scholarly journals Assessing the viability of Legionella pneumophila in environmental samples: regarding the filter application of Ethidium Monoazide Bromide

2021 ◽  
Vol 71 (1) ◽  
Author(s):  
Michela Consonni ◽  
Anna Grassi ◽  
Stefania Scuri ◽  
Maria Gori ◽  
Elisabetta Tanzi ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Water Samples ◽  
Environmental Samples ◽  
Pure Water ◽  
Dna Amplification ◽  
Culture Method ◽  
Ethidium Monoazide ◽  
Cultivable Bacteria ◽  
Source Of Infection ◽  
Commercial Kit

Abstract Purpose Analyses of 34 water samples from 13 healthcare structures revealed how culture method and quantitative PCR (qPCR) often differ in the detection of Legionella pneumophila (Lp). With these considerations in hand, culture method, PCR and Ethidium Monoazide Bromide (EMA) qPCR have all been compared in order to detect Lp in water samples, identify a method able to speed up the procedures, detect the “viable but not cultivable” bacteria (VBNC) and exclude non-viable bacteria using a commercial kit for extraction and amplification as well as modification of the protocol. Methods Pure water samples artificially spiked with viable, non-viable and VBNC Lp ATCC 33152 were analyzed using a commercial kit for both qPCR and EMA-qPCR, while ISO 11731-2-2004 was used for culture method. Results Only 35% (12/34) of the environmental samples were positive in both culture and qPCR methods. With regard to EMA-qPCR, results showed the absence of dye toxicity on viable and VBNC strains and an incomplete effectiveness on the non-viable ones. In both viable and VBNC strains, a decrease of bacterial DNA amplification was recorded as a function of sample dilution but not of EMA concentration. Conclusions Discrepancies between culture method and EMA-qPCR were observed and may be due to different causes such as membrane-dye interactions, presence of interfering compounds and the sensitivity of the kit used. Study significance and impact In the presence of one or more suspected cases of nosocomial legionellosis, the application of a rapid molecular method able to identify only the viable and VBNC Lp would be useful in order to quickly identify the source of infection and to intervene with sanitation treatments. However, seeing that in our experience EMA pretreatment on the filter membrane did not come up with the expected results, it would be necessary to proceed with other experiments and/or different dyes. Graphical Abstract

scholarly journals Assessment of Viability of Legionella Pneumophila in Environmental Samples: On Filter Application of Ethidium Monoazide Bromide

2021 ◽  
Author(s):  
Michela Consonni ◽  
Anna Grassi ◽  
Stefania Scuri ◽  
Maria Gori ◽  
Elisabetta Tanzi ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Water Samples ◽  
Environmental Samples ◽  
Dna Amplification ◽  
Culture Method ◽  
Environmental Water ◽  
Ethidium Monoazide ◽  
Cultivable Bacteria ◽  
Source Of Infection ◽  
Commercial Kit

Abstract Purpose: Culture method, Real-Time PCR (qPCR) and Ethidium Monoazide Bromide (EMA) qPCR have been compared in order to detect Legionella pneumophila (Lp) in water samples, to identify a method able to speed up the procedures, detect the “viable but not cultivable” bacteria (VBNC) and exclude dead bacteria using a commercial kit for extraction and amplification and modifying the protocol.Methods: Using these three methods, 34 environmental water samples and a series of samples artificially spiked with alive, dead and VBNC Lp ATCC 33152 were analysed. ISO 11731-2-2004 culture method was applied, whereas a commercial kit was selected for both qPCR and EMA qPCR pretreatment.Results: only 35% (12/34) of the environmental samples were positive in both culture and qPCR methods. With regard to EMA qPCR, results showed the absence of dye toxicity on viable and VBNC strains and an incomplete effectiveness on the dead ones. In both viable and VBNC strains a decrease of bacterial DNA amplification was recorded as a function of sample dilution but not of EMA concentration.Conclusions: Discrepancies between culture method and EMA-qPCR were observed and could be due to different causes as membrane-dye interactions, presence of interfering compounds and the relatively low sensitivity of the kit used.Significance and Impact of the Study: In presence of one or more suspected cases of nosocomial legionellosis, the application of a rapid molecular method able to identify only the viable and VBNC Lp would be useful in order to quickly identify the source of infection and to intervene with sanitation treatments. However, because in our experience EMA pretreatment on filter membrane has not given the expected results, it would be necessary to proceed with other experiments and different dyes.

