Legionella In Water Samples Research Articles

Ethambutol inhibited the growth of acid-fast bacteria and enhanced the detection of Legionella in environmental water samples.

The growth of acid-fast bacteria often hinders the detection of Legionella in water samples on agar plates by the plate culture method. We studied whether anti-tubercular agents inhibit acid-fast bacteria growth on agar plates. First, the antimicrobial activities of isoniazid, ethionamide, and ethambutol were evaluated against Mycobacterium and Legionella. We found that ethambutol at ≥ 100 μg/mL completely inhibited Mycobacterium growth, but ethambutol at 1,000 μg/mL did not inhibit Legionella growth. Next, the effect of ethambutol dissolved in acid buffer was examined. Cell suspensions of L. pneumophila and Mycobacterium spp. were mixed, and ethambutol-acid buffer was added. After 5 min, mixtures were inoculated on GVPC agar plates and incubated at 36℃ for 6 d. We found that ethambutol inhibited Mycobacterium growth on agar plates, but the Legionella colonies recovered. The effect of ethambutol was also significant in the evaluation using bathwaters. Comparing 1,302 bathwaters, the addition of ethambutol reduced the detection rate of acid-fast bacteria from 30.6% to 0% and increased the detection rate of Legionella from 7.1% to 7.5%. Ethambutol, which selectively inhibited acid-fast bacteria growth, enhanced the detection of Legionella on agar plates and will contribute to improving the accuracy of Legionella testing by the plate culture method.

Environmental Monitoring of Legionella in Hospitals in the Campania Region: A 5-Year Study

Legionella is a pathogen that colonizes soils, freshwater, and building water systems. People who are most affected are those with immunodeficiencies, so it is necessary to monitor its presence in hospitals. The purpose of this study was to evaluate the presence of Legionella in water samples collected from hospitals in the Campania region, Southern Italy. A total of 3365 water samples were collected from January 2018 to December 2022 twice a year in hospital wards from taps and showers, tank bottoms, and air-treatment units. Microbiological analysis was conducted in accordance with the UNI EN ISO 11731:2017, and the correlations between the presence of Legionella and water temperature and residual chlorine were investigated. In total, 708 samples (21.0%) tested positive. The most represented species was L. pneumophila 2-14 (70.9%). The serogroups isolated were 1 (27.7%), 6 (24.5%), 8 (23.3%), 3 (18.9%), 5 (3.1%), and 10 (1.1%). Non-pneumophila Legionella spp. represented 1.4% of the total. Regarding temperature, the majority of Legionella positive samples were found in the temperature range of 26.0-40.9 °C. An influence of residual chlorine on the presence of the bacterium was observed, confirming that chlorine disinfection is effective for controlling contamination. The positivity for serogroups other than serogroup 1 suggested the need to continue environmental monitoring of Legionella and to focus on the clinical diagnosis of other serogroups.

Legionella Species Isolated in the Water Systems of Ships and Their Molecular Characterization

