Legionella Pneumophila In Water Samples Research Articles

Concordance between IDEXX Legiolert® (liquid culture assay) and plate culture (ISO 11731:2017) for the detection and quantification of Legionella pneumophila in water samples

Legionella pneumophila is a water-borne bacterium that can cause Legionnaires' disease. Legiolert® (IDEXX, USA) is a low-labour liquid culture assay for the detection and enumeration of L.pneumophila (SG1-15) from water. To analyse concordance between Legiolert and ISO 11731:2017 plate culture method (membrane filtration and culture on selective agars) using hospital water samples (N = 100). Incubation was at 39°C and 36°C, respectively, for seven days, followed by most-probable enumeration for Legiolert and subculturing and serogrouping of suspected Legionella colonies, with plate culture. L.pneumophila (SG1-15) was isolated from 25 out of 100 samples when using Legiolert or plate culture. Fourteen additional Legiolert samples tested positive for L.pneumophila; analysis of the same samples by plate culture was negative (12 out of 14) or yielded only Legionella rubrilucens (two out of 14; confirmed via matrix-assisted ionization/desorption time-of-flight mass spectrometry). L.pneumophila was not captured from Quanti-Tray/Legiolert pouch wells of these positive samples after subculture of puncture aliquots on buffered charcoal yeast-extract agar. Both methods in concordance did not detect L.pneumophila in 61 out of 100 samples. Legiolert and plate culture are both satisfactory methods to detect L.pneumophila from water samples, and both to detect isolated L.pneumophila in 25% of the sample population. Legiolert provides a faster time to result, and is less resource-demanding and labour-intensive; however, there may be a low risk of cross-reactivity with other organisms. Both methods are suitable for the analysis of water in healthcare settings, where the monitoring of L.pneumophila is imperative in preventing cases of Legionnaires' disease.

Design of an optical nanobiosensor for detection of Legionella pneumophila in water samples.

Legionella spp. is a causative agent of Legionnaires' disease that creates public health problems. Isolation of these bacteria from water sources is essential to identify outbreak origins and prevent disease. Diagnostic biosensors for water quality control to protect consumers from water-borne infections can predict many outbreaks. Gold nanoparticles conjugated probes are a new generation of diagnostic tools. In this study, an optical nano biosensor was designed and characterized to detect Legionella pneumophila in water samples rapidly. Thiolated probes designed for the mip gene were attached to gold nanoparticles and then water samples containing Legionella pneumophila were examined. The limit of detection for PCR and biosensor was 104 and 103 copy numbers/μl, respectively. Biosensor sensitivity and PCR were reported to be 90% (18 out of 20) and 85% (17 out of 20), respectively. Specificity 100% has been reported for both methods. According to the obtained results, this method has the potential to diagnose L. pneumophila with high sensitivity and specificity. This system can be employed as a practical tool for rapid, accurate, high-sensitivity, and acceptable detection of Legionella pneumophila in contaminated water, which is cost-effective in terms of cost and time.

Factors Influencing Legionella Contamination of Domestic Household Showers.

Legionnaires’ disease is a potentially fatal pneumonia like infection caused by inhalation or aspiration of water particles contaminated with pathogenic Legionella spp. Household showers have been identified as a potential source of sporadic, community-acquired Legionnaires’ disease. This study used qPCR to enumerate Legionella spp. and Legionella pneumophila in water samples collected from domestic showers across metropolitan Adelaide, South Australia. A survey was used to identify risk factors associated with contamination and to examine awareness of Legionella control in the home. The hot water temperature was also measured. A total of 74.6% (50/68) and 64.2% (43/68) showers were positive for Legionella spp. and L. pneumophila, respectively. Statistically significant associations were found between Legionella spp. concentration and maximum hot water temperature (p = 0.000), frequency of shower use (p = 0.000) and age of house (p = 0.037). Lower Legionella spp. concentrations were associated with higher hot water temperatures, showers used at least every week and houses less than 5 years old. However, examination of risk factors associated with L. pneumophila found that there were no statistically significant associations (p > 0.05) with L. pneumophila concentrations and temperature, type of hot water system, age of system, age of house or frequency of use. This study demonstrated that domestic showers were frequently colonized by Legionella spp. and L. pneumophila and should be considered a potential source of sporadic Legionnaires’ disease. Increasing hot water temperature and running showers every week to enable water sitting in pipes to be replenished by the municipal water supply were identified as strategies to reduce the risk of Legionella in showers. The lack of public awareness in this study identified the need for public health campaigns to inform vulnerable populations of the steps they can take to reduce the risk of Legionella contamination and exposure.