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

Pathogens ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 690 ◽  
Author(s):  
Maria Scaturro ◽  
Matteo Buffoni ◽  
Antonietta Girolamo ◽  
Sandra Cristino ◽  
Luna Girolamini ◽  
...  
Keyword(s):  
Risk Assessment ◽  
Legionella Pneumophila ◽  
Water Samples ◽  
Gold Standard ◽  
Liquid Culture ◽  
Potable Water ◽  
Culture Method ◽  
Alternative Methods ◽  
Significant Difference

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.

scholarly journals Nosocomial legionella pneumonia: demonstration of potable water as the source of infection

1988 ◽  
Vol 101 (3) ◽  
pp. 647-654 ◽  
Author(s):  
B. Ruf ◽  
D. Schürmann ◽  
I. Horbrach ◽  
K. Seodel ◽  
H. D. Pohle
Keyword(s):  
Monoclonal Antibodies ◽  
Water Supply ◽  
Legionella Pneumophila ◽  
Water Samples ◽  
Potable Water ◽  
Supply System ◽  
University Hospital ◽  
Environmental Isolates ◽  
Legionella Pneumonia ◽  
Source Of Infection

SUMMARYFrom January 1983 until December 1985, 35 cases of sporadic nosocomial legionella pneumonia, all caused byLegionella pneumophila, were diagnosed in a university hospital.L. pneumophilaserogroup (SG) 1 was cultured from 12 of the 35 cases and compared to correspondingL. pneumophilaSG 1 isolates from water outlets in the patients' immediate environment by subtyping with monoclonal antibodies. The corresponding environmental isolates were identical to 9 out of 12 (75%) of those from the cases. However, even in the remaining three cases identical subtypes were found distributed throughout the hospital water supply. From the hospital water supply four different subtypes ofL. pneumophilaSG 1 were isolated, three of which were implicated in legionella pneumonia. Of 453 water samples taken during the study 298 (65.8%) were positive for legionellae. Species ofLegionellaother thanL. pneumophilahave not been isolated. This may explain the exclusiveness ofL.pneumophilaas the legionella pneumonia-causing agent. Our results suggest that the water supply system was the source of infection.

scholarly journals Helicobacter pullorum in broiler chickens and the farm environment: A one health approach

2019 ◽  
pp. 20-25
Author(s):  
Soe Soe Wai ◽  
Saleha Abdul-Aziz ◽  
Asinamai Athliamai Bitrus ◽  
Zakaria Zunita ◽  
Jalila Abu
Keyword(s):  
Food Chain ◽  
Water Samples ◽  
Environmental Samples ◽  
Broiler Chickens ◽  
One Health ◽  
House Flies ◽  
Source Of Infection ◽  
Negative Conclusion ◽  
Campylobacter Spp

Aim: This study aimed to investigate the occurrence of Helicobacter pullorum in broiler chickens and their farm environment. Materials and Methods: The ceca from 100 broiler chickens from ten farms were sampled from processing sites or markets. The cecal contents were aseptically collected from each cecum and cultured. The farms were visited, and environmental samples were collected which included water, house flies, floor swabs and soils in chicken houses. Results: H. pullorum was present in 51% of the broilers; 17.5% of the flies were found to carry H. pullorum and Campylobacter spp., 30% of house floors were positive, while all water samples were negative. Conclusion: Flies could have picked up the organisms from the chickens' feces and/or the environment of the chicken houses or they could be one of the sources in the spread of the organisms. This study also showed that broiler chickens are potential reservoirs for H. pullorum and may serve as a source of infection for humans through the food chain.

scholarly journals Contamination of Hospital Water Supplies in Gilan, Iran, withLegionella pneumophila, Escherichia coli,andPseudomonas aeruginosa

2015 ◽  
Vol 2015 ◽  
pp. 1-7 ◽  
Author(s):  
Masoumeh Ahmadi Jalali Moghadam ◽  
Hamidreza Honarmand ◽  
Sajad Asfaram Meshginshahr
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Legionella Pneumophila ◽  
Water Samples ◽  
Bacterial Contamination ◽  
Multiple Drug Resistance ◽  
Multiple Drug ◽  
Water Supplies ◽  
E Coli ◽  
Contamination Degree ◽  
Commercial Kit

