The detection of Vibrio cholerae 01 from the aquatic environment of Daro and Bintulu in Sarawak was carried out following an outbreak of cholera. Conventional culture methods and detection of ctx gene by polymerase chain reaction technique were carried out on 80 water samples. Only one sample was positive by culture methods while 8 were positive by PCR. DNA finger printing by pulsed-field gel electrophoresis showed that the clinical isolates in Daro and Bintulu were genetically identical while the environmental isolate was closely related. Recovery of Vibrio cholerae by culture method is poor and newer methods of detection should be developed.
Vibrio cholerae O139 isolated from different countries, as well as from different locations within a country, were examined using macrorestriction DNA analysis to determine the clonality of the O139 strains. NotI digests of genomic DNA of representative strains from Nepal, India, Bangladesh, China, Thailand, and Malaysia revealed very similar but not identical patterns. Examinations of the banding patterns generated by pulsed-field gel electrophoresis of strains isolated within countries revealed complete homogeneity. These results further reiterate the spread of an identical clone of V. cholerae O139 although it appears that genetic polymorphism among the O139 strains is becoming apparent.
The importance of bacteria-suspending media and fingertip positions on the survival of Vibrio cholerae on human fingertips were examined. Vibrios were suspended in phosphate-buffered saline (PBS), PBS with albumin, and PBS with agarose. Each type of preparation was inoculated on the fingerpads, the hyponychia, or the eponychia and lateral nail grooves of the fourth, third and second fingers of a volunteer's hand. The last finger inoculated was immediately washed with PBS and the washing collected for examination ("0 minute" exposure). The third and fourth inoculated fingers were likewise washed for examination 2 and 5 minutes later, respectively. The vibrios obtained from the washings were enumerated by culture. For each of the different groups, which consisted of a different inoculated fingertip position, bacteria-suspending medium and exposure period of 2 or 5 minutes, the proportion of replicate inoculated fingers which retained viable vibrios (isolation rate) and the mean number of surviving vibrios, as a percentage of the inoculated vibrios at "0 minute exposure" (survival rate) were as follows: finger pads: vibrios in PBS, 2 minutes post-inoculation (isolation rate, 25%; mean survival rate, 0.002%); 5 minutes post-inoculation (isolation rate, 0%; mean survival rate, 0%). PBS-albumin: 2 minutes post-inoculation (60%, 0.004%); 5 minutes post-inoculation (40%, 0.03%). PBS-agarose: 2 minutes post-inoculation (100%, 24%); 5 minutes post-inoculation (38%, 0.005%). Lateral nail grooves and eponychia: PBS: 2 minutes post-inoculation (100%, 2.2%); 5 minutes post-inoculation (44%, 0.2%). PBS-agarose: 2 minutes post-inoculation (100%, 32%); 5 minutes post-inoculation (100%, 0.7%). Hyponychia: PBS: 2 minutes post-inoculation (100%, 8%); 5 minutes post-inoculation (100%, 0.2%). PBS-agarose: 2 minutes post-inoculation (100%, 46%); 5 minutes post-inoculation (100%, 8%). The results show that vibrios in moisture-retaining medium (PBS-agarose) and inoculated on a sheltered fingertip locations (hyponychium) have the best survival rates. However, the high survival rate was maintained briefly.
Four Vibrio cholerae O139 Bengal strains isolated from surface water were characterized by antibiotic resistance, plasmid profile, presence of cholera toxin gene and random amplification of polymorphic DNA (RAPD) analysis. All four strains exhibit multiple resistance towards the antibiotics tested with a multiple antibiotic resistance index of 0.5-0.66, and harboured a 2.0 MDa non-conjugative plasmid. The Vibrio cholerae O139 Bengal were positive for the cholera toxin gene. Antibiotyping and random amplification of polymorphic DNA analysis with four primers proved to be useful in discriminating the isolates. RAPD proved to be more sensitive. These results reveal that there is significant genetic diversity among the Vibrio cholerae O139 Bengal strains studied.
