scholarly journals RNA Colony Blot Hybridization Method for Enumeration of Culturable Vibrio cholerae and Vibrio mimicus Bacteria

2009 ◽  
Vol 75 (17) ◽  
pp. 5439-5444 ◽  
Author(s):  
Christopher J. Grim ◽  
Young-Gun Zo ◽  
Nur A. Hasan ◽  
Afsar Ali ◽  
Wasimul B. Chowdhury ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Sensitivity And Specificity ◽  
Water Samples ◽  
Blot Hybridization ◽  
False Negative ◽  
Sodium Dodecyl ◽  
Environmental Water ◽  
Health Clinic ◽  
Vibrio Mimicus ◽  
Hybridization Protocol

ABSTRACT A species-specific RNA colony blot hybridization protocol was developed for enumeration of culturable Vibrio cholerae and Vibrio mimicus bacteria in environmental water samples. Bacterial colonies on selective or nonselective plates were lysed by sodium dodecyl sulfate, and the lysates were immobilized on nylon membranes. A fluorescently labeled oligonucleotide probe targeting a phylogenetic signature sequence of 16S rRNA of V. cholerae and V. mimicus was hybridized to rRNA molecules immobilized on the nylon colony lift blots. The protocol produced strong positive signals for all colonies of the 15 diverse V. cholerae-V. mimicus strains tested, indicating 100% sensitivity of the probe for the targeted species. For visible colonies of 10 nontarget species, the specificity of the probe was calculated to be 90% because of a weak positive signal produced by Grimontia (Vibrio) hollisae, a marine bacterium. When both the sensitivity and specificity of the assay were evaluated using lake water samples amended with a bioluminescent V. cholerae strain, no false-negative or false-positive results were found, indicating 100% sensitivity and specificity for culturable bacterial populations in freshwater samples when G. hollisae was not present. When the protocol was applied to laboratory microcosms containing V. cholerae attached to live copepods, copepods were found to carry approximately 10,000 to 50,000 CFU of V. cholerae per copepod. The protocol was also used to analyze pond water samples collected in an area of cholera endemicity in Bangladesh over a 9-month period. Water samples collected from six ponds demonstrated a peak in abundance of total culturable V. cholerae bacteria 1 to 2 months prior to observed increases in pathogenic V. cholerae and in clinical cases recorded by the area health clinic. The method provides a highly specific and sensitive tool for monitoring the dynamics of V. cholerae in the environment. The RNA blot hybridization protocol can also be applied to detection of other gram-negative bacteria for taxon-specific enumeration.

scholarly journals Rapid and Sensitive Quantification of Vibrio cholerae and Vibrio mimicus Cells in Water Samples by Use of Catalyzed Reporter Deposition FluorescenceIn SituHybridization Combined with Solid-Phase Cytometry

2012 ◽  
Vol 78 (20) ◽  
pp. 7369-7375 ◽  
Author(s):  
Sonja Schauer ◽  
Regina Sommer ◽  
Andreas H. Farnleitner ◽  
Alexander K. T. Kirschner
Keyword(s):  
Vibrio Cholerae ◽  
Water Samples ◽  
Solid Phase ◽  
Soda Lake ◽  
Environmental Water ◽  
Relative Precision ◽  
Content Type ◽  
Vibrio Mimicus ◽  
Catalyzed Reporter Deposition ◽  
Card Fish

ABSTRACTA new protocol for rapid, specific, and sensitive cell-based quantification ofVibrio cholerae/Vibrio mimicusin water samples was developed. The protocol is based on catalyzed reporter deposition fluorescencein situhybridization (CARD-FISH) in combination with solid-phase cytometry. For pure cultures, we were able to quantify down to 6V. choleraecells on one membrane with a relative precision of 39% and down to 12 cells with a relative precision of 17% after hybridization with the horseradish peroxidase (HRP)-labeled probe Vchomim1276 (specific forV. choleraeandV. mimicus) and signal amplification. The corresponding position of the probe on the 16S rRNA is highly accessible even when labeled with HRP. For the first time, we were also able to successfully quantifyV. cholerae/V. mimicusvia solid-phase cytometry in extremely turbid environmental water samples collected in Austria. Cell numbers ranged from 4.5 × 101cells ml−1in the large saline lake Neusiedler See to 5.6 × 104cells ml−1in an extremely turbid shallow soda lake situated nearby. We therefore suggest CARD-FISH in combination with solid-phase cytometry as a powerful tool to quantifyV. cholerae/V. mimicusin ecological studies as well as for risk assessment and monitoring programs.