Legionella species are generally found in nature and in water resources, and they are gram negative bacilli that can cause pneumonia by being transmitted from water systems to humans via aerosol or aspiration. Legionnaires' disease caused by this agent continues to be a public health problem in cruise ships. In this study, it was aimed to determine the prevalence of the colonization of Legionella species by culture method and to determine the molecular characterization of the isolated Legionella in water samples taken from the water systems of the ships docking in Mersin International Port. A total of 158 cold water samples were taken from 18 ferry and/or cargo ships docking in Mersin International Port between December 2014 and June 2015. Fifty-four of the samples were obtained from tanks, 68 from taps and 36 from shower heads. All samples were centrifuged and inoculated from the pellet onto "Buffered Coal Yeast Extract" (BCYE) (Oxoid, CM0655, UK) agar medium supplemented with iron pyrophosphate, L-cysteine and α-ketoglutarate (Oxoid, SR0110, UK). The culture plates were incubated for 10-15 days in microaerophilic environment in a desiccator at 37°C. The suspicious colonies grown in cultures were serogrouped by latex agglutination test (Oxoid, DR0800M, UK) and fluorescent antibody method (m-Tech Monoclonal Technologies, Inc., USA). For the molecular analysis of Legionella species grown in culture, DNA isolation was made from Legionella colonies and then polymerase chain reaction amplification was performed using specific primer sequences targeting the rpoB gene region of the Legionella genome. Direct DNA sequencing of rpoB gene products was performed in the "ABI PRISM 3130XL Genetic Analyzer" (Applied Biosystems, USA). The DNA sequences were typed by BLAST analysis and the determined types, and NCBI (National Center for Biotechnology Information) reference Legionella sequences were phylogenetically compared with the Neighbor-Joining comparison method by using the Mega 7 program. Legionella spp. was isolated in 18 (11.4%) of 158 samples. Of these, four (7.4%, 4/54) were detected from the tank, 11 (16.2%, 11/68) from the tap and three (8.33%, 3/36) from the shower head. After the latex agglutination test performed from the growing bacterial colonies, five (27.8%) were serogrouped as Legionella spp., four (22.2%) as Legionella pneumophila sg 5, two (11.1%, each) as L.pneumophila sg 1,L.pneumophila sg 8 and Legionella bozemanii and one (5.6%) as L.pneumophila sg 3. Two (11.1%) of the isolates grown in culture could not be serogrouped. Molecular characterization of 12 Legionella isolates could be performed. One of them was serologically serogrouped as L.bozemanii, and it was found to be 99% similar to Legionella rubrilucens when compared with NCBI Legionella sequence data in the BLAST program. One isolate that could not be differentiated by serogrouping was identified as Legionella erytra in the BLAST program after DNA sequence analysis. The remaining 10 isolates (55.6%, n= 18) were confirmed as L.pneumophilia after the comparison with reference NCBI sequences. In this study, it was determined that 11.4% of the water samples collected from the water systems of the ships docking in Mersin International Port were contaminated with Legionella species. The detected Legionella species have an important potential source of infection for the captain, ship workers and passengers travelling on the ships. In this respect, this study reveals the necessity of establishing studies to improve the risk management of Legionella in the water systems of ships.

Comparative study of Legiolert with ISO 11731-1998 standard method-conclusions from a Public Health Laboratory

BackgroundLegionella pneumophila (L. pneumophila) is responsible for 96% of Legionnaires' disease (LD) and 10% of all worldwide pneumonia cases. Legiolert™, a liquid culture method for most probable number (MPN) enumeration of L. pneumophila, was developed by IDEXX Laboratories. The method detects all serogroups of L. pneumophila in potable and non-potable water samples. ObjectiveThe goal of this study is to establish that Legiolert is a suitable alternative method to meet testing requirements in Spain for the enumeration of Legionella in water samples. MethodologyThe laboratory analyzed 118 environmental water samples from the Barcelona region (56 potable and 62 non-potable) in parallel by the Standard method for detection and enumeration of Legionella (ISO 11731:1998) and by Legiolert. Comparison of the recovery of the alternative method (Legiolert) and the Standard was made using ISO 17994:2014 and McNemar's binomial test statistical methods. Results44 samples were positive for Legionella (36 potable and 8 non-potable). Legiolert and the Standard method detected a similar percentage of positive samples, with Legiolert being slightly higher (31 vs 30%) and detecting higher concentrations of Legionella within the samples. ISO 17994:2014 analysis of the potable water samples found Legiolert was more sensitive than the Standard at detecting Legionella, even when complete Legionella species (L. spp.) results were considered for both methods. The two methods also demonstrated equivalent detection of L. spp. according to the McNemar's test. The comparison is significantly more in favor of Legiolert when only L. pneumophila results are considered. Each confirmation run with material extracted from positive Legiolert wells contained L. pneumophila, giving the method a specificity of 100%. Although statistical results for non-potable waters are not included because of the low number of samples, the two methods trended towards equivalence. ConclusionsRelative to the Standard method, Legiolert has a greater sensitivity and selectivity, and appears to have higher recovery for L. pneumophila, and equivalent recovery when L. spp. is included in the comparison. Legiolert also has high specificity. The procedural advantages of Legiolert allow laboratories to save on resources, costs, and time and consequently to test more frequently. In conclusion, the study finds IDEXX Legiolert a suitable alternative to ISO 11731:1998.