Vibrio cholerae is a human pathogen that causes mild to severe diarrheal illnesses and has major public health significance. Herein, we present a thermostabilized electrochemical genosensing assay combining the use of magnetic beads as a biorecognition platform and gold nanoparticles as a hybridization tag for the detection and quantification of V. cholerae lolB gene single-stranded asymmetric PCR amplicons as an alternative to the time-consuming classical isolation method. This thermostabilized, pre-mixed, pre-aliquoted and ready-to-use magnetogenosensing assay simplified the procedures and permitted the reaction to be conducted at room temperature. The asymmetric PCR amplicons were hybridized to a magnetic bead-functionalized capture probe and a fluorescein-labeled detection probe followed by tagging with gold nanoparticles. Electrochemical detection of the chemically dissolved gold nanoparticles was performed using the differential pulse anodic stripping voltammetry method. The real-time stability evaluation of thermostabilized assay was found to be stable for at least 180 days at room temperature (25-30°C). The analytical specificity of the assay was 100%, while its analytical sensitivity was linearly related to different concentrations of 200-mer synthetic target, purified genomic DNA, and bacterial culture with a limit of detection (LoD) of 3.9nM, 5pg/µl, and 10(3)CFU/ml, respectively. The clinical applicability of the assay was successfully validated using spiked stool samples with an average current signal-to-cut-off ratio of 10.8. Overall, the precision of the assay via relative standard deviation was <10%, demonstrating its reliability and accuracy.
A disposable horseradish peroxidase (HRP)-based electrochemical genosensor was developed for chronoamperometric detection of single-stranded asymmetric lolB gene PCR amplicon (118 bp in length) of the food-borne pathogen, Vibrio cholerae. A two-step sandwich-type hybridization strategy using two specific probes was employed for specific detection of the target single-stranded DNA (ssDNA). The analytical performances of the detection platform have been evaluated using a synthetic ssDNA (ST3) which was identical to the target single-stranded amplicon and a total of 19 bacterial strains. Under optimal condition, ST3 was calibrated with a dynamic range of 0.4883-15.6250 nM. By coupling asymmetric PCR amplification, the probe-based electrochemical genosensor was highly specific to the target organism (100% specificity) and able to detect as little as 0.85 ng/μl of V. cholerae genomic DNA.
The genetic diversity or clonality among Vibrio cholerae O1, O139 and non-O1/ non-O139 of clinical and environmental origin using ribotyping and PFGE was performed in order to ascertain the public health implications of the different genotypes circulating within the Malaysian environment. Using an in-house typing scheme, of the 214 strains included, 202 strains were isolated locally between 1992 and 1998, seven were obtained from Bangladesh and five were reference strains. Amongst the 176 El Tor O1 strains, 152 clinical strains demonstrated five ribotypes--E1a, E1b, E2a, E3 and E1c. E1b was the most predominant ribotype demonstrated by 84% of the El Tor O1 strains and was present in all years demonstrating that this strain was intrinsic to Malaysia. PFGE analysis of these strains demonstrated minimal variation amongst the 15 PFGE profiles obtained. Ribotpye E2a amongst five clinical and two environmental O1 strains, were from one location and had previously been reported in Indonesia and the Philippines, thus demonstrating strong evidence that these strains may have been imported into Malaysia. Among Vibrio cholerae O139 strains, 91.7% were of ribotype A1a similar to the original O139, while two others were of ribotype A1b and one of A1e, corresponding to ribotypes 1, 2 and 3 of Dalsgaard and colleagues' scheme for O139 strains. PFGE analysis demonstrated that 89% of ribotype A1a could be differentiated into three PFGE genotypes which were very closely related. The eight non-O1/non-O139 serogroup strains were heterogeneous in both ribotype and PFGE patterns.