New system for the detection of Legionella pneumophila in water samples

Waterborne pathogens are a global concern for public health worldwide. Despite continuing efforts to maintain water safety, water quality is still affected by deterioration and pollution. Legionella pneumophila colonizes man-made water systems and can infect humans causing Legionnaire's disease (LD), pneumonia. The prevention of LD is a public health issue and requires specific systems to control and detect these microorganisms. Culture plate is the only technique currently approved, but requires more than 10 days to obtain results. A rapid test that inform in hours about the presence of Legionella pneumophila in water samples will improve the control of this pathogen colonization. In order to control colonization by L. pneumophila we developed a membrane filter method to capture and immunodetect this microorganism in water samples. This membrane filter is used to retain the bacteria using a nitrocellulose disc inside a home-made cartridge. Subsequently we perform the immunodetection of the bacteria retained in the nitrocellulose (blocking, antibody incubation, washings and developing). On comparing our test with the gold-standard, the most important finding is the considerably reduction in time maintaining the same detection limit. This rapid test is easily automated for L. pneumophila detection allowing a comprehensive surveillance of L. pneumophila in water facilities and reducing the variability in the analyses due to the low need for manipulation. Moreover, corrective measures may be applied the same day of the analysis.This method considerably reduces the detection time compared with the conventional, gold-standard detection culture method that requires more than 10 days, being decisive to prevent outbreaks.

Detection of Legionella spp. and Legionella pneumophila in water samples of Spain by specific real-time PCR

Legionella pneumophila is the primary cause of the legionellosis diseases (90%) (Yu et al. in J Infect Dis 186:127-128, 2002; Doleans et al. in J Clin Microbiol 42:458-460, 2004; Den Boer et al. in Clin Microbiol Infect 14:459-466, 2008). In this study, methodologies based on molecular biology were developed in order to provide a quick diagnosis of the bacterial presence in water samples of Spain. Multiplex real-time polymerase chain reaction assays were realized to target the 16S rRNA and macrophage infectivity potentiator (mip) genes of, respectively, Legionella spp. and L. pneumophila including in the design of an internal control. The results obtained by the culture and the gene amplification methods agreed in 94.44% for the 16S rRNA gene, and a concordance of 66.67% of the cases was obtained for the mip gene.

Fast immunosensing technique to detect Legionella pneumophila in different natural and anthropogenic environments: comparative and collaborative trials

BackgroundLegionellosis is an uncommon form of pneumonia. After a clinical encounter, the necessary antibiotic treatment is available if the diagnosis is made early in the illness. Before the clinical encounter, early detection of the main pathogen involved, Legionella pneumophila, in hazardous environments is important in preventing infectious levels of this bacterium. In this study a qualitative test based on combined magnetic immunocapture and enzyme-immunoassay for the fast detection of Legionella pneumophila in water samples was compared with the standard method, in both comparative and collaborative trials. The test was based on the use of anti-Legionella pneumophila antibodies immobilized on magnetic microspheres. The final protocol included concentration by filtration, resuspension and immunomagnetic capture. The whole assay took less than 1 hour to complete.ResultsA comparative trial was performed against the standard culture method (ISO 11731) on both artificially and naturally contaminated water samples, for two matrices: chlorinated tap water and cooling tower water. Performance characteristics of the test used as screening with culture confirmation resulted in sensitivity, specificity, false positive, false negative, and efficiency of 96.6%, 100%, 0%, 3.4%, and 97.8%, respectively. The detection limit at the level under which the false negative rate increases to 50% (LOD50) was 93 colony forming units (CFU) in the volume examined for both tested matrices. The collaborative trial included twelve laboratories. Water samples spiked with certified reference materials were tested. In this study the coincidence level between the two methods was 95.8%.ConclusionResults demonstrate the applicability of this immunosensing technique to the rapid, simple, and efficient detection of Legionella pneumophila in water samples. This test is not based on microbial growth, so it could be used as a rapid screening technique for the detection of L. pneumophila in waters, maintaining the performance of conventional culture for isolation of the pathogen and related studies.