This study is designed to determine the contamination degree of hospital water supplies withPseudomonas aeruginosa, Legionella pneumophila, andE. coliin Gilan, Iran. Samples were collected directly into sterile containers and concentrated by centrifuge. Half part of any sample transferred to yeast extract broth and the second part transferred to Trypticase Soy Broth and incubated for 3 days. DNA was extracted by using commercial kit. Four rounds of PCR were performed as follows: multiplex PCR for detectingPseudomonas aeruginosa, Integron 1, and Metallo-β-lactamases gene; PCR for detectingLegionella pneumophilaandmipgene separately; PCR for detectingE. coli; and another PCR for detecting whole bacterial presence. Contamination rates of cold, warm, and incubator water samples withP. aeruginosa, were 16.6%, 37.5%, and 6.8% consequently. Degrees of contamination withL. pneumophilawere 3.3%, 9.3%, and 10.9% and withE. coliwere zero, 6.2%, and zero. Total bacterial contamination of cold, warm, and incubator water samples was 93.3%, 84.4%, and 89.0% consequently. Metallo-β-lactamases gene was found in 20.0% of all samples. Contamination degree withP. aeruginosawas considerable and withL. pneumophilawas moderate. Metallo-β-lactamases gene was found frequently indicating widespread multiple drug resistance bacteria. We suggest using new decontamination method based on nanotechnology.

scholarly journals Clusters of Healthcare-Associated Legionnaires’ Disease in Two Hospitals of Central Greece

2018 ◽  
Vol 2018 ◽  
pp. 1-5 ◽  
Author(s):  
Maria A. Kyritsi ◽  
Varvara A. Mouchtouri ◽  
Anna Katsiafliaka ◽  
Foteini Kolokythopoulou ◽  
Elias Plakokefalos ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Environmental Samples ◽  
Distribution Systems ◽  
Water Distribution ◽  
Control Measures ◽  
Aerosol Therapy ◽  
Environmental Investigation ◽  
Source Of Infection ◽  
Legionnaires Disease ◽  
Healthcare Associated

Healthcare-associated Legionnaires’ disease often leads to fatal respiratory tract infection among hospitalized patients. In this report, three cases of Legionnaires’ disease among patients in two different hospitals (Hospital A and Hospital B) were investigated. After conducting an epidemiologic and environmental investigation, the water distribution systems (WDSs) were identified as the possible source of infection, as Legionella pneumophila serogroup 1 (Lp1) was isolated from both clinical and environmental samples. Patients received aerosol therapy with nebulizers during their hospitalization. Based on the results of the investigation, the hospitals’ infection control committees reviewed their policies for Legionnaires’ disease prevention and implemented control measures focusing on using sterile fluids for aerosol treatments.

scholarly journals Targeting Species-Specific Low-Affinity 16S rRNA Binding Sites by Using Peptide Nucleic Acids for Detection of Legionellae in Biofilms

2006 ◽  
Vol 72 (8) ◽  
pp. 5453-5462 ◽  
Author(s):  
Sandra A. Wilks ◽  
C. William Keevil
Keyword(s):  
16S Rrna ◽  
Legionella Pneumophila ◽  
Environmental Samples ◽  
Culture Method ◽  
Dna Probes ◽  
Culture Methods ◽  
Variable Regions ◽  
Standard Culture ◽  
Species Specific

ABSTRACT Using fluorescence in situ hybridization to detect bacterial groups has several inherent limitations. DNA probes are generally used, targeting sites on the 16S rRNA. However, much of the 16S rRNA is highly conserved, with variable regions often located in inaccessible areas where secondary structures can restrict probe access. Here, we describe the use of peptide nucleic acid (PNA) probes as a superior alternative to DNA probes, especially when used for environmental samples. A complex bacterial genus (Legionella) was studied, and two probes were designed, one to detect all species and one targeted to Legionella pneumophila. These probes were developed from existing sequences and are targeted to low-binding-affinity sites on the 16S rRNA. In total, 47 strains of Legionella were tested. In all cases, the Legionella spp. PNA probe labeled cells strongly but did not bind to any non-Legionella species. Likewise, the specific L. pneumophila PNA probe labeled only strains of L. pneumophila. By contrast, the equivalent DNA probes performed poorly. To assess the applicability of this method for use on environmental samples, drinking-water biofilms were spiked with a known concentration of L. pneumophila bacteria. Quantifications of the L. pneumophila bacteria were compared using PNA hybridization and standard culture methods. The culture method quantified only 10% of the number of L. pneumophila bacteria found by PNA hybridization. This illustrates the value of this method for use on complex environmental samples, especially where cells may be in a viable but noncultivable state.

scholarly journals Comparison of Updated Methods for Legionella Detection in Environmental Water Samples

2021 ◽  
Vol 18 (10) ◽  
pp. 5436
Author(s):  
Daniela Toplitsch ◽  
Sabine Platzer ◽  
Romana Zehner ◽  
Stephanie Maitz ◽  
Franz Mascher ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Water Samples ◽  
Limit Of Detection ◽  
Culture Method ◽  
Environmental Water ◽  
Microbial Flora ◽  
Culture Methods ◽  
Outbreak Investigations ◽  
Standard Culture ◽  
Selection Of

