Comparative characteristics of the preparations of hemolysin of ctx+ and ctx- Vibrio cholerae eltor and Vibrio cholerae O139 strains

Hemolysin of ctx+ Vibrio cholerae strains was obtained and studied. Ctx+ Vibrio cholerae O1 and O139 strains produced the hemolysin during cultivation in triptone medium without FeCl3. Mol.wt. and the spectrum of lytic activities of hemolysins of ctx+ Vibrio cholerae did not differ from hemolysins of ctx- strains.     

Comparative study of the diagnostic value of new cholera eltor bacteriophages ctx+, ctx-, and KhDF-3, 4, and 5

The comparative evaluation of the diagnostic value of new cholera eltor bacteriophages ctx+ and ctx-, as well as monophages X[symbol: see text]-3, 4, 5, demonstrated their high activity and specificity. Using of these bacteriophages epidemic potential of 95% Vibrio cholerae eltor strains ctx+ and 84.5% of V. cholerae eltor stains ctx- was determined. Commercial monophages X[symbol: see text]-3, 4, 5 were inferior to bacteriophages ctx+ and ctx- in their diagnostic value: only 55% of strains having gene ctxAB were found to be epidemically dangerous, i.e. 45% of strains capable of causing the disease were not detected. On the basis of the results obtained in this investigation cholera eltor bacteriophages ctx+ and ctx- were recommended for introduction into practical use, while further production of cholera diagnostic monophages X[symbol: see text]-3, 4, 5 was recommended to be stopped.     

Multiplex polymerase chain reaction to detect toxigenic Vibrio cholerae and to biotype Vibrio cholerae O1

A multiplex polymerase chain reaction (PCR) was developed to identify cholera toxin-producing Vibrio cholerae and to biotype V. cholerae O1. Enterotoxin-producing V. cholerae strains were identified with a primer pair that amplified a fragment of the ctxA2-B gene. Vibrio cholerae O1 strains were simultaneously differentiated into biotypes with three primers specified for the hlyA gene in the same reaction. The hlyA amplicon in the multiplex PCR serves as an internal control when testing toxin-producing strains, as hlyA gene sequences exist in all tested V. cholerae strains. Enrichment of V. cholerae present on oysters for 6 h in alkaline peptone water before detection by a nested PCR with internal primers for ctxA2-B gene yielded a detection limit lower than 3 colony-forming units (cfu) per gram of food.     

Study of the epidemic significance of noncultivated forms of Vibrio cholerae by the polymerase chain reaction]

A specific method of the isolation of the cholera toxin gene by the directional amplification of DNA in the polymerase chain reaction (PCR) has been developed. The product of this reaction has a molecular weight of 440 sequence pairs and is a DNA fragment located on the A-subunit of V. cholerae gene vct. The sensitivity of the method permits the detection of one bacterial cell in the reaction mixture. The method is effective when V. cholerae purified DNA, cell lysates and the DNA of total microflora isolated from the water of natural springs are used. The study of water samples from natural water bodies by the method of PRC has revealed cholera toxin genes of V. cholerae noncultivated forms ni 5 out of 7 water samples taken from natural water bodies at the regions of Azerbaijan endemic for cholera and made it possible to evaluate the number of V. cholerae. The prospects of using PCR for the control of the epidemiological situation in regions endemic for cholera are discussed.     

Diversity of DNA sequences among Vibrio cholerae O1 and non-O1 isolates detected by whole-cell repetitive element sequence-based polymerase chain reaction

Vibrio cholerae strains isolated from patient, food and environmental sources in Taiwan and reference V. cholerae strains were examined by repetitive element sequence-based PCR (rep-PCR). Specimens from broth cultures were used directly in the PCR mixture with three different primers. The PCR fingerprinting profiles of toxigenic 01 isolates were not only homogeneous with primers from enterobacterial repetitive intergenic consensus (ERIC) sequences, but also allowed the differentiation from non-toxigenic O1 and non-O1 strains. Toxigenic 01 strains were further differentiated into El Tor and classical biotypes with primers designed from ERIC-related sequences of V. cholerae. Primers from the other V. cholerae repetitive DNA sequences, VCR, separated toxigenic El Tor strains into six groups and a unique pattern was also obtained in 16 isolates from imported cases of cholera and imported seafood. The results indicated that rep-PCR can be used to identify and differentiate different toxigenic 01, non-toxigenic 01 and non-O1 V. cholerae isolates.     

