The aim of this study was to evaluate the frequency of Legionella pneumophila in three hot spring water recreation areas by conventional culture on GVPC and by Real Time-PCR. No legionellae were isolated from the spring water upstream the distribution system suggesting strongly that the thermal area was the probable source of these pathogens. L. pneumophila was present at high level in some samples, reaching values of 8.2 × 10(3) CFU/l at the site C. We observed a low diversity among the 18 isolated Legionella strains according to 16S DNA analysis, since only L. pneumophila and Legionella londiniensis were identified. All of the L. pneumophila strains belong to five serogroups (1, 4, 5, 6 and 8). Pulsed-field gel electrophoresis analysis identified various genotypes among these serogroups and showed that these strains had pulsotypes distinct from L. pneumophila present in the CNRL Legionella-database. These environmental strains showed higher tolerance to heat shock stress at 48 °C than L. pneumophila clinical strain, which may explain their presence in warm waters. Our data suggested that potential pathogen legionellae are present in Tunisian medicalized thermal spas and emphasized the importance of adopting control measures to prevent infection.

The aim of this study was to determine the survival of human pathogens (Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa) in five natural mineral waters (NMWs) with different properties and mineralization levels. Five NMWs from four Spanish spas with different dry residue at 110 °C were used: A = 76,935 mg/L; B = 1,827 mg/L; C = 808.4 mg/L; D = 283.8 mg/L; and E = 170.4 mg/L. An initial inoculum of 1 × 10(6) colony forming units (cfu)/mL was used for survival studies. Distilled water, chlorinated tap water and Mueller-Hinton broth were used as controls. Colony counts in all different waters were lower than those achieved with Mueller-Hinton broth over all incubation periods. A direct effect between the bacterial survival and the level of mineralization water was observed. The NMW E with low mineralization level along with the radioactive properties showed the highest antibacterial activity among all NMWs.

This work explores the factors associated with contamination of public spas by Legionella spp., Pseudomonas aeruginosa and Escherichia coli. Physicochemical and microbiological parameters were measured in water samples from 95 spas inQuébec, Canada. Spa maintenance was documented by a questionnaire. Legionella spp. were detected in 23% of spas, P. aeruginosa in 41% and E. coli in 2%. Bacteria were found in concerning concentrations (Legionella spp. ≥ 500 CFU/l, P. aeruginosa ≥ 51 CFU/100 ml or E. coli ≥ 1 CFU/100 ml) in 26% ofspas. Observed physicochemical parameters frequently differed from recommended guidelines. The following factors decreased the prevalence of concerning microbial contamination: a free chlorine concentration ≥ 2 mg/l or total bromine ≥ 3 mg/l (p = 0.001), an oxidation-reduction potential (ORP) > 650 mV (p = 0.001), emptying and cleaning the spa at least monthly (p = 0.019) and a turbidity ≤ 1 NTU (p = 0.013). Proper regulations and training of spa operators are critical for better maintenance of these increasingly popular facilities.

The fact itself that thermo-mineral waters and mud have healing effects has always attracted attention throughout the history to exploit, explore and study their benefit on the human body. Modern lifestyle and the speed of life endanger man's psycho-physical health. This is why people more often return to old time proven values, the nature and natural health resorts.To establish hydro-geological conditions for the formation of mineral water and to summarize their balneological characteristics in spas, i.e., in rehabilitation centres of the Pannonian basin of the Republic of Serbia, where underground waters are still actively exploited for balneotherapy.By retrospective descriptive analysis, a recapitulation of hydro-geological conditions for the formation of mineral waters was made and their balneological characteristics were established in eight spas of the Pannonian basin.The healing spas of the Pannonian basin are predominated by HCO3 (2.9 g/l - 4.6% milival), iodic (up to 6.5 mg/l), slightly alkaline (pH up to 8.1) thermal-mineral water (temperature up to 72 degrees C, bounty to 36.6 l/s) with a significant content of Br (up to 8.1 mg/l), Fe (to 6.0 mg/l), metaboron (up to 60 mg/l) and metasilicon acid (up to 95 mg/l). They are used for external application, bathing and showering.Once the Pannonian Sea (the Paratethys), today a wide plain terrain is a tectonic depression of the lower Pannonian pont age with compact type aquifers. In the geological column of sedimentary rocks a large underground aquifer was formed with free water of high mineralization, high temperature and geothermal properties above the average in relation to the European hydrogeological standards. Therefore, the Pannonian basin can be rightly called a thermal valley with the predominance of sodium hydro-carbonate (alkaline) iodine healing water of enviable abundance and reserves.