Are pathogenic Legionella non-pneumophila a common bacteria in Water Distribution Networks?

The present study analyzes at the national level, the presence of circulating Legionella in the artificial aquatic systems of different facilities of all of them state-owned centers throughout Spain for 12 months. 1754 water samples from various state-owned centers were collected from January to December 2014. Samples were collected from the cooling towers and evaporative condensers (CTC), and water distribution networks such as domestic hot water (DHW), cold water for human consumption (CW), sprinkler irrigation systems (SIS), fire sprinkler systems (FSS), and water from decorative fountains (DF). All these facilities are considered, according to current regulations, as potential amplifying systems for bacteria and possible sources of infection by the generation of droplets and aerosols. The isolation and counting of Legionella in water samples was carried out using microbiological culture following the international normative UNE-EN-ISO 11,731:2007 (ISO 11,731:1998) and UNE-EN ISO 8199:2008 (ISO 8199:2005).The quantification of Legionella colonization, the annual distribution, and the geographical distribution of the Legionella isolates recovered in the water were analyzed. Besides, molecular techniques were used for the characterization of the Legionella non-pneumophila isolates. Legionella was recovered from 15.79% of the analyzed water samples. High colonization was more frequently detected in water samples from CTC, DHW, CW, and DF. Regarding the geographic distribution, positive samples of Legionella were obtained in 14 of the 18 Spanish locations analyzed. Legionella non-pneumophila was the most prevalent and was isolated from water samples from 13 different geographical locations (72%). Legionella anisa and Legionella jordanis were the most frequently non-pneumophila species isolated. Legionella donaldsonii was isolated for the first time in the water distribution networks in Spain. Legionella pneumophila sg 2–14 was detected in 13 locations and Legionella pneumophila sg 1 in 11 locations. Therefore, our study concludes that the presence of Legionella pneumophila and Legionella non-pneumophila species in these systems can be a potential threat to public health and should be examined thoroughly with complementary techniques, such as molecular techniques as a screen for routine diagnosis.

Performance of Legiolert Test vs. ISO 11731 to Confirm Legionella pneumophila Contamination in Potable Water Samples.

Detection and enumeration of Legionella in water samples is of great importance for risk assessment analysis. The plate culture method is the gold standard, but has received several well-known criticisms, which have induced researchers to develop alternative methods. The purpose of this study was to compare Legionella counts obtained by the analysis of potable water samples through the plate culture method and through the IDEXX liquid culture Legiolert method. Legionella plate culture, according to ISO 11731:1998, was performed using 1 L of water. Legiolert was performed using both the 10 mL and 100 mL Legiolert protocols. Overall, 123 potable water samples were analyzed. Thirty-seven (30%) of them, positive for L. pneumophila, serogroups 1 or 2–14 by plate culture, were used for comparison with the Legiolert results. The Legiolert 10 mL test detected 34 positive samples (27.6%) and the Legiolert 100 mL test detected 37 positive samples, 27.6% and 30% respectively, out of the total samples analyzed. No significant difference was found between either the Legiolert 10 mL and Legiolert 100 mL vs. the plate culture (p = 0.9 and p = 0.3, respectively) or between the Legiolert 10 mL and Legiolert 100 mL tests (p = 0.83). This study confirms the reliability of the IDEXX Legiolert test for Legionella pneumophila detection and enumeration, as already shown in similar studies. Like the plate culture method, the Legiolert assay is also suitable for obtaining isolates for typing purposes, relevant for epidemiological investigations.