Detection of luciferase gene sequence in nonluminescent Vibrio cholerae by colony hybridization and polymerase chain reaction.

Bioluminescence is a trait observed among approximately 10% of Vibrio cholerae isolates. We have demonstrated that not only do some strains of V. cholerae produce low levels of light, undetectable by the human eye, but the luciferase gene sequence is present in strains of V. cholerae which emit no detectable light, evidenced by hybridization with a luciferase DNA probe. Comparisons of the amino acid sequences of luciferase enzymes of marine species have shown that these proteins have diverged to the point where they have only short regions of amino acid identity. The polymerase chain reaction method of DNA amplification with oligonucleotide primers based on these regions was used to isolate a region of the luxA gene from both luminescent and nonluminescent V. cholerae strains. The nucleotide sequence of this region was determined and reveals that nonluminescent V. cholerae have 99.7% nucleotide sequence similarity in this region with the luminescent biovar V. cholerae bv. albensis as well as significant similarity to other species of bioluminescent bacteria, a finding that is in accord with the hypothesis that these species have a common luminescent ancestor, most probably from the marine environment.     

Detection of luciferase gene sequence in nonluminescent Vibrio cholerae by colony hybridization and polymerase chain reaction.

Bioluminescence is a trait observed among approximately 10% of Vibrio cholerae isolates. We have demonstrated that not only do some strains of V. cholerae produce low levels of light, undetectable by the human eye, but the luciferase gene sequence is present in strains of V. cholerae which emit no detectable light, evidenced by hybridization with a luciferase DNA probe. Comparisons of the amino acid sequences of luciferase enzymes of marine species have shown that these proteins have diverged to the point where they have only short regions of amino acid identity. The polymerase chain reaction method of DNA amplification with oligonucleotide primers based on these regions was used to isolate a region of the luxA gene from both luminescent and nonluminescent V. cholerae strains. The nucleotide sequence of this region was determined and reveals that nonluminescent V. cholerae have 99.7% nucleotide sequence similarity in this region with the luminescent biovar V. cholerae bv. albensis as well as significant similarity to other species of bioluminescent bacteria, a finding that is in accord with the hypothesis that these species have a common luminescent ancestor, most probably from the marine environment.     

Discrimination of gamma-irradiated and nonirradiated Vibrio vulnificus by using real-time polymerase chain reaction

Aims: To develop a PCR strategy for Vibrio vulnificus in irradiated foods. Methods and Results: Real‐time PCR was used to discriminate between DNA from γ‐irradiated and nonirradiated cells. γ‐Irradiation at 1·08 KGy and above of cell suspensions containing 1 × 10⁶ CFU ml^?1 resulted in 0 CFU ml^?1. Real‐time PCR was able to detect 86·6% destruction by 1·08 KGy, while ethidium bromide monoazide (EMA) real‐time PCR was able to detect 93·2% destruction at this dose. With 3·0 and 5·0 KGy, EMA real‐time PCR was able to detect 99·3% and 100% destruction, respectively. Conclusions: The inability to detect via PCR extensively degraded DNA resulting from γ‐irradiation can be taken as evidence of cell death. The increased ability of EMA to further reduce the detectable number of target sequences via PCR, with DNA from cells exposed to increased doses of γ‐irradiation, can be considered to reflect the accompanying increase in membrane damage which allows EMA to penetrate the cells. Significance and impact of the study: This is the first study that has made use of the PCR to discriminate between γ‐irradiated and nonirradiated bacterial cells and has led to insights regarding the ability of the PCR to discriminate between nonirradiated and γ‐irradiation destroyed cells.     