Bacterial resistance to various antimicrobial agents is common in area with high usage of antibiotics. In this study, the data on antimicrobial susceptibility patterns of Vibrio cholerae O1 from patients during an outbreak period was found to be high but variable rates of multidrug resistance. Thirty-two of 33 V. cholerae isolates harboured the tcp, ctx, zot and ace genes, suggesting their possible roles in the outbreak cases. We analyzed the molecular diversity of a total of 33 strains of V. cholerae O1 isolated from 33 patients between November 1997 and April 1998 using random amplified polymorphic DNA (RAPD) analysis. The 30 typable isolates could be separated into four major clusters containing 5, 17, 2 and 6 isolates, respectively. However, no particular RAPD pattern was predictive of a particular pattern of antibiotic susceptibility. The findings of this study showed that multiple clones seemed to be responsible for cases in the outbreaks in the study area.
Live eels and processed fish products from Malaysia are routinely checked for microbial pathogens before export to Japan. The eels and water from the ponds are screened for Vibrio cholerae and Salmonella spp, whereas the processed fish products are tested for microbial contamination (aerobic plate count), coliforms, E. coil and Vibrio cholerae. Results showed that live eels and water samples were negative for Vibrio cholerae but Salmonella spp were isolated occasionally. Various types of processed fish products had counts below 1.0 x 10(5) whilst coliforms, E. coli and Vibrio cholerae were absent. Records available showed that procedures involved in the production and transportation of live eel, preparation and processing of fish products have resulted in relatively safe food products.
This paper describes a new bacterial white spot syndrome (BWSS) in cultured tiger shrimp Penaeus monodon. The affected shrimp showed white spots similar to those caused by white spot syndrome virus (WSSV), but the shrimp remained active and grew normally without significant mortalities. The study revealed no evidence of WSSV infection using electron microscopy, histopathology and nested polymerase chain reaction. Electron microscopy indicated bacteria associated with white spot formation, and with degeneration and discoloration of the cuticle as a result of erosion of the epicuticle and underlying cuticular layers. Grossly the white spots in BWSS and WSS look similar but showed different profiles under wet mount microscopy. The bacterial white spots were lichen-like, having perforated centers unlike the melanized dots in WSSV-induced white spots. Bacteriological examination showed that the dominant isolate in the lesions was Bacillus subtilis. The occurrence of BWSS may be associated with the regular use of probiotics containing B. subtilis in shrimp ponds. The externally induced white spot lesions were localized at the integumental tissues, i.e., cuticle and epidermis, and connective tissues. Damage to the deeper tissues was limited. The BWS lesions are non-fatal in the absence of other complications and are usually shed through molting.
The importance of house fly (Musca domestica L) wings in mechanical transmission of bacteria was studied. A droplet of phosphate-buffered saline containing Vibrio cholerae was rolled along one wing of each house fly. None adhered to the wings but small proportions of the bacterium were isolated from about half the wings. Vibrio cholerae was spread onto the ventral wing surfaces of each unconscious house fly which then was placed inside a bottle. When it regained consciousness, the types of activity it performed over five minutes were noted before the house fly was killed and the bacteria on its wings numerated. Control were house flies killed before inoculation. The proportion of house flies with bacteria on their wings and the mean number of bacteria remaining were significantly less on live house flies than killed controls. Among the live house flies, bacteria were detected on fewer house flies which flew (25%) than those which did not fly (81%). In addition, the mean number of bacteria on the former was significantly less than the latter (5 against 780 colonies). However, both these parameters were not significantly different between the group which performed and the group which did not perform wing grooming; takeoff and alighting over short distances, and somersaulting. Wings of unconscious house flies tethered by their thoraxes were inoculated with V. cholerae. After regaining consciousness, the house flies were allowed to move their wings in flight motions for up to 30 seconds. Small proportions of bacteria remained on all the house flies. House flies were placed in a chamber containing a liquid bait spiked with V. cholerae. After two hours, 10 were removed sequentially and cultured for V. cholerae. The bacterium was isolated from four house flies: two from the legs, and two others from their bodies minus legs and wings. In conclusion, house fly wings do not play an important role in mechanical transmission of bacteria suspended in a non-adhering liquid medium because of the low transfer rate of the bacteria to the wings and poor retention of bacteria on the wings during normal house fly activities.
We report a highly sensitive and selective multiplex assay by empowering an electrochemical DNA sensor with isothermal rolling circle amplification. The assay could simultaneously detect and discriminate three common entero-pathogens in a single reaction, with femtomolar sensitivity. It is useful for field- or resource-limited settings.