scholarly journals Isolation of viable but nonculturable Vibrio cholerae O1 from environmental water samples in Kolkata, India, in a culturable state

2014 ◽  
Vol 3 (2) ◽  
pp. 239-246 ◽  
Author(s):  
Mitsutoshi Senoh ◽  
Jayeeta Ghosh‐Banerjee ◽  
Tamaki Mizuno ◽  
Sumio Shinoda ◽  
Shin‐ichi Miyoshi ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Water Samples ◽  
Environmental Water ◽  
Environmental Water Samples ◽  
Viable But Nonculturable ◽  
Vibrio Cholerae O1

scholarly journals Detection of Vibrio cholerae O1 and O139 in Environmental Water Samples by an Immunofluorescent-Aggregation Assay

2010 ◽  
Vol 76 (16) ◽  
pp. 5520-5525 ◽  
Author(s):  
Duochun Wang ◽  
Xuebin Xu ◽  
Xiaoling Deng ◽  
Changyi Chen ◽  
Baisheng Li ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Real Time ◽  
Water Samples ◽  
Real Time Pcr ◽  
High Specificity ◽  
Culture Method ◽  
Environmental Water ◽  
Estuarine Water ◽  
Conventional Culture ◽  
Conventional Culture Method

ABSTRACT Environmental waters are an important reservoir for Vibrio cholerae, and effective surveillance of the pathogen can help to warn of and prevent infection with this potentially fatal pathogen. An immunofluorescent-aggregation (IFAG) assay to detect V. cholerae O1 and O139 was established and evaluated with estuarine water samples. The practical application of this assay was compared with the conventional culture method and real-time PCR. The IFAG method had a sensitivity of 103 CFU/ml for detection of V. cholerae O1 and O139 strains in a suspension containing 10 different species of enterobacterial strains (total, 105 CFU/ml). Ten fluorescent bacterial aggregate colonies were randomly picked and tested positive in serum agglutination tests for the V. cholerae O1 and O139 strains, showing a high specificity. The enrichment broths of 146 samples of estuarine water were tested, and the percentage positive by the IFAG assay was 19.9% (29/146), which was significantly higher than that of the conventional culture method (10.3%, 15/146; P < 0.01) but lower than that of real-time PCR (29.5%, 43/146; P < 0.01). The coincidence rates of real-time PCR and IFAG detection were decreased with the reduction of the V. cholerae concentration. The IFAG method, with a high specificity and a relatively high sensitivity, may be used for detection and isolation of V. cholerae in environmental water samples.

scholarly journals Variants of ctxB alleles of Vibrio cholerae O1 caused sequential cholera outbreaks in the tribal areas of Odisha, India

2021 ◽  
Author(s):  
Bibhuti Bhusan Pal ◽  
Smruti Ranjan Nayak ◽  
Ashish Kumar Nayak ◽  
Dipti Ranjan Behera ◽  
Swatishree Pany ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Water Samples ◽  
Environmental Water ◽  
High Morbidity ◽  
Rectal Swabs ◽  
Outbreak Areas ◽  
Vibrio Cholerae O1 ◽  
El Tor ◽  
Tribal Areas ◽  
The Relationship

Abstract Cholera localized outbreaks/epidemics accounting for high morbidity and mortality have been reported in different years both from the coastal and tribal districts of Odisha. In the present study, the emergence and spread of two sequential cholera outbreaks reported in July to October 2012 from Rayagada and Kalahandi districts of Odisha was investigated. Environmental water samples from different sources and rectal swabs from diarrhoea patients were analysed for identification, antibiogram profiles and molecular studies using DMAMA-PCR assays. The pulsed field gel electrophoresis (PFGE) was done on some selected Vibrio cholerae O1 strains isolated from these cholera outbreak areas. Results showed 42% of rectal swabs and 2.3% of water samples collected from both the districts were positive for Vibrio cholerae O1 Ogawa biotype El Tor carrying both ctxB1 and ctxB7 genotypes. The common resistance profile of V. cholerae O1 strains was ampicillin, nalidixic acid, furazolidone and co-trimoxazole. The PFGE analysis on selected V. cholerae O1 strains of ctxB1 and ctxB7 genotypes showed three pulsotypes with 96% similarity matrix exhibiting the relationship with their respective water sources. Hence, continuous surveillance is highly essential to monitor the antibiogram profile and changing pattern of ctxB genotypes of V. cholerae O1 in this region.

