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.

scholarly journals Rapid Method for Enumeration of Viable Legionella pneumophila and Other Legionella spp. in Water

2005 ◽  
Vol 71 (7) ◽  
pp. 4086-4096 ◽  
Author(s):  
Pilar Delgado-Viscogliosi ◽  
Tristan Simonart ◽  
Virginie Parent ◽  
Grégory Marchand ◽  
Marie Dobbelaere ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Limit Of Detection ◽  
Culture Method ◽  
Epifluorescence Microscopy ◽  
Specific Method ◽  
Double Staining ◽  
Culture Methods ◽  
Experimental Conditions ◽  
Short Period ◽  
Standard Culture

ABSTRACT A sensitive and specific method has been developed to enumerate viable L. pneumophila and other Legionella spp. in water by epifluorescence microscopy in a short period of time (a few hours). This method allows the quantification of L. pneumophila or other Legionella spp. as well as the discrimination between viable and nonviable Legionella. It simultaneously combines the specific detection of Legionella cells using antibodies and a bacterial viability marker (ChemChrome V6), the enumeration being achieved by epifluorescence microscopy. The performance of this immunological double-staining (IDS) method was investigated in 38 natural filterable water samples from different aquatic sources, and the viable Legionella counts were compared with those obtained by the standard culture method. The recovery rate of the IDS method is similar to, or higher than, that of the conventional culture method. Under our experimental conditions, the limit of detection of the IDS method was <176 Legionella cells per liter. The examination of several samples in duplicates for the presence of L. pneumophila and other Legionella spp. indicated that the IDS method exhibits an excellent intralaboratory reproducibility, better than that of the standard culture method. This immunological approach allows rapid measurements in emergency situations, such as monitoring the efficacy of disinfection shock treatments. Although its field of application is as yet limited to filterable waters, the double-staining method may be an interesting alternative (not equivalent) to the conventional standard culture methods for enumerating viable Legionella when rapid detection is required.

Comparison of Automated BAX PCR and Standard Culture Methods for Detection of Listeria monocytogenes in Blue Crabmeat (Callinectus sapidus) and Blue Crab Processing Plants

2011 ◽  
Vol 74 (11) ◽  
pp. 1930-1933 ◽  
Author(s):  
SIVARANJANI PAGADALA ◽  
SALINA PARVEEN ◽  
JURGEN G. SCHWARZ ◽  
THOMAS RIPPEN ◽  
JOHN B. LUCHANSKY
Keyword(s):  
Listeria Monocytogenes ◽  
Blue Crab ◽  
Environmental Samples ◽  
Culture Method ◽  
Culture Methods ◽  
Enrichment Broth ◽  
Significant Difference ◽  
Processing Plants ◽  
Standard Culture

This study compared the automated BAX PCR with the standard culture method (SCM) to detect Listeria monocytogenes in blue crab processing plants. Raw crabs, crabmeat, and environmental sponge samples were collected monthly from seven processing plants during the plant operating season, May through November 2006. For detection of L. monocytogenes in raw crabs and crabmeat, enrichment was performed in Listeria enrichment broth, whereas for environmental samples, demi-Fraser broth was used, and then plating on both Oxford agar and L. monocytogenes plating medium was done. Enriched samples were also analyzed by BAX PCR. A total of 960 samples were examined; 59 were positive by BAX PCR and 43 by SCM. Overall, there was no significant difference (P ≤0.05) between the methods for detecting the presence of L. monocytogenes in samples collected from crab processing plants. Twenty-two and 18 raw crab samples were positive for L. monocytogenes by SCM and BAX PCR, respectively. Twenty and 32 environmental samples were positive for L. monocytogenes by SCM and BAX PCR, respectively, whereas only one and nine finished products were positive. The sensitivities of BAX PCR for detecting L. monocytogenes in raw crabs, crabmeat, and environmental samples were 59.1, 100, and 60%, respectively. The results of this study indicate that BAX PCR is as sensitive as SCM for detecting L. monocytogenes in crabmeat, but more sensitive than SCM for detecting this bacterium in raw crabs and environmental samples.

scholarly journals Direct detection of Mycobacterium avium in environmental water and scale samples by loop-mediated isothermal amplification

2013 ◽  
Vol 12 (2) ◽  
pp. 211-219 ◽  
Author(s):  
Yukiko Nishiuchi ◽  
Aki Tamaru ◽  
Yasuhiko Suzuki ◽  
Seigo Kitada ◽  
Ryoji Maekura ◽  
...  
Keyword(s):  
Mycobacterium Avium ◽  
Environmental Samples ◽  
Direct Detection ◽  
Culture Method ◽  
Environmental Water ◽  
Isothermal Amplification ◽  
Lamp Method ◽  
Culture Methods ◽  
Loop Mediated Isothermal Amplification ◽  
Conventional Culture