SPR based immunosensor for detection of Legionella pneumophila in water samples

Detection of legionellae by water sampling is an important factor in epidemiological investigations of Legionnaires' disease and its prevention. To avoid labor-intensive problems with conventional methods, an alternative, highly sensitive and simple method is proposed for detecting L. pneumophila in aqueous samples. A compact Surface Plasmon Resonance (SPR) instrumentation prototype, provided with proper microfluidics tools, is built. The developed immunosensor is capable of dynamically following the binding between antigens and the corresponding antibody molecules immobilized on the SPR sensor surface. A proper immobilization strategy is used in this work that makes use of an important efficient step aimed at the orientation of antibodies onto the sensor surface. The feasibility of the integration of SPR-based biosensing setups with microfluidic technologies, resulting in a low-cost and portable biosensor is demonstrated.

Rapid and quantitative detection of Legionella pneumophila applying immunomagnetic separation and flow cytometry

Legionella is a pathogenic bacterium that establishes and proliferates well in water storage and distribution systems. Worldwide it is responsible for numerous outbreaks of legionellosis, which can be fatal. Despite recent advances in molecular and immunological methods, the official, internationally accepted detection method for Legionella spp. in water samples (ISO 11371) is still based on cultivation. This method has major disadvantages such as a long assay time of 10 days and the detection of cultivable cells only. Therefore, we developed a cultivation-independent, quantitative, and fast detection method for Legionella pneumophila in water samples. It consists of four steps, starting with (1) a concentrating step, in which cells present in one litre of water are concentrated into 5 ml by filtration (pore size 0.45 microm), (2) then cells are resuspended with sterile filtered buffer and double-stained with FITC- and Alexa-conjugated Legionella-specific antibodies, (3) subsequently, the cells are immunomagnetically caught, and (4) finally, fluorescently labeled Legionella cells were flow cytometrically detected and quantified. The efficiency of each step was tested separately. The whole method allows detection of L. pneumophila in 180 min with a detection limit of around 500 cells/l and a recovery of Legionella cells of 52.1 % out of spiked tap water. Fluorescence microscopy and flow cytometric cell-counting correlated well.

The rapid and specific real-time detection of Legionella pneumophila in water samples using Optical Waveguide Lightmode Spectroscopy

The detection of Legionella pneumophila in water samples using standard microbiological culture techniques is both prolonged and problematic. The bacterium is slow-growing and nutritionally fastidious, such that other indigenous species can out-compete the Legionella even when using antibiotic supplemented media. Optical Waveguide Lightmode Spectroscopy (OWLS) is a real-time analytical system whereby a change to a higher coupling angle where the refractive index of a bacterial cell is higher than that of the covering medium. In this study an aqueous suspension of L. pneumophila was passed across the surface of waveguides functionalised with a specific anti- Legionella antibody. The binding between the bacterial cells and the antibody specific for that cell resulted in an increase in the refraction indices of the transverse electric and transverse magnetic photoelectric currents. We report the optimisation of a rapid and sensitive (1.3 × 10 4 CFU mL − 1 ) detection method for L. pneumophila contamination in a water sample in less than 25 min. This is a significant reduction in the time taken to determine the presence of the bacterium which with conventional techniques normally takes up to fourteen days. In addition, the specificity of the technique to L. pneumophila was demonstrated. The OWLS results were validated by conventional microbiology screening and atomic force microscopy of the surface of the waveguide, showing its species specificity and potential applications in environmental and clinical analysis.

Detection of Escherichia coli O157:H7, Salmonella typhimurium, and Legionella pneumophila in Water Using a Flow-Through Chemiluminescence Microarray Readout System

