Detection of &lt;i&gt;Legionella pneumophila&lt;/i&gt; in bioaerosols by polymerase chain reaction

Pascual, Leonor; Pérez-Luz, Sara; Amo, A. Del; Moreno, Carmen; Apráiz, D.; Catalán, Vicente

doi:10.1139/cjm-47-4-341

Cited by 24 publications

(15 citation statements)

References 28 publications

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“…Legionnaires' disease (LD) is caused mainly by inhalation of aerosols generated from soil or aquatic environments contaminated with Legionella (Pascual et al, 2001). Outbreaks of Legionellosis occur throughout the world affecting public health as well as various industrial, tourist, and social activities (Sabria and Campins, 2003).…”

Section: Introductionmentioning

confidence: 99%

Quantification of viable Legionella pneumophila cells using propidium monoazide combined with quantitative PCR

Yáñez¹,

Nocker

Soria-Soria³

et al. 2011

Journal of Microbiological Methods

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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.

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Section: Introductionmentioning

confidence: 99%

Quantification of viable Legionella pneumophila cells using propidium monoazide combined with quantitative PCR

Yáñez¹,

Nocker

Soria-Soria³

et al. 2011

Journal of Microbiological Methods

Self Cite

150

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“…[ 14 ] In the study of Pascual et al ., (2001), in a WWTP; three samples from the pretreatment point (30%) and one from the biological treatment (11%) were positive for Legionella . [ 9 ] However, both studies found Legionella in air samples with only PCR, but not with a culture. Although, culture on a selective medium has commonly been used for detection of Legionella spp.…”

Section: Discussionmentioning

confidence: 99%

“…in the wastewater of various units of WWTPs. [ 1 2 3 9 10 ] Therefore, Legionella aerosols can be produced whenever aeration or mechanical agitation is used in wastewater treatment processes. There is evidence that the presence of Legionella in wastewater has been linked with increased levels of antibodies among wastewater irrigation workers.…”

Section: Introductionmentioning

confidence: 99%

“…in bio -aerosols generated from WWTPs. [ 2 7 9 13 ] However, the presence of Legionella in bio -aerosols of WWTPs could be influenced by several parameters, including, the concentration of these bacteria in the wastewater, characteristics of WWTP units, and the aerosolization at the different steps of treatment and weather conditions. [ 11 14 ] This study was carried out to detect Legionella spp.…”

Section: Introductionmentioning

confidence: 99%

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Detection of Legionella spp. by a nested-PCR assay in air samples of a wastewater treatment plant and downwind distances in Isfahan

et al. 2015

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Background:Wastewater contains a variety of pathogens and bio -aerosols generated during the wastewater treatment process, which could be a potential health risk for exposed individuals. This study was carried out to detect Legionella spp. in the bio -aerosols generated from different processes of a wastewater treatment plant (WWTP) in Isfahan, Iran, and the downwind distances.Materials and Methods:A total of 54 air samples were collected and analyzed for the presence of Legionella spp. by a nested- polymerase chain reaction (PCR) assay. A liquid impingement biosampler was used to capture bio -aerosols. The weather conditions were also recorded.Results:Legionella were detected in 6% of the samples, including air samples above the aeration tank (1/9), belt filter press (1/9), and 250 m downwind (1/9).Conclusion:The result of this study revealed the presence of Legionella spp. in air samples of a WWTP and downwind distance, which consequently represent a potential health risk to the exposed individuals.

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“…Air samples were collected using impact method at two sampling sites, which were selected with respect to the highest expected emissions of the bioaerosol, wind velocity, and direction on the sampling days. To detect the total counts of Enterobacteriaceae bacteria in the air by cultivation method, total number of bacteria by 4′,6-diamidino-2-phenylindole (DAPI) staining, and number of Enterobacteriaceae by in situ hybridization FISH, air samples were collected with the MAS-100 Eco (Merck) using parallel two Petri dishes, each containing 30 ml of phosphate buffered saline (PBS) according to Pascual et al (2001) with own modification. The 4,000 l of air samples was impacted onto the PBS, and afterwards, the PBS was transferred to sterile screw-capped tubes.…”

Section: Methodsmentioning

confidence: 99%

Culture-Dependent and Culture-Independent Methods in Evaluation of Emission of Enterobacteriaceae from Sewage to the Air and Surface Water

Korzeniewska

Harnisz

2012

Water Air Soil Pollut

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The number of Enterobacteriaceae, with particular attention given to the presence of Escherichia coli and Klebsiella pneumoniae, was determined in hospital effluents and municipal wastewater after various stages of purification. The emission of these microorganisms to the ambient air near wastewater treatment plant (WWTP) facilities and to the river water, which is a receiver of the WWTP effluent, was also studied using fluorescence in situ hybridization (FISH) and cultivation methods. The number of Enterobacteriaceae determined by cultivation and fluorescence methods in different kinds of sewage sample ranged from 0.5 × 103 to 2.9 × 106 CFU/ml and from 2.2 × 105 to 1.3 × 108 cells/ml, respectively. Their removal rates during treatment processes were close to 99 %, but the number of these bacteria in the WWTP outflow was quite high and ranged from 5.9 × 103 to 3.5 × 104 CFU/ml and from 1.1 × 105 to 6.1 × 105 cells/ml, respectively. In the river water and the air samples, the number of Enterobacteriaceae was also high and ranged from 4.1 × 103 to 7.9 × 103 CFU/ml and from 3 to 458 CFU/m3, respectively. The numbers of these microorganisms obtained from fluorescence and cultivation methods were statistically and significantly correlated; however, the analysis of the studied samples indicated that the FISH method gave values up to 103-fold times greater than those obtained by the cultivation method. From a sanitary point of view, this means that the number of viable fecal bacteria is systematically underestimated by traditional culture-based methods. Thus, the FISH proves to be a method that could be used to estimate bacterial load, particularly in air samples and less contaminated river water.

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Detection of <i>Legionella pneumophila</i> in bioaerosols by polymerase chain reaction

Cited by 24 publications

References 28 publications

Quantification of viable Legionella pneumophila cells using propidium monoazide combined with quantitative PCR

Quantification of viable Legionella pneumophila cells using propidium monoazide combined with quantitative PCR

Detection of Legionella spp. by a nested-PCR assay in air samples of a wastewater treatment plant and downwind distances in Isfahan

Culture-Dependent and Culture-Independent Methods in Evaluation of Emission of Enterobacteriaceae from Sewage to the Air and Surface Water

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