We previously demonstrated the colonization of Mycobacterium avium complex in bathrooms by the conventional culture method. In the present study, we aimed to directly detect M. avium organisms in the environment using loop-mediated isothermal amplification (LAMP), and to demonstrate the efficacy of LAMP by comparing the results with those obtained by culture. Our data showed that LAMP analysis has detection limits of 100 fg DNA/reaction for M. avium. Using an FTA® elute card, DNA templates were extracted from environmental samples from bathrooms in the residences of 29 patients with pulmonary M. avium disease. Of the 162 environmental samples examined, 143 (88%) showed identical results by both methods; 20 (12%) and 123 (76%) samples were positive and negative, respectively, for M. avium. Of the remaining 19 samples (12%), seven (5%) and 12 (7%) samples were positive by the LAMP and culture methods, respectively. All samples that contained over 20 colony forming units/primary isolation plate, as measured by the culture method, were also positive by the LAMP method. Our data demonstrate that the combination of the FTA elute card and LAMP can facilitate prompt detection of M. avium in the environment.

scholarly journals VIDAS® Enzyme-Linked Immunofluorescent Assay for Detection of Listeria in Foods: Collaborative Study

2000 ◽  
Vol 83 (4) ◽  
pp. 903-918 ◽  
Author(s):  
Vidhya Gangar ◽  
Michael S Curiale ◽  
Armando D’Onorio ◽  
Ann Schultz ◽  
Ronald L Johnson ◽  
...  
Keyword(s):  
False Positive Rate ◽  
Collaborative Study ◽  
False Negative ◽  
Traditional Culture ◽  
Culture Method ◽  
Contamination Level ◽  
Culture Methods ◽  
Positive Rate ◽  
Standard Culture ◽  
Lis Method

Abstract The VIDAS LIS method and the traditional culture methods for detection of Listeria species in food were evaluated in a multilaboratory comparative study. The 6 foods tested were either naturally contaminated or inoculated with 3 different concentrations of Listeria. Results for each food and each contamination level with the VIDAS LIS method were as good as or better than those obtained with the traditional culture method. Of 1558 samples tested, 935 were positive: 839 by the VIDAS method and 809 by standard culture methods. Overall false negative rates were 10.3 and 13.5% for the VIDAS LIS and culture methods, respectively. The false positive rate for the VIDAS LIS assay was 1.4% based on 9 VIDAS LIS positive assays that did not confirm positive by isolation of Listeria. The agreement between the VIDAS LIS and culture methods for all samples tested was 86%.

scholarly journals Integrated Real-Time PCR for Detection and Monitoring of Legionella pneumophila in Water Systems

2006 ◽  
Vol 73 (5) ◽  
pp. 1452-1456 ◽  
Author(s):  
Diaraf Farba Yaradou ◽  
Sylvie Hallier-Soulier ◽  
Sophie Moreau ◽  
Florence Poty ◽  
Yves Hillion ◽  
...  
Keyword(s):  
Real Time ◽  
Legionella Pneumophila ◽  
Cooling Tower ◽  
Water System ◽  
Culture Method ◽  
Hot Water ◽  
Pcr Assay ◽  
Standard Culture ◽  
Serial Samples ◽  
Over Time

ABSTRACT We evaluated a ready-to-use real-time quantitative Legionella pneumophila PCR assay system by testing 136 hot-water-system samples collected from 55 sites as well as 49 cooling tower samples collected from 20 different sites, in parallel with the standard culture method. The PCR assay was reproducible and suitable for routine quantification of L. pneumophila. An acceptable correlation between PCR and culture results was obtained for sanitary hot-water samples but not for cooling tower samples. We also monitored the same L. pneumophila-contaminated cooling tower for 13 months by analyzing 104 serial samples. The culture and PCR results were extremely variable over time, but the curves were similar. The differences between the PCR and culture results did not change over time and were not affected by regular biocide treatment. This ready-to-use PCR assay for L. pneumophila quantification could permit more timely disinfection of cooling towers.

