Wadowsky-Yee-Okuda (WYO) Medium: A New Selective Medium for the Isolation of Legionellae Strains from Environmental Water Specimens

Okuda, K; Ikedo, Masanari; Yabuuchi, Eiko

doi:10.11150/kansenshogakuzasshi1970.58.1073

Cited by 14 publications

(7 citation statements)

References 25 publications

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“…Pretreatment with acid buffer to kill other microbes in the samples may also kill some legionellae in the samples (4). In addition, some Legionella species are sensitive to selective agents in the medium (15,24). Another reason why the detection rate by the culture method was lower (79.5%) than that by the seminested PCR assay (91.8%) may be due to the viability of cells.…”

Section: Discussionmentioning

confidence: 99%

Development of a new seminested PCR method for detection of Legionella species and its application to surveillance of legionellae in hospital cooling tower water

Miyamoto

Yamamoto

Arima

et al. 1997

Appl Environ Microbiol

165

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The presence of PCR inhibitors in water samples is well known and contributes to the fact that a practical PCR assay has not been developed for legionella surveillance. In this study, we devised a new seminested PCR assay for detection of Legionella spp. in water samples as a means of overriding the PCR inhibitors without loss of sensitivity. The seminested PCR assay utilized primers to amplify the 16S rRNA gene (LEG primers) of 39 Legionella spp. The assay was specific to legionellae, and the sensitivity was 1 fg of extracted Legionella DNA in laboratory examination. To evaluate the feasibility and sensitivity of the PCR assay in identifying the presence of legionellae, it was used to survey Legionella contamination in the water of 49 cooling towers of 32 hospitals. A commercially available EnviroAmp Legionella kit and a culture method were also used in the survey for comparison with the seminested PCR assay. The detection rates of legionellae in the samples were 91.8% (45 of 49) by the PCR assay and 79.5% (39 of 49) by the culture method. The EnviroAmp kit revealed that 30.6% of the water samples (15 of 49) contained inhibitors of the PCR amplification. However, the seminested PCR assay could produce the Legionella-specific DNA bands in 14 of the 15 samples. Although 8 of the 14 samples were positive in the first-step PCR, 6 of the 14 samples became positive in the second-step PCR. These results suggest that the effect of PCR inhibitors in samples, if any, can be reduced because of the dilution of the sample in the second-step PCR and that sensitivity of detection can be increased by the second-step PCR. Thus, the seminested PCR assay with LEG primers to amplify the 16S rRNA gene of 39 Legionella spp. was a practical and sensitive method to detect Legionella spp. in water samples.

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Section: Discussionmentioning

confidence: 99%

Development of a new seminested PCR method for detection of Legionella species and its application to surveillance of legionellae in hospital cooling tower water

Miyamoto

Yamamoto

Arima

et al. 1997

Appl Environ Microbiol

165

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“…Even if a sample is treated with acid-buffer and inoculated on a selective medium, some fast-growing bacteria or fungi disturb counting, thereby interfering with the detection of legionellae, such as the case of the plate count for the G-16 sample (Table 2). In addition, some Legionella species are sensitive to selective agents in the medium (14). These may be caused by lower numbers of legionellae in colony counts.…”

Section: Discussionmentioning

confidence: 99%

Comparison of Detection Methods for Legionella Species in Environmental Water by Colony Isolation, Fluorescent Antibody Staining, and Polymerase Chain Reaction

Yamamoto

Hashimoto

Ezaki

1993

Microbiology and Immunology

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Three detection methods for Legionella species in water samples from cooling towers and a river were examined. Direct counting of bacteria stained with fluorescent antibody (FA) for L. pneumophila (serogroups 1 to 6) could detect the cell of 104 to 106 cell/100 ml in all 14 samples, while colony counting method detected 10 to 103 CFU/100 ml only in 8 samples from cooling towers. Polymerase chain reaction (PCR) assay with primers to amplify 16S ribosomal DNA sequence of most Legionella species (LEG primer) detected legionellae in 13 samples, while species-specific primers for L. pneumophila detected the DNAs from 3 samples. In laboratory examination, LEG primers could amplify DNAs of 29 species of genus Legionella with high sensitivity, even from 1 cell of L. pneumophila GIFU 9134. The PCR assay with LEG primers was specific and sensitive methods to be satisfied the survey of legionellae. Thus, PCR assay is a suitable method to detect and monitor Legionella species in an environment.

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“…Selective agar plates contain media that inhibit the growth of nontarget microorganisms and facilitate the growth of target microorganisms. The selective agar plates for Legionella are generally non-selective BCYEα (Edelstein, 1981) agar plates containing vancomycin and polymyxin B to inhibit the growth of bacteria other than Legionella, and cycloheximide (Dennis et al, 1984a) , amphotericin B (Okuda et al, 1984) or natamycin (Edelstein and Edelstein, 1996) to inhibit the growth of fungi. Various selective agar plates are available as commercial products, but differences in the growth of Legionella often occur depending on the manufacturer even if the medium is supposedly the same.…”

Section: Types Of Selective Agar Platesmentioning

confidence: 99%

Interpreting the Results of the Conventional Plate Culture and Gene Detection Methods for <i>Legionella</i> Detection in Environmental Water Samples

Inoue¹

2020

Biocontrol Sci.

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Wadowsky-Yee-Okuda (WYO) Medium: A New Selective Medium for the Isolation of Legionellae Strains from Environmental Water Specimens

Cited by 14 publications

References 25 publications

Development of a new seminested PCR method for detection of Legionella species and its application to surveillance of legionellae in hospital cooling tower water

Development of a new seminested PCR method for detection of Legionella species and its application to surveillance of legionellae in hospital cooling tower water

Comparison of Detection Methods for Legionella Species in Environmental Water by Colony Isolation, Fluorescent Antibody Staining, and Polymerase Chain Reaction

Interpreting the Results of the Conventional Plate Culture and Gene Detection Methods for <i>Legionella</i> Detection in Environmental Water Samples

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