reported in Japan in the last few years. Although the mode of transmission is unclear, the presence of Legionella in hot springs may discourage hot springs resort visits by the general public. An environmental survey was conducted to determine the presence of Legionella in hot springs in Taiwan. In total, 55 water samples were collected from 19 hot springs resorts; 21% (4/19) of the hot spring resorts sampled yielded L. pneumophila in the public hot springs bath. Legionella pneumophila serogroups 1 and 6, L. pneumophila serogroup 3, L. pneumophila serogroup 5, and L. pneumophila serogroup 7 were isolated from four different resort spas, respectively. The total sample positivity rate for L. pneumophila was 11% (6/55). The risk of occurrences of legionnaires' disease outbreaks associated with hot springs water in general public is unknown, and epidemiologic investigations should be conducted for locating the potential sources of Legionella for those cases of community-acquired legionnaires' disease. Disinfection of hot springs for Legionella may be necessary if the risk of contracting legionnaires' disease from hot springs can be validated by an evidence-based approach.

Characteristics of Cooling Towers and Evaporative Condensers and Presence of Legionella in Water

P-721 Introduction: Cooling towers and evaporative condensers are elements associated with cooling and refrigeration systems. Many legionella outbreaks have been linked to this type of installations. To minimise the risk of legionella transmission, the Spanish Ministry of Health and Consumption published Royal Decree 865/2003, which establishes the preventive design and surveillance system to be fulfilled by these installations. With the aim of assessing the degree of fulfilment (infrastructure, maintenance, cleaning, disinfection) of the mentioned installations in Gipuzkoa (the Basque Country, Spain), they were characterised and the water in the cooling circuits was checked for legionella. Methods: Over the period 2002–2004, 297 cooling circuits were visited and characterised. A survey gathered information on three variables: infrastructure, maintenance of circuits and water quality control. The sampling and analysis of the water was carried out in two points of the cooling circuit (the tray and the bleeder valve). The Legionella analysis was conducted according to the ISO 11734 standard. Results: 260 (87.5%) of installations carried out maintenance operations; 243 (81.8%) had a continuous treatment of the water in the circuits and 270 (90.9%) cleaned and disinfected the circuits periodically. Out of the 200 samples taken, 72 (36%) were positive (detection limit 100 u.f.c/l) for Legionella spp and 56 (28%) were positive for L. pneumophila. The cooling towers which did not incorporate a stop mode proved to be more risky for Legionella spp. (O.R: 4.89; IC: 1.17–20.38) and for L. pneumophila (O.R: 3.56; IC: 0.86–14.85)than those which incorporated this mode. Four factors were identified as protective against Legionella spp.: collecting the water from the municipal network (O.R: 0.46; IC: 0.24–0.86); having circuits which are accessible for cleaning (O.R: 0.32; IC: 0.10–0.94); conducting periodical cleaning and shock disinfections in the circuits (O.R: 0.17; IC: 0.04–0.64), and carrying out a continuous treatment of the water by using a disinfectant with residual power (O.R: 0.40; IC:0.15–0.83). In the case of L. pneumophila, two protective factors were identified: collecting the water from the municipal network (O.R: 0.35; IC: 0.18–0.68), and carrying out a continuous treatment of the water by using a disinfectant with residual power (O.R: 0.25; IC:0.08–0.0.83) Discussion and Conclusions: Designing the installations properly, using water coming from the municipal net work, periodically cleaning and disinfecting the circuits and treating the water they contain by means of disinfectant products with residual power reduces the prevalence of Legionella in water.

Characteristics of Domestic Hot Water Networks in Establishments Considered to be Risky and Presence of Legionella in Water

Characteristics of Domestic Hot Water Networks in Establishments Considered to be Risky and Presence of Legionella in Water

Influence of amoebae and physical and chemical characteristics of water on presence and proliferation of Legionella species in hospital water systems