Fast, sensitive, and especially, multianalyte test systems are currently of high interest for the monitoring and quality control of drinking water, since traditional microbiological methods are labor intensive and can take days until a response is achieved. In this study, the first flow-through chemiluminescence microarray was developed and characterized for the rapid and simultaneous detection of Escherichia coli O157:H7, Salmonella typhimurium, and Legionella pneumophila in water samples using a semiautomated readout system. Therefore, antibody microarrays were produced on poly(ethylene glycol)-modified glass substrates by means of a contact arrayer. For capturing bacteria, species-specific polyclonal antibodies were used. Cell recognition was carried out by binding of species-specific biotinylated antibodies. Chemiluminescence detection was accomplished by a streptavidin-horseradish peroxidase (HRP) catalyzed reaction of luminol and hydrogen peroxide. The chemiluminescence reaction that occurred was recorded by a sensitive charge-coupled device (CCD) camera. The overall assay time was 13 min, enabling a fast sample analysis. In multianalyte experiments, the detection limits were 3 x 10(6), 1 x 10(5), and 3 x 10(3) cells/mL for S. typhimurium, L. pneumophila, and E. coli O157:H7, respectively. Quantification of samples was possible in a wide concentration range with good recoveries. The presented system is well suited for quick and automatic water analysis.

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.

Development and evaluation of a Taqman duplex real-time PCR quantification method for reliable enumeration of Legionella pneumophila in water samples

This study describes the development and evaluation of a specific Legionella pneumophila Taqman duplex real-time PCR (qPCR) for fast and reliable quantification of this human pathogen in suspected man-made water systems. The qPCR assay was 100% specific for all L. pneumophila serogroups 1–15 with a sensitivity of 60 genome units/l and an amplification efficiency of 98%. Amplification inhibitors were detected via an exogenous internal positive control, which was amplified simultaneously with L. pneumophila DNA using its own primer and probe set. Mean recovery rates of the qPCR assay for tap water and cooling circuit water, spiked with a known number L. pneumophila bacteria, were 93.0% and 56.3%, respectively. Additionally, by using the Ultraclean™ Soil DNA isolation kit, we were able to remove amplification inhibitors ubiquitously present in cooling water. The practical value of our qPCR assay was evaluated through analysis of 30 water samples from showers, taps, eyewash stations, fire sprinklers and recirculation loops with qPCR and traditional culture. In conclusion, the described L. pneumophila Taqman duplex real-time assay proved to be specific, sensitive and reproducible. This makes it a promising method complementing the current time-consuming culture standard method.

Detection of Legionella pneumophila in water samples by species-specific real-time and nested PCR assays

Legionella pneumophila is a contaminant of man-made water systems, including potable water, cooling towers, water systems of large buildings, etc. It is the most common causative agent of legionellosis, a respiratory infection, which may give rise to restricted outbreaks. To survey environmental water samples from hospitals and private habitations in Bologna, we developed a species-specific nested and a TaqMan real-time PCR for the detection of L. pneumophila. We compared the two assays and both to cultural isolation. The targeted gene was macrophage infectivity potentiator (mip), conserved in L. pneumophila, and divergent in other legionellae. One assay was based on a nested PCR and the other on a TaqMan real-time PCR protocol. Their sensitivities were 14 % or 5% higher than that of cultural isolation respectively. The detection limits were 1-2 genome equivalents per 50-microl reaction. Specificity was assessed using DNA from nine target and 20 nontarget organisms. When applied to water samples, both assays detected L. pneumophila at 80% or higher frequency. The species-specific molecular diagnosis of L. pneumophila by means of nested PCR does not require a specific instrumentation, exhibits a high sensitivity, and is advantageous over the cultural isolation and real-time PCR detection. It allows to quickly monitor water samples for the risk assessment of environmental contaminations.

Detection of Legionella pneumophila in water samples by quantitative culture and an antigen detection assay

We evaluated the new Legionella pneumophila antigen detection assay Binax EquateTM for quantitative determination of legionellae in potable water samples. Seventy-seven water samples from different sources were investigated by Binax EquateTM and quantitative culture. Our culture assay is able to detect 20 to 40 cfu per 100 ml water. The rates of detection of legionellae were 1% (1 of 77) for the antigen detection assay and 25% (19 of 77) by culture. We were able to detect antigen in one water sample with 28 cfu per ml L. pneumophila serogroup 1. In in-vitro experiments the antigen assay had a sensitivity of about 333 cfu per ml when the bacteria were added directly to the test tubes and about 1000 cfu per ml when a simulated water sample was investigated. None of the water samples positive for L. pneumophila serogroup 2 to 14 was positive in the Binax EquateTM. The new antigen assay proved to be a valuable tool for investigating heavy L. pneumophila Serogroup 1 contamination in potable water systems but lacks sufficient sensitivity to be used in the surveillance of water supplies.