One of the greatest challenges of implementing fast molecular detection methods as part of Legionella surveillance systems is to limit detection to live cells. In this work, a protocol for sample treatment with propidium monoazide (PMA) in combination with quantitative PCR (qPCR) has been optimized and validated for L. pneumophila as an alternative of the currently used time-consuming culture method. Results from PMA-qPCR were compared with culture isolation and traditional qPCR. Under the conditions used, sample treatment with 50 μM PMA followed by 5 min of light exposure were assumed optimal resulting in an average reduction of 4.45 log units of the qPCR signal from heat-killed cells. When applied to environmental samples (including water from cooling water towers, hospitals, spas, hot water systems in hotels, and tap water), different degrees of correlations between the three methods were obtained which might be explained by different matrix properties, but also varying degrees of non-culturable cells. It was furthermore shown that PMA displayed substantially lower cytotoxicity with Legionella than the alternative dye ethidium monoazide (EMA) when exposing live cells to the dye followed by plate counting. This result confirmed the findings with other species that PMA is less membrane-permeant and more selective for the intact cells. In conclusion, PMA-qPCR is a promising technique for limiting detection to intact cells and makes Legionella surveillance data substantially more relevant in comparison with qPCR alone. For future research it would be desirable to increase the method's capacity to exclude signals from dead cells in difficult matrices or samples containing high numbers of dead cells.

Legionella pneumophila, aerosolized from numerous indoor facilities (e.g., shower heads, hot tubs, spas), may cause Pontiac fever (PF) and lethal pneumonia named Legionnaires' disease (LD) in humans. Reliable methods on quantitative exposure assessment of this bioaerosol are essential for the prevention of PF and LD. Coupled with culture, ethidium monoazide with qPCR, and qPCR assays, the collection efficiency for culturable, viable, and total L. pneumophila was assessed by means of filtration sampling (IOM with gelatin filter and cassette with polycarbonate filter) and liquid-based sampling methods (BioSampler, AGI-30, MAS-100 sampler with Tween mixture and deionized water (DW)). Results show IOM/gelatin filter was comparable to cassette/polycarbonate filter (P = 0.33) and performed greater than all of tested liquid-based methods for total cell collection. On the other hand, IOM/gelatin filter obtained greater efficiencies than cassette/polycarbonate filter by a factor of 3.8-8.6 for viable cells (P = 0.0006) and two orders of magnitude for culturable cells (P = 0.00002). Further comparison between liquid impingement and filtration methods indicates the sampling by IOM/gelatin filter, AGI-30, and BioSampler with DW were the most appropriate for viable cells, while culturable cells were collected most efficiently by BioSampler/DW with periodical replenishment during the sampling. PRACTICAL

IMPLICATIONS:

This study recommends the most suitable methodologies for quantifying culturable, viable, and total Legionella pneumophila in indoor air. By using appropriate sampling and analytical methods, the residents and building owners are able to obtain the reliable data and further characterize the exposure risk and/or intervention efficacy against L. pneumophila. Moreover, the adoption of suitable monitoring methods also assists the investigators to explore the sources linked to PF and LD during the outbreaks. Considering reliable microbial monitoring is fundamental for epidemiological survey and risk assessment, the present information should be taken into account in assessing L. pneumophila indoors.

Legionella bacteria ubiquitously colonize natural freshwater and are responsible for legionellosis in humans. Several cases of legionellosis have been associated in particular with the use of whirlpool spas. The objective of this study was to verify whether real-time PCR is applicable for the quantification of Legionella spp. in spa water.The study compared concentrations obtained by real-time PCR vs that obtained by conventional culture for 101 spa water samples. For the culture method, Legionella spp. were detected and quantified in 14 of 101 samples with measured concentrations ranging from 250 to 3.5 × 10(5) CFU l(-1). With the real-time PCR method, Legionella spp. were detected and quantified in 42 of 101 samples with concentrations ranging from 1000 to 6.1 × 10(7) GU l(-1). Results revealed a significant but weak correlation (r(2) = 0.1867) between the two methods. The positive predictive value (35%) of the PCR method compared to conventional culture herein was low. In contrast, the negative predictive value was excellent, reaching 93%.Real-time PCR could be used as a screening tool to rapidly ascertain the absence of Legionella spp. in spa water. However, a positive result involves the need to resort to conventional culture.Data of this study highlighted the pros and cons of quantification of Legionella spp. in spa water with real-time PCR using a commercial quantitative PCR kit in a routine laboratory, when compared to conventional culture.