The reservoir for hospital-acquired Legionnaires' disease has been shown to be the potable water distribution system. The objectives of the present study were as follows: (1) to examine the possible relationship between physical-chemical characteristics of water such as temperature, pH, hardness, conductivity, and residual chlorine and the presence of amoebae as growth-promoting factors for Legionella species and (2) to determine eradication measures for water distribution systems to seek ways of reducing the risk of legionellosis. Ten hospitals in southwest France took part in this study. Water samples were collected from 106 hot water faucets, showers, hot water tanks, and cooling towers. Two analyses were performed to analyze the association between water characteristics and (1) the presence of Legionella species and (2) the proliferation of Legionella species. Of the 106 water samples examined, 67 (63.2%) were positive for Legionella species. Amoebae were detected in 73 of 106 (68.9%) samples and in 56 of 67 (86.6%) Legionella species-positive samples (P < 10(-6)). In these positive samples, conductivity was lower than 500 microOmega(-1).cm(-1) in 58.2% (P = .026), temperature was below 50 degrees C in 80.6% (P = .004), and hardness was significantly higher (P = 002) than in Legionella species-negative samples. Neither Legionella species nor amoebae were isolated from any sampling point in which the water temperature was above 58.8 degrees C. Multivariate analysis shows that high hardness and presence of amoebae were strongly correlated statistically with the presence of Legionella when showers, tanks, pH, and temperature promoted their proliferation. This study shows the importance of water quality evaluation in assessing environmental risk factors and in selecting the most appropriate prevention and control measures in hospital water systems.

Effect of flow regimes on the presence of Legionella within the biofilm of a model plumbing system

Stagnation is widely believed to predispose water systems to colonization by Legionella. A model plumbing system was constructed to determine the effect of flow regimes on the presence of Legionella within microbial biofilms. The plumbing model contained three parallel pipes where turbulent, laminar and stagnant flow regimes were established. Four sets of experiments were carried out with Reynolds number from 10,000 to 40,000 and from 355 to 2,000 in turbulent and laminar pipes, respectively. Legionella counts recovered from biofilm and planktonic water samples of the three sampling pipes were compared with to determine the effect of flow regime on the presence of Legionella. Significantly higher colony counts of Legionella were recovered from the biofilm of the pipe with turbulent flow compared with the pipe with laminar flow. The lowest counts were in the pipe with stagnant flow. We were unable to demonstrate that stagnant conditions promoted Legionella colonization. Plumbing modifications to remove areas of stagnation including deadlegs are widely recommended, but these modifications are tedious and expensive to perform. Controlled studies in large buildings are needed to validate this unproved hypothesis.

The Role of Flushing Dental Water Lines for the Removal of Microbial Contaminants

This study was designed to determine the role of flushing dental water lines for the removal of heterotrophic plate count bacteria, Legionella spp., and free-living protozoa. Forty dental offices were surveyed in the study. An initial sample and a sample taken after three minutes of flushing were obtained from the air/water syringe at each location. All samples were quantitatively analyzed for heterotrophic bacteria using three bacteriological procedures. The samples were analyzed for the presence of Legionella spp. using cultural, immunological, and molecular procedures and for the occurrence of free-living protozoa using a killed bacteria plate procedure. The flushing process reduced the level of heterotrophic plate count bacteria by 1.1 to 1.5 log10 CFU/ml. Compliance with recommendations for bacterial levels varied depending on the methodology employed in the analysis. The flushing process did not reduce the occurrence of Legionella spp. or free-living protozoa. The results support recent U.S. Centers for Disease Control and Prevention recommendations that the process of flushing dental water lines cannot be relied upon as a sole means of reliably improving the quality of water used in dental treatment.

Biodiversity of Amoebae and Amoeba-Resisting Bacteria in a Hospital Water Network

Free-living amoebae (FLA) are ubiquitous organisms that have been isolated from various domestic water systems, such as cooling towers and hospital water networks. In addition to their own pathogenicity, FLA can also act as Trojan horses and be naturally infected with amoeba-resisting bacteria (ARB) that may be involved in human infections, such as pneumonia. We investigated the biodiversity of bacteria and their amoebal hosts in a hospital water network. Using amoebal enrichment on nonnutrient agar, we isolated 15 protist strains from 200 (7.5%) samples. One thermotolerant Hartmannella vermiformis isolate harbored both Legionella pneumophila and Bradyrhizobium japonicum. By using amoebal coculture with axenic Acanthamoeba castellanii as the cellular background, we recovered at least one ARB from 45.5% of the samples. Four new ARB isolates were recovered by culture, and one of these isolates was widely present in the water network. Alphaproteobacteria (such as Rhodoplanes, Methylobacterium, Bradyrhizobium, Afipia, and Bosea) were recovered from 30.5% of the samples, mycobacteria (Mycobacterium gordonae, Mycobacterium kansasii, and Mycobacterium xenopi) were recovered from 20.5% of the samples, and Gammaproteobacteria (Legionella) were recovered from 5.5% of the samples. No Chlamydia or Chlamydia-like organisms were recovered by amoebal coculture or detected by PCR. The observed strong association between the presence of amoebae and the presence of Legionella (P < 0.001) and mycobacteria (P = 0.009) further suggests that FLA are a reservoir for these ARB and underlines the importance of considering amoebae when water control measures are designed.