Impact of temperature on Legionella pneumophila, its protozoan host cells, and the microbial diversity of the biofilm community of a pilot cooling tower

Legionella pneumophila is a waterborne bacterium known for causing Legionnaires' Disease, a severe pneumonia. Cooling towers are a major source of outbreaks, since they provide ideal conditions for L. pneumophila growth and produce aerosols. In such systems, L. pneumophila typically grow inside protozoan hosts. Several abiotic factors such as water temperature, pipe material and disinfection regime affect the colonization of cooling towers by L. pneumophila. The local physical and biological factors promoting the growth of L. pneumophila in water systems and its spatial distribution are not well understood. Therefore, we built a lab-scale cooling tower to study the dynamics of L. pneumophila colonization in relationship to the resident microbiota and spatial distribution. The pilot was filled with water from an operating cooling tower harboring low levels of L. pneumophila. It was seeded with Vermamoeba vermiformis, a natural host of L. pneumophila, and then inoculated with L. pneumophila. After 92 days of operation, the pilot was disassembled, the water was collected, and biofilm was extracted from the pipes. The microbiome was studied using 16S rRNA and 18S rRNA genes amplicon sequencing. The communities of the water and of the biofilm were highly dissimilar. The relative abundance of Legionella in water samples reached up to 11% whereas abundance in the biofilm was extremely low (≤0.5%). In contrast, the host cells were mainly present in the biofilm. This suggests that L. pneumophila grows in host cells associated with biofilm and is then released back into the water following host cell lysis. In addition, water temperature shaped the bacterial and eukaryotic community of the biofilm, indicating that different parts of the systems may have different effects on Legionella growth.

Environmental Surveillance of Legionellosis within an Italian University Hospital-Results of 15 Years of Analysis.

Legionnaires’ disease is normally acquired by inhalation of legionellae from a contaminated environmental source. Water systems of large and old buildings, such as hospitals, can be contaminated with legionellae and therefore represent a potential risk for the hospital population. In this study, we demonstrated the constant presence of Legionella in water samples from the water system of a large university hospital in Messina (Sicily, Italy) consisting of 11 separate pavilions during a period of 15 years (2004–2018). In total, 1346 hot water samples were collected between January 2004 and December 2018. During this period, to recover Legionella spp. from water samples, the standard procedures reported by the 2000 Italian Guidelines were adopted; from May 2015 to 2018 Italian Guidelines revised in 2015 (ISS, 2015) were used. Most water samples (72%) were positive to L. pneumophila serogroups 2–14, whereas L. pneumophila serogroup 1 accounted for 18% and non-Legionella pneumophila spp. Accounted for 15%. Most of the positive samples were found in the buildings where the following critical wards are situated: (Intensive Care Unit) ICU, Neurosurgery, Surgeries, Pneumology, and Neonatal Intensive Unit Care. This study highlights the importance of the continuous monitoring of hospital water samples to prevent the potential risk of nosocomial legionellosis.

Legionella pneumophila in Thermal Spa – Methodological Aspects of Sampling and Analysis

The aim of the study was to check the occurrence of Legionella pneumophila in thermal water and to compare different analytical methods of its determination and/or quantification used in different laboratories. The research has shown that there are noticeable differences between results from culture method, qPCR, and FISH. Significant influence on the results has got analysts qualification, samples preparation, and equipment parameters. Very important is also the homogeneousness of the samples. qPCR or FISH method can be used as a preliminary, quick study, for identification of Legionella in water samples but the results obtained should be confirmed by culture method.

A novel method for a quick detection of legionella in water samples

A novel method for a quick detection of legionella in water samples

Presencia de Legionella spp. en depósitos domiciliarios de agua potable en Resistencia, Chaco, Argentina. Informe preliminar

Legionella spp. is an environmental bacterium that can survive in a wide range of physicochemical conditions and may colonize distribution systems of drinking water and storage tanks. Legionella pneumophila is the major waterborne pathogen that can cause 90% of Legionnaires’ disease cases. The aim of this study was to detect the presence of Legionella spp. in household drinking water tanks in the city of Resistencia, Chaco. The detection of Legionella in water samples was performed by culture methods as set out in ISO 11731:1998. Thirty two water samples were analyzed and Legionella spp. was recovered in 12 (37.5%) of them. The monitoring of this microorganism in drinking water is the first step towards addressing the control of its spread to susceptible hosts.