scholarly journals Particle Diffusometry: An Optical Detection Method for Vibrio cholerae Presence in Environmental Water Samples

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Katherine N. Clayton ◽  
Taylor J. Moehling ◽  
Dong Hoon Lee ◽  
Steven T. Wereley ◽  
Jacqueline C. Linnes ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Water Samples ◽  
Detection Method ◽  
Optical Detection ◽  
Environmental Water ◽  
Environmental Water Samples

scholarly journals Molecular-Beacon Multiplex Real-Time PCR Assay for Detection of Vibrio cholerae

2006 ◽  
Vol 72 (9) ◽  
pp. 6424-6428 ◽  
Author(s):  
Aneta J. Gubala ◽  
David F. Proll
Keyword(s):  
Vibrio Cholerae ◽  
Real Time ◽  
Water Samples ◽  
Real Time Pcr ◽  
Molecular Beacon ◽  
Environmental Water ◽  
Molecular Beacons ◽  
Pcr Assay ◽  
Specificity And Sensitivity ◽  
Pcr Assays

ABSTRACT A multiplex real-time PCR assay was developed using molecular beacons for the detection of Vibrio cholerae by targeting four important virulence and regulatory genes. The specificity and sensitivity of this assay, when tested with pure culture and spiked environmental water samples, were high, surpassing those of currently published PCR assays for the detection of this organism.

scholarly journals Quorum-sensing autoinducers resuscitate dormant Vibrio cholerae in environmental water samples

2013 ◽  
Vol 110 (24) ◽  
pp. 9926-9931 ◽  
Author(s):  
S. M. N. Bari ◽  
M. K. Roky ◽  
M. Mohiuddin ◽  
M. Kamruzzaman ◽  
J. J. Mekalanos ◽  
...  
Keyword(s):  
Quorum Sensing ◽  
Vibrio Cholerae ◽  
Water Samples ◽  
Environmental Water ◽  
Environmental Water Samples

scholarly journals Detection of Vibrio cholerae by Real-Time Nucleic Acid Sequence-Based Amplification

2007 ◽  
Vol 73 (5) ◽  
pp. 1457-1466 ◽  
Author(s):  
Else M. Fykse ◽  
Gunnar Skogan ◽  
William Davies ◽  
Jaran Strand Olsen ◽  
Janet M. Blatny
Keyword(s):  
Nucleic Acid ◽  
Vibrio Cholerae ◽  
Real Time ◽  
Water Samples ◽  
Environmental Water ◽  
Nucleic Acid Sequence ◽  
Environmental Water Samples ◽  
Dnase Treatment ◽  
Nasba Assay ◽  
Toxigenic Strains

ABSTRACT A multitarget molecular beacon-based real-time nucleic acid sequence-based amplification (NASBA) assay for the specific detection of Vibrio cholerae has been developed. The genes encoding the cholera toxin (ctxA), the toxin-coregulated pilus (tcpA; colonization factor), the ctxA toxin regulator (toxR), hemolysin (hlyA), and the 60-kDa chaperonin product (groEL) were selected as target sequences for detection. The beacons for the five different genetic targets were evaluated by serial dilution of RNA from V. cholerae cells. RNase treatment of the nucleic acids eliminated all NASBA, whereas DNase treatment had no effect, showing that RNA and not DNA was amplified. The specificity of the assay was investigated by testing several isolates of V. cholerae, other Vibrio species, and Bacillus cereus, Salmonella enterica, and Escherichia coli strains. The toxR, groEL, and hlyA beacons identified all V. cholerae isolates, whereas the ctxA and tcpA beacons identified the O1 toxigenic clinical isolates. The NASBA assay detected V. cholerae at 50 CFU/ml by using the general marker groEL and tcpA that specifically indicates toxigenic strains. A correlation between cell viability and NASBA was demonstrated for the ctxA, toxR, and hlyA targets. RNA isolated from different environmental water samples spiked with V. cholerae was specifically detected by NASBA. These results indicate that NASBA can be used in the rapid detection of V. cholerae from various environmental water samples. This method has a strong potential for detecting toxigenic strains by using the tcpA and ctxA markers. The entire assay including RNA extraction and NASBA was completed within 3 h.

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