Cholera, caused by Vibrio cholerae is a foodborne disease that frequently reported in food and water related outbreak. Rapid diagnosis of cholera infection is important to avoid potential spread of disease. Among available diagnostic platforms, loop-mediated isothermal amplification (LAMP) is regarded as a potential diagnostic tool due to its rapidity, high sensitivity and specificity and independent of sophisticated thermalcycler. However, the current LAMP often requires multiple pipetting steps, hence is susceptible to cross contamination. Besides, the strict requirement of cold-chain during transportation and storage make its application in low resource settings to be inconvenient. To overcome these problems, the present study is aimed to develop an ambient-temperature-stable and ready-to-use LAMP assay for the detection of toxigenic Vibrio cholerae in low resource settings. A set of specific LAMP primers were designed and tested against 155 V. cholerae and non-V. cholerae strains. Analytical specifity showed that the developed LAMP assay detected 100% of pathogenic V. cholerae and did not amplified other tested bacterial strains. Upon testing against stool samples spiked with toxigenic V. cholerae outbreak isolates, the LAMP assay detected all of the spiked samples (n = 76/76, 100%), in contrast to the conventional PCR which amplified 77.6% (n = 59/76) of the tested specimens. In term of sensitivity, the LAMP assay was 100-fold more sensitive as compared to the conventional PCR method, with LOD of 10 fg per μL and 10 CFU per mL. Following lyophilisation with addition of lyoprotectants, the dry-reagent LAMP mix has an estimated shelf-life of 90.75 days at room temperature.
A PCR assay has been developed based on a lolB (hemM) gene, which was found to be highly conserved among the Vibrio cholerae species but non-conserved among the other enteric bacteria. The lolB PCR detected all O1, O139 and non-O1/non-O139 serogroup and biotypes of V. cholerae. The analytical specificity of this assay was 100% while the analytical sensitivity was 10 pg/microL and 10(3) CFU/mL at DNA and bacterial level respectively. The diagnostic sensitivity and specificity was 98.5% and 100% respectively.
In this study, we developed a nucleic acid-sensing platform in which a simple, dry-reagent-based nucleic acid amplification assay is combined with a portable multiplex electrochemical genosensor. Preparation of an amplification reaction mix targeting multiple DNA regions of interest is greatly simplified because the lyophilized reagents need only be reconstituted with ultrapure water before the DNA sample is added. The presence of single or multiple target DNAs causes the corresponding single-stranded DNA (ssDNA) amplicons to be generated and tagged with a fluorescein label. The fluorescein-labeled ssDNA amplicons are then analyzed using capture probe-modified screen-printed gold electrode bisensors. Enzymatic amplification of the hybridization event is achieved through the catalytic production of electroactive α-naphthol by anti-fluorescein-conjugated alkaline phosphatase. The applicability of this platform as a diagnostic tool is demonstrated with the detection of toxigenic Vibrio cholerae serogroups O1 and O139, which are associated with cholera epidemics and pandemics. The platform showed excellent diagnostic sensitivity and specificity (100%) when challenged with 168 spiked stool samples. The limit of detection was low (10 colony-forming units/ml) for both toxigenic V. cholerae serogroups. A heat stability assay revealed that the dry-reagent amplification reaction mix was stable at temperatures of 4-56 °C, with an estimated shelf life of seven months. The findings of this study highlight the potential of combining a dry-reagent-based nucleic acid amplification assay with an electrochemical genosensor in a more convenient, sensitive, and sequence-specific detection strategy for multiple target nucleic acids.
A chromatographic immunoassay cholera antigen detection kit, the Cholera Spot test, was evaluated. The test was found to be specific with a sensitivity of 10(6) cfu/ml for the direct detection of V. cholerae in simulated stool specimens and 10 cfu/ml in simulated cotton-tipped swab specimens after overnight incubation in alkaline peptone water. This enables early recognition of cholera cases and their contacts so that prevention and control measures can be promptly instituted.