Legionella in water samples: How can you interpret the results obtained by quantitative PCR?

Evaluation of the potential risk associated with Legionella has traditionally been determined from culture-based methods. Quantitative polymerase chain reaction (qPCR) is an alternative tool that offers rapid, sensitive and specific detection of Legionella in environmental water samples.In this study we compare the results obtained by conventional qPCR (iQ-Check™ Quanti Legionella spp.; Bio-Rad) and by culture method on artificial samples prepared in Page's saline by addiction of Legionella pneumophila serogroup 1 (ATCC 33152) and we analyse the selective quantification of viable Legionella cells by the qPCR-PMA method.The amount of Legionella DNA (GU) determined by qPCR was 28-fold higher than the load detected by culture (CFU).Applying the qPCR combined with PMA treatment we obtained a reduction of 98.5% of the qPCR signal from dead cells. We observed a dissimilarity in the ability of PMA to suppress the PCR signal in samples with different amounts of bacteria: the effective elimination of detection signals by PMA depended on the concentration of GU and increasing amounts of cells resulted in higher values of reduction.Using the results from this study we created an algorithm to facilitate the interpretation of viable cell level estimation with qPCR-PMA.

English

Infections caused by Legionella spp. are considered an emerging public health problem and are linked to high rates of mortality and morbidity, if not properly treated. This study analyzes 76 samples of water in Guangzhou with the aim of obtaining a more efficient means based on culture to isolate the genus Legionella. A comparative assessment of techniques recommended was performed on monitoring Legionella spp. in environmental water samples. Fourteen (14) sampling methods included in four categories based on concentration (filtration or centrifugation), acid buffer treatment (no acid treatment, treatment for 10 min), thermal treatment (no thermal treatment or treated for 30 min in 50°C) and heat enrich the samples in 37°C for 72 h were tested to identify their sensitivity. The four categories methodologies were assembled by different procedures and 14 methods were obtained to compare the efficiency for isolating Legionella in 76 water samples and in which Legionella were isolated from 42 water samples through these 14 methods. Merit and demerit of each single method was evaluated. Results suggest that a single category methodology for water sampling was insufficient to isolate Legionella spp. from water samples. Some Legionella positive water samples were missed through a single category methodology which made the results mistranslation. PSF category methods in this study were recommended methods and they could isolate Legionella species from 78.6% Legionella positive water samples; and if time was enough, PSF category methods combined with ERCT category methods to isolate Legionella from water samples are strongly recommended. Legionella were isolated from 41 water samples through these two category methods, it only missed 2.4% positive water sample. The results obtained, highlighted the importance of improving culture method for the identification of Legionella in water samples.   Key words: Legionella, environmental water samples, sampling methods. &nbsp

Evaluation of a Legionella urinary antigen enzyme immunoassay for rapid detection of Legionella pneumophila in water samples

Despite advances in medium formulations and pretreatment techniques, recovery of Legionella from water samples can still be quite low, difficult and time consuming. The aim of this study was to evaluate the utility of a Legionella urinary antigen enzyme immunoassay (Bartels ELISA, Trinity Biotech, Ireland) for the detection of Legionella in water samples. Reference ATCC Legionella strains were used to spike water samples to a final concentration of 10 4–10 5 cfu/ml. The lower detection limit of the test for all Legionella pneumophila serogroups was assessed by serial dilutions of spiked water samples. Legionella antigen was detected in all filtered samples except for those spiked with L. bozemanii and L. longbeachae. The lower detection limit for soluble L. pneumophila serogroup 1 antigen was 780 cfu/ml. Bartels ELISA could be a useful method for antigen detection in water samples when a high recovery of L. pneumophila is suspected. The test could be used as a rapid screening method for the detection of Legionella in a large number of samples. However, the low sensitivity of the test requires to keep on performing conventional culture for isolation and for further studies on isolated bacteria.