A detection method for Legionella spp in (cooling) water: fluorescent in situ hybridisation (FISH) on whole bacteria

2003 ◽  
Vol 47 (3) ◽  
pp. 143-146 ◽  
Author(s):  
P. Declerck ◽  
L. Verelst ◽  
L. Duvivier ◽  
A. Van Damme ◽  
F. Ollevier
Keyword(s):  
16S Rrna ◽  
In Situ Hybridisation ◽  
Cooling Water ◽  
Culture Methods ◽  
Rrna Sequence ◽  
16S Rrna Sequence ◽  
Fluorescent In Situ Hybridisation ◽  
Background Staining ◽  
Legionella Species

Although traditional culture methods are appropriate for detection of Legionella species, such culture takes several days. Rapid detection (&lt;24 h) of individual Legionella is possible using fluorescent in situ hybridisation (FISH) on whole bacteria. Water samples were filtered and the concentrated bacteria were immediately detected (without culture) with a fluorescence microscope following appropriate labelling. The detection level was very high and quantification was possible. For the detection of all Legionella spp. the probe LEG705 was used, complementary to a 16S rRNA sequence conserved in all Legionella spp. For specific detection of L. pneumophila the probe LEGPNE1 was used. This probe is designed against a variable domain of the 16S rRNA sequence from L. pneumophila. CY3 and FLUOS labels were tested and CY3 showed clearly detectable bacteria with minimum background staining. This FISH technique is very sensitive, fast, reliable and individual bacteria are easily detected.

scholarly journals Rapid Detection and Enumeration of Legionella pneumophila in Hot Water Systems by Solid-Phase Cytometry

2004 ◽  
Vol 70 (3) ◽  
pp. 1651-1657 ◽  
Author(s):  
Helena Aurell ◽  
Philippe Catala ◽  
Pierre Farge ◽  
France Wallet ◽  
Matthieu Le Brun ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Natural Waters ◽  
Solid Phase ◽  
Tap Water ◽  
Culture Method ◽  
Water Systems ◽  
Cross Reactivity ◽  
Hot Water ◽  
Standard Culture ◽  
Direct Counts

ABSTRACT A new method for the rapid and sensitive detection of Legionella pneumophila in hot water systems has been developed. The method is based on an IF assay combined with detection by solid-phase cytometry. This method allowed the enumeration of L. pneumophila serogroup 1 and L. pneumophila serogroups 2 to 6, 8 to 10, and 12 to 15 in tap water samples within 3 to 4 h. The sensitivity of the method was between 10 and 100 bacteria per liter and was principally limited by the filtration capacity of membranes. The specificity of the antibody was evaluated against 15 non-Legionella strains, and no cross-reactivity was observed. When the method was applied to natural waters, direct counts of L. pneumophila were compared with the number of CFU obtained by the standard culture method. Direct counts were always higher than culturable counts, and the ratio between the two methods ranged from 1.4 to 325. Solid-phase cytometry offers a fast and sensitive alternative to the culture method for L. pneumophila screening in hot water systems.

Validation of the ANSR®Listeria Method for Detection of Listeria spp. in Selected Foods

2015 ◽  
Vol 98 (5) ◽  
pp. 1290-1300 ◽  
Author(s):  
Oscar Caballero ◽  
Susan Alles ◽  
Michael Wendorf ◽  
R Lucas Gray ◽  
Kayla Walton ◽  
...  
Keyword(s):  
Culture Method ◽  
Cross Reactivity ◽  
Single Step ◽  
Probability Of Detection ◽  
Nucleic Acid Amplification ◽  
Culture Methods ◽  
Detection Model ◽  
Smoked Salmon ◽  
Standard Culture ◽  
Nicking Enzyme

Abstract ANSR®Listeria was previously certified as Performance Tested MethodSM 101202 for detection of Listeria spp. on selected environmental surfaces. This study proposes a matrix extension to the method for detection of Listeria spp. in selected food matrixes. The method is an isothermal nucleic acid amplification assay based on the nicking enzyme amplification reaction technology. Following single-step sample enrichment for 16–24 h, the assay is completed in less than 50 min, requiring only simple instrumentation. Inclusivity testing was performed using a panel of 51 strains of Listeria spp., representing the species L. grayi, L. innocua, L. ivanovii, L. monocytogenes, L. seeligeri, and L. welshimeri. All strains tested were detected by the ANSR assay. Exclusivity testing of 30 strains representing non-Listeria Gram-positive bacteria yielded no evidence of cross-reactivity. Performance of the ANSR method for detection of Listeria spp. was compared to that of reference culture procedures for pasteurized liquid egg, pasteurized 2% milk, Mexican-style cheese, ice cream, smoked salmon, lettuce, cantaloupe, and guacamole. Data obtained in these unpaired studies and analyzed using a probability of detection model demonstrated that there were no statistically significant differences in results between the ANSR and reference culture methods, except for milk at 16 h and cantaloupe. In milk and smoked salmon, ANSR sensitivity was low at 16 h and therefore the recommended incubation time is 24 h. In cantaloupe, ANSR was found to be more sensitive than the reference culture method at both 16 and 24 h in independent aboratory testing. The ANSR Listeria method can be used as an accurate, rapid, and simple alternative to standard culture methods for detection of Listeria spp. in selected food types.

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