In situ identification of Legionellaceae using 16S rRNA-targeted oligonucleotide probes and confocal laser scanning microscopy

Manz, Werner; Amann, Rudolf; Szewzyk, Regine; Szewzyk, Ulrich; Stenström, Thor-Axel; Hutzler, Peter; Schleifer, Karl‐Heinz

doi:10.1099/00221287-141-1-29

Cited by 98 publications

(78 citation statements)

References 52 publications

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“…The rapid diagnostic tests used for detection of Legionella pneumonia are based on the direct fluorescent antibody (DFA) staining technique, on the polymerase chain reaction (PCR) [16][17][18][19] and on fluorescence in situ hybridisation (FISH) of whole cells with 16S rRNA-targeted oligonucleotide probes [20][21][22].…”

Section: Introductionmentioning

confidence: 99%

Identification of Legionella spp. in Environmental Water Samples by ScanVIT-Legionella™ Method in Spain

Grúas

Alvarez²,

Lara

et al. 2013

Indian J Microbiol

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Rapid and more sensitive methods for the detection and quantification of viable Legionella cells have been developed. In this paper, a comparative analysis of environmental water samples using the ScanVIT-Legionella TM method and the traditional ''gold standard'' method of culturing is realised indicating the usefulness of the ScanVIT method. The ScanVIT-Legionella TM method was performed on environmental water samples from different locations of Huesca region (Spain). Legionella micro-colonies should appear green colour and Legionella pneumophila micro-colonies appear red. Twenty-one environmental water samples analysed by standard culture plus five control samples (Two sterile water samples with Legionella as positive controls and three sterile water samples as negative controls). All of them were used to apply ScanVIT-Legionella TM method. From of 21 environmental samples eleven were positive, six negative with both methods and four samples were negative for culture method and positive for ScanVIT-Legionella TM method. The positive control samples were positive and the negative were negative for both methods. A comparative analysis of the results obtained with two methods showed a strong positive determination coefficient (R 2 = 0.99753). The results demonstrate the usefulness of the ScanVITLegionella TM method as a rapid diagnostic tool in order to provide a diagnosis as quick as possible. ScanVITLegionella TM method offers a series of advantages such as quickly diagnosis, higher sensitivity and the possibility to identify Legionella spp. and L. pneumophila simultaneously.

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Section: Introductionmentioning

confidence: 99%

Identification of Legionella spp. in Environmental Water Samples by ScanVIT-Legionella™ Method in Spain

Grúas

Alvarez²,

Lara

et al. 2013

Indian J Microbiol

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“…Laser scanning confocal microscopy (LSCM) was used to determine whether fluorescent bacteria were intracellular. That combination of methods previously has been used to visualize Legionella invasion of a protozoan species (23). This study is the first to use them to demonstrate what appears to be an intracellular microbial community within buccal epithelial cells.…”

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confidence: 97%

Intracellular Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis in Buccal Epithelial Cells Collected from Human Subjects

Rudney¹,

Chen²,

2001

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The mouth may provide an accessible model for studying bacterial interactions with human cells in vivo. Using fluorescent in situ hybridization and laser scanning confocal microscopy, we found that human buccal epithelial cells from 23 of 24 subjects were infected with intracellular bacteria, including the periodontal pathogens Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis, as well as other species which have yet to be identified. Buccal cell invasion may allow fastidious anaerobes to establish themselves in aerobic sites that otherwise present an unfavorable environment. Exfoliated buccal epithelial cells might provide a protected route for bacterial transmission between different oral sites within and between hosts.

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“…For fluorescence in situ hybridization (FISH), ENT1probe (5Ј-CCGCTTGC TCTCGCGAG-3Ј), labeled with Cy3 at the 5Ј end and specific for Enterobacteriaceae 16S rRNA, was used to detect E. coli (22). The FISH protocol was adapted from that of Manz et al (24). Each sample (1 to 10 ml) was filtered through a 25-mm-diameter, 0.2-m-pore-size white polycarbonate membrane (Millipore) and was fixed with 3.7% (vol/vol) formaldehyde for 30 min.…”

Section: Production Of Aom (I) Algal Culturementioning

confidence: 99%

Escherichia coli Behavior in the Presence of Organic Matter Released by Algae Exposed to Water Treatment Chemicals

Bouteleux

Saby²,

Tozza

et al. 2005

Appl Environ Microbiol

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When exposed to oxidation, algae release dissolved organic matter with significant carbohydrate (52%) and biodegradable (55 to 74%) fractions. This study examined whether algal organic matter (AOM) added in drinking water can compromise water biological stability by supporting bacterial survival. Escherichia coli (1.3 ؋ 10 5 cells ml ؊1 ) was inoculated in sterile dechlorinated tap water supplemented with various qualities of organic substrate, such as the organic matter coming from chlorinated algae, ozonated algae, and acetate (model molecule) to add 0.2 ؎ 0.1 mg of biodegradable dissolved organic carbon (BDOC) liter ؊1 . Despite equivalent levels of BDOC, E. coli behavior depended on the source of the added organic matter. The addition of AOM from chlorinated algae led to an E. coli growth equivalent to that in nonsupplemented tap water; the addition of AOM from ozonated algae allowed a 4-to 12-fold increase in E. coli proliferation compared to nonsupplemented tap water. Under our experimental conditions, 0.1 mg of algal BDOC was sufficient to support E. coli growth, whereas the 0.7 mg of BDOC liter ؊1 initially present in drinking water and an additional 0.2 mg of BDOC acetate liter ؊1 were not sufficient. Better maintenance of E. coli cultivability was also observed when AOM was added; cultivability was even increased after addition of AOM from ozonated algae. AOM, likely to be present in treatment plants during algal blooms, and thus potentially in the treated water may compromise water biological stability.In spite of the disinfection processes, bacterial proliferation can be observed in drinking water distribution network systems (20,26,37,42) proving that certain bacterial populations are able to adapt, transiently or permanently, to the oligotrophic conditions of distribution networks (16,17). This phenomenon has been described for fecal indicators such as coliform bacteria, particularly Escherichia coli (16), and poses the problem of compliance with water quality health regulations.Although multiple factors certainly affect microbial growth phenomena, organic matter in treated waters, mainly the biodegradable fraction, has a determining effect since it provides a carbon and energy source essential to the growth of heterotrophic bacteria, including coliforms (16,19,25,38,39,42). Atypical events (algal bloom, swelling, rainfall, etc.) capable of modifying the quality of the organic matter in raw and treated waters have been suggested to be a catalyst for coliform growth (3, 18, 23). Lake et al. (15) showed that there is a strong link between the end of the algal bloom and the presence of coliforms in the distribution system, leading to a situation incompatible with health standards. Algal products in the treated water were suspected of providing a good nutritional source for bacterial regrowth in the distribution system. In addition to the natural capacity of algae to secrete organic compounds, massive contamination at the inlet of a treatment plant using preoxidation can lead to algal cell lysis and re...

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In situ identification of Legionellaceae using 16S rRNA-targeted oligonucleotide probes and confocal laser scanning microscopy

Cited by 98 publications

References 52 publications

Identification of Legionella spp. in Environmental Water Samples by ScanVIT-Legionella™ Method in Spain

Identification of Legionella spp. in Environmental Water Samples by ScanVIT-Legionella™ Method in Spain

Intracellular Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis in Buccal Epithelial Cells Collected from Human Subjects

Escherichia coli Behavior in the Presence of Organic Matter Released by Algae Exposed to Water Treatment Chemicals

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