Evaluation of standardized scored inspections for Legionnaires' disease prevention, during the Athens 2004 Olympics.

The study was designed to determine the contribution of standardized scored inspections implemented during the Athens 2004 Pre-Olympic and Olympic period, in assessing the presence of Legionella spp. in water sites. Inspection grading scores of 477 water supply systems, 127 cooling towers and 134 decorative fountains were associated with the corresponding microbiological test results of 2514 samples for Legionella spp. Nine violations of water supply systems and nine of cooling towers significantly associated with positive microbiological test results, and four violations of water supply systems and one of cooling towers were among those designated as 'critical' water safety hazards in the inspection reports. The study documents a strong correlation [water supply systems (RR 1.92), cooling towers (RR 1.94)] between unsatisfactory inspection scoring results and Legionella-positive microbiological test results (in excess of 10,000 c.f.u./l) and suggests the utility of inspection scoring systems in predicting Legionella proliferation of water systems and in preventing Legionnaires' disease.

ISOLATION OF LEGIONELLA FROM COOLING TOWERS AND POTABLE WATER SYSTEMS IN HOSPITAL AND NON-MEDICAL BUILDINGS IN A UNIVERSITY CAMPUS

Six cooling towers and eleven sources of potable water in a universitycampus in Kuala Lumpur were surveyed for the presence of legionellae. One nonmedical building cooling tower and three hot water systems from one ward of the hospital tested positive for Legionella, two of which contained Legionella pneumophila serogroup 1. The identification of Legionella was based on isolation, immunofluorescence and polymerase chain reaction (PCR).

No evidence of Legionella infection in general practice patients presenting with acute respiratory infections in The Netherlands

The role of Legionella spp. in the aetiology of acute respiratory infections (ARIs) is largely unknown. In this case‐control study, conducted in a general practitioner setting during 2000 and 2001, nose and throat samples from patients presenting with ARIs (n = 230) and controls (n = 200) were analysed for the presence of Legionella spp. by real‐time PCR. Legionella DNA was not detected in any of the cases or controls. Thus, Legionella spp. do not seem to play a role in patients presenting with ARIs, nor were they present in patients who visited their general practitioner for complaints other than ARIs.

Presence and Persistence of Legionella spp. in Groundwater

Groundwater samples (111) from six different boreholes located in two geographical areas were examined for the presence of legionellae over a 7-year period. The number of Legionella isolates detected was generally low. The colonization of the aquifers was not uniform, and the persistence of Legionella was independent of the hydraulic pumps and the plumbing system present in the borehole. A total of 374 isolates identified by fatty acid methyl ester analysis belonged to Legionella pneumophila, L. oakridgensis, L. sainthelensi, and L. londiniensis. In area 1, L. oakridgensis constituted the major population detected, exhibiting only one random amplified polymorphic DNA (RAPD)-PCR profile. L. sainthelensi strains were less frequently isolated and also displayed a single RAPD profile, while L. pneumophila was only sporadically detected. In contrast, L. pneumophila comprised the vast majority of the isolates in area 2 and exhibited six distinct RAPD patterns, indicating the presence of different genetic groups; three L. londiniensis RAPD types were also detected. Two of the L. pneumophila and one of the L. londiniensis RAPD types were persistent in this environment for at least 12 years. The genetic structure of L. pneumophila groundwater populations, inferred from rpoB and dotA gene sequences, was peculiar, since the majority of the isolates were allied in a discrete group different from the lineages containing most of the type and reference strains of the three subspecies of L. pneumophila. Furthermore, gene exchange events related to the dotA allele could be envisioned.