The difficulty of cultivation of Legionella spp. from water samples remains a strenuous task even for experienced laboratories. The long incubation periods for Legionellae make isolation difficult. In addition, the water samples themselves are often contaminated with accompanying microbial flora, and therefore require complex cultivation methods from diagnostic laboratories. In addition to the recent update of the standard culture method ISO 11731:2017, new strategies such as quantitative PCR (qPCR) are often discussed as alternatives or additions to conventional Legionella culture approaches. In this study, we compared ISO 11731:2017 with qPCR assays targeting Legionella spp., Legionella pneumophila, and Legionella pneumophila serogroup 1. In samples with a high burden of accompanying microbial flora, qPCR shows an excellent negative predictive value for Legionella pneumophila, thus making qPCR an excellent tool for pre-selection of negative samples prior to work-intensive culture methods. This and its low limit of detection make qPCR a diagnostic asset in Legionellosis outbreak investigations, where quick-risk assessments are essential, and are a useful method for monitoring risk sites.

Detection and quantification ofLegionella pneumophilain water samples using competitive PCR

2006 ◽  
Vol 52 (6) ◽  
pp. 584-590 ◽  
Author(s):  
Priscilla Declerck ◽  
Jonas Behets ◽  
Elke Lammertyn ◽  
Ilya Lebeau ◽  
Jozef Anné ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Water Samples ◽  
Cooling Tower ◽  
Tap Water ◽  
Cost Effective ◽  
Culture Method ◽  
Environmental Water ◽  
Competitive Pcr ◽  
Promising Alternative ◽  
Standard Culture

The presence of high levels of Legionella pneumophila in man-made aquatic systems correlates with the incidence of nosocomial Legionnaires' disease. This requires a rapid, reliable, and sensitive quantification of L. pneumophila concentrations in suspected water systems. In this research, a homologous competitor was developed and evaluated in a L. pneumophila competitive polymerase chain reaction (cPCR) to quantify this human pathogen in a quick, cost-effective, and reliable way. Accuracy of cPCR was evaluated by analyzing cooling tower and tap water samples spiked with known concentrations of L. pneumophila bacteria, in parallel with the standard culture method. Legionella pneumophila amounts detected and calculated from cPCR and culture correlated very well: r = 0.998, P = 0.002 for tap water and r = 0.990, P = 0.009 for cooling tower water. Nevertheless, for both kinds of water samples, mean numbers of L. pneumophila calculated from cPCR results were always higher than those obtained by culture. This study makes it clear that the rapid, sensitive, and cost-effective L. pneumophila cPCR is a promising alternative to the standard time-consuming culture method and expensive real-time PCR to enumerate L. pneumophila bacteria in environmental water samples.Key words: Legionella pneumophila, competitive PCR, cost-effective, cooling tower water, tap water, sensitive detection.

Validation of the Legipid® Bioalarm Legionella Assay

2012 ◽  
Vol 95 (5) ◽  
pp. 1440-1451 ◽  
Author(s):  
Guillermo Rodríguez Albalat ◽  
Begoña Bedrina Broch ◽  
Marisa Jiménez Bono
Keyword(s):  
Legionella Pneumophila ◽  
Water Samples ◽  
False Positive Rate ◽  
False Negative ◽  
False Negative Rate ◽  
Reference Method ◽  
Culture Method ◽  
Magnetic Microspheres ◽  
Test Volume ◽  
Significant Difference

Abstract Legipid® Bioalarm Legionella is a test based on combined magnetic immunocapture and enzyme-immunoassay (CEIA) for the detection of Legionella pneumophila in water. Anti-L. pneumophila antibodies are immobilized on magnetic microspheres. Immunomagnetic analysis is applied to preconcentrated water samples in a final test volume of 9 mL. The method was compared with the standard culture method on both spiked and naturally contaminated water samples. The test was evaluated in potable, industrial, and natural water matrixes, according to the scope of the ISO 11731 reference method. These waters were tested with the target at levels ranging from low (10–99 CFU/mL) to high (100–999 CFU/mL); a Chi-square value of 1.8 indicated that there was no significant difference between the test and the reference method. The false-positive rate was 7%, and the false-negative rate 2%. For the inclusivity study, all 17 strains of L. pneumophila of different serogroups reacted with the test. For the exclusivity study, 17 strains of other Legionella species and 16 non-Legionella strains were tested. There were no cross-reactions with non-Legionella strains. L. beliardensis, L. adelaidensis, and one environmentally isolated Legionella sp. produced a positive result at high concentrations of 1800, 230, and 3900 CFU/mL, respectively. Agreement between the two methods was 95.9%.

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