Evaluation of nested PCR assays for the detection of Legionella pneumophila in a wide range of aquatic samples

Devos, Liesbet; Clymans, Koen; Boon, Nico; Verstraete, Willy

doi:10.1111/j.1365-2672.2005.02668.x

Cited by 16 publications

(13 citation statements)

References 27 publications

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“…In contrast, variations in the Legionella population structure were observed over time, with significant changes occurring at the end of June, following a stable phase from mid-May to midJune. In a previous study of microbial communities in a wide range of aquatic samples containing L. pneumophila, no relationship was found between the occurrence of L. pneumophila and the associated microbiota (15). It was observed, however, that the occurrence of L. pneumophila was possible within a certain range of species richness and diversity.…”

Section: Discussionmentioning

confidence: 98%

Dynamics of Legionella spp. and Bacterial Populations during the Proliferation of L. pneumophila in a Cooling Tower Facility

Wéry

Bru-Adan

Minervini

et al. 2008

Appl Environ Microbiol

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The dynamics of Legionella spp. and of dominant bacteria were investigated in water from a cooling tower plant over a 9-month period which included several weeks when Legionella pneumophila proliferated. The structural diversity of both the bacteria and the Legionella spp. was monitored by a fingerprint technique, single-strand conformation polymorphism, and Legionella spp. and L. pneumophila were quantified by real-time quantitative PCR. The structure of the bacterial community did not change over time, but it was perturbed periodically by chemical treatment or biofilm detachment. In contrast, the structure of the Legionella sp. population changed in different periods, its dynamics at times showing stability but also a rapid major shift during the proliferation of L. pneumophila in July. The dynamics of the Legionella spp. and of dominant bacteria were not correlated. In particular, no change in the bacterial community structure was observed during the proliferation of L. pneumophila. Legionella spp. present in the cooling tower system were identified by cloning and sequencing of 16S rRNA genes. A high diversity of Legionella spp. was observed before proliferation, including L. lytica, L. fallonii, and other Legionella-like amoebal pathogen types, along with as-yet-undescribed species. During the proliferation of L. pneumophila, Legionella sp. diversity decreased significantly, L. fallonii and L. pneumophila being the main species recovered.

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Section: Discussionmentioning

confidence: 98%

Dynamics of Legionella spp. and Bacterial Populations during the Proliferation of L. pneumophila in a Cooling Tower Facility

Wéry

Bru-Adan

Minervini

et al. 2008

Appl Environ Microbiol

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“…Strains used in the course of this study were L. pneumophila sg1 (ATCC 33152) and P. putida UWC3. Legionella pneumophila was grown aerobically in Buffered Yeast Extract broth for 72 h at 37°C and supplemented with additional nutrients (Devos et al , 2005b). Pseudomonas putida was grown aerobically in Luria Bertani (LB) broth (Oxoid, Basingstoke, UK) for 24 h at 28°C.…”

Section: Methodsmentioning

confidence: 99%

Introduction of a boost of Legionella pneumophila into a stagnant-water model by heat treatment

Vervaeren

Temmerman

Devos³

et al. 2006

FEMS Microbiology Ecology

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An environmentally representative stagnant-water model was developed to monitor the growth dynamics of Legionella pneumophila. This model was evaluated for three distinct water treatments: untreated tap water, heat-treated tap water, and heat-treated tap water supplemented with Pseudomonas putida, a known biofilm-forming bacterium. Bringing heat-treated tap water after subsequent cooling into contact with a densely formed untreated biofilm was found to promote the number of L. pneumophila by 4 log units within the biofilm, while the use of untreated water only sustained the L. pneumophila levels. Subsequent colonization of the water phase by L. pneumophila was noticed in the heat-treated stagnant-water models, with concentrations as high as 1 x 10(10) mip gene copies L(-1) stagnant water. Denaturing gradient gel electrophoresis in combination with clustering analysis of the prokaryotic community in the water phase and in the biofilm phase suggests that the different water treatments induced different communities. Moreover, boosts of L. pneumophila arising from heat treatment of water were accompanied by shifts to a more diverse eukaryotic community. Stimulated growth of L. pneumophila after heating of the water may explain the rapid recolonization of L. pneumophila in water systems. These results highlight the need for additional or alternative measures to heat treatment of water in order to prevent or abate potential outbreaks of L. pneumophila.

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“…Wellinghausen et al (2001) showed that DNA extraction enabled the detection of legionellae in free-living amoeba, but culturing methods could not detect amoeba. Furthermore, standard culture techniques based on ISO 11731 have numerous limitations (Behets et al, 2007) and several factors complicate the interpretation of plate-counting results (Devos et al, 2005). Cultures can be fastidious due to various factors; for example: the legionellae growth requirements necessitate prolonged incubation periods; the legionellae are difficult to isolate in samples contaminated with high levels of other microbiota, particularly environmental samples; the pre-treatment by acid or heating can lead to underestimates of the number of viable legionellae; viable, but non-culturable bacteria cannot be detected (Catalan et al, 1997;Wellinghausen et al, 2001;Yañez et al, 2005); and Legionella spp.…”

Section: Discussionmentioning

confidence: 99%

Comparison of conventional culture and real-time quantitative PCR using SYBR Green for detection of <i>Llegionella pneumophila</i> in water samples

Fittipaldi¹,

Codony²,

Morató³

2010

WSA

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The genus Legionella comprises more than 40 species and 64 serogroups with approximately half of those species associated with human diseases. Legionella pneumophila Serogroup 1 is the most common pathogenic species and is responsible for up to 80% of legionellosis cases in the world. Legionella levels in water are assessed routinely by culture on a selective medium, but its slow growth is a serious drawback, given that at least 10 days are required to obtain results. In an attempt to provide a simple screening method for Legionella pneumophila in water systems samples a real time PCR assay using SYBR Green was developed. A total of 50 samples from cooling towers and hot tap water systems were analysed by DNA amplification using 2 pairs of primers targeting the mip and dot genes. Legionella pneumophila Serogroup 1 (NCTC12821) was used as a reference strain and to evaluate real-time PCR performance. The assays were successful with both primer sets; good and similar amplification efficiencies were achieved. In addition, high sensitivity was obtained; the method proved to allow for the detection of fewer than 10 gene copies per reaction. Results of real-time PCR were compared to conventional analysis based on culture. Although no strong correlation was observed between both methods and consequently real-time PCR could not substitute for the reference method, it represents a powerful screening tool. The inexpensive, sensitive and rapid real-time PCR based in SYBR Green method is of interest in monitoring Legionella pneumophila contamination, especially in environmental samples, and should be economical for large-scale routine tests.

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Evaluation of nested PCR assays for the detection of Legionella pneumophila in a wide range of aquatic samples

Cited by 16 publications

References 27 publications

Dynamics of Legionella spp. and Bacterial Populations during the Proliferation of L. pneumophila in a Cooling Tower Facility

Dynamics of Legionella spp. and Bacterial Populations during the Proliferation of L. pneumophila in a Cooling Tower Facility

Introduction of a boost of Legionella pneumophila into a stagnant-water model by heat treatment

Comparison of conventional culture and real-time quantitative PCR using SYBR Green for detection of <i>Llegionella pneumophila</i> in water samples

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