Some species of Legionella are recognized as opportunistic potential human pathogens, such as Legionella pneumophila, which causes legionnaires disease. Indeed, outbreaks of legionellosis are frequently reported in areas in which the organism has been spread via aerosols from contaminated institutional water systems. Contamination in hot tubs, spas and public baths are also possible. As a result, in this study, we investigated the distribution of Legionella at six hot spring recreation areas throughout Taiwan. Legionella were detected in all six hot spring recreation areas, as well as in 20 of the 72 samples that were collected (27.8%). Seven species of Legionella identified from samples by the direct DNA extraction method were unidentified Legionella spp., Legionella anisa, L. pneumophila, Legionella erythra, Legionella lytica, Legionella gresilensis and Legionella rubrilucen. Three species of Legionella identified in the samples using the culture method were L. pneumophila, unidentified Legionella spp. and L. erythra. Legionella species were found in water with temperatures ranging from 22.7 °C to 48.6 °C. The optimal pH appeared to range from 5.0 to 8.0. Taken together, the results of this survey confirmed the ubiquity of Legionella in Taiwan spring recreational areas. Therefore, a long-term investigation of the health of workers at hot spring recreational areas and the occurrence of Legionella in hot spring recreational areas throughout Taiwan are needed.

Traditional geothermally heated therapeutic spas, widely distributed in Europe, use water that is not disinfected. The colonization of therapeutic spas by Legionella spp. has been reported and several outbreaks and sporadic cases of legionellosis have been associated with the use of these facilities. We tested the effectiveness of hyperchlorination and the combination of hyperchlorination and ultraviolet light (UV) disinfection against legionellae which were persistently detected in the water used to supply the therapeutic spa. The hyperchlorination of the water distribution system was performed with a free residual chlorine concentration of 50 mg/l for 4h. An initial reduction in the numbers of legionellae was determined, but it rapidly increased to the original values. A UV unit was installed at the entrance to the spa's water distribution system and a new hyperchlorination procedure was performed immediately before UV irradiation (40 mJ/cm(2)), which was in use continuously for a nine-month period. While legionellae were not recovered during the irradiation period, these organisms were detected immediately after deactivation of the UV unit. We demonstrated that UV disinfection provides effective control of Legionella spp., with the advantage of being a method that, unlike chemical disinfectants, does not affect the physicochemical composition of the water. These findings suggest that UV irradiation, applied at key points in therapeutic spas, can be used to control colonization of water distribution systems by Legionella spp.

Recreational waters are a source of infection by several microorganisms causing acute gastrointestinal, cutaneous and respiratory illnesses. Cryptosporidium, noroviruses and enteropathogenic Escherichia coli strains are the most important causes of diarrhea, while Pseudomonas and Staphylococcus aureus are the main causes of cutaneous infections, and Legionella is the major cause of acute lower respiratory disease. Approximately 90% of outbreaks occur in treated recreational waters (swimming pools, spas and recreational parks), while the remaining 10% arise from natural waters used for leisure (bathing in rivers, beaches, etc). In spas, most infections are caused by thermophilic bacteria, such as Pseudomonas and Legionella, since overgrowth of these bacteria is facilitated by the direct effect of temperature and, indirectly, by the evaporation of the disinfectant. Outbreaks related to recreational waters usually reflect deficient control of the system: a low level of disinfectant, or the use of an inappropriate disinfectant, insufficient maintenance and cleaning of the installation, higher than recommended usage, and failure of the disinfectant dosage system. The correct design, maintenance and use of these facilities drastically lower the risk of infections from recreational waters. Thus, other key actions to minimize this risk are the existence of, and compliance with, regulatory rules, as well as educational campaigns on good hygiene practices directed at users. Rapid etiologic diagnosis of affected patients, together with an epidemiological survey and detection of the pathogen implicated in water samples are the keys to outbreak control.