Characterization of Vibrio cholerae O1 isolates responsible for cholera outbreaks in Kenya between 1975 and 2017

Kenya is endemic for cholera with different waves of outbreaks having been documented since 1971. In recent years, new variants of Vibrio cholerae O1 have emerged and have replaced most of the traditional El Tor biotype globally. These strains also appear to have increased virulence, and it is important to describe and document their phenotypic and genotypic traits. This study characterized 146 V. cholerae O1 isolates from cholera outbreaks that occurred in Kenya between 1975 and 2017. Our study reports that the 1975–1984 strains had typical classical or El Tor biotype characters. New variants of V. cholerae O1 having traits of both classical and El Tor biotypes were observed from 2007 with all strains isolated between 2015 and 2017 being sensitive to polymyxin B and carrying both classical and El Tor type ctxB. All strains were resistant to Phage IV and harbored rstR, rtxC, hlyA, rtxA and tcpA genes specific for El Tor biotype indicating that the strains had an El Tor backbone. Pulsed field gel electrophoresis (PFGE) genotyping differentiated the isolates into 14 pulsotypes. The clustering also corresponded with the year of isolation signifying that the cholera outbreaks occurred as separate waves of different genetic fingerprints exhibiting different genotypic and phenotypic characteristics. The emergence and prevalence of V. cholerae O1 strains carrying El Tor type and classical type ctxB in Kenya are reported. These strains have replaced the typical El Tor biotype in Kenya and are potentially more virulent and easily transmitted within the population.     

Detection and differentiation of the gene for toxin co-regulated pili (tcpA) in Vibrio cholerae non-O1 using the polymerase chain reaction

Abstract The polymerase chain reaction has been used to differentiate the gene which encodes the toxin co-regulated pili ( tcpA ) of the El Tor and classical biotypes of Vibrio cholerae O1. The same PCR primers were applied to strains belonging to non-O1 serogroups that produced cholera toxin. The size of fragment amplified was either identical to the tcpA of biotype El Tor (471 bp) or to the tcpA of biotype classical (617 bp). All strains belonging to the novel epidemic serogroup O139 generated a 471-bp fragment identical to El Tor tcpA . The present study suggests that there may be an association between non-O1 serogroup and tcpA type.     

Molecular evolution of the seventh-pandemic clone of Vibrio cholerae and its relationship to other pandemic and epidemic V. cholerae isolates.

Genetic variation and molecular evolution within the seventh-pandemic clone of Vibrio cholerae O1 and its relationship to other V. cholerae isolates were examined by studying 58 clinical isolates that were epidemiologically unassociated and isolated from patients in different countries over 62 years (1931 to 1993). The sample consisted of 45 isolates from the seventh cholera pandemic (1961 to the present), 3 from the sixth pandemic, 3 from sporadic El Tor outbreaks prior to the seventh pandemic, 2 from the U.S. Gulf Coast, and 5 O139 Bengal isolates. Ribotyping detected 11 polymorphic restriction sites within the seventh-pandemic isolates and showed major differences in ribotypes in comparison with sixth- and pre-seventh-pandemic isolates. O139 isolates were very similar to isolates from the start of the seventh pandemic, differing at only two sites. The majority of seventh-pandemic isolates fall into two groups, the first present from 1961 to the present and found only in Asia and the second arising in 1966 and spreading worldwide. Both groups underwent change over time, allowing a provisional estimate for the nucleotide substitution rate within the seventh pandemic clone.     

Adhesive and other properties of Vibrio cholerae tcp+ ctx- isolated from environmental objects in the Rostov region in 2002

The adhesive, hemolytic and triacylglycerol lipase activity of 4 V. cholerae ctp+ ctx- cultures, sensitive to bacteriophage ctx+, isolated from the Don and sewage water were tested. All these cultures were found to induce hemolysis of sheep red blood cells in the Greig test in 2 hours, possessed triacylglycerol lipase activity, but had no adhesive properties. By these parameters--atoxigenicity and the absence of adhesive properties--the isolated V. cholerae strains were characterized as avirulent and epidemically safe.     

Molecular typing of epidemic and nonepidemic Vibrio cholerae isolates and differentiation of V. cholerae and V. mimicus isolates by PCR-single-strand conformation polymorphism analysis

AIMS: To examine the utility of polymerase chain reaction (PCR)-single-strand conformation polymorphism (SSCP) analysis to differentiate epidemic and nonepidemic Vibrio cholerae isolates as well as to differentiate V. cholerae and Vibrio mimicus isolates. METHODS AND RESULTS: By both PCR-restriction fragment length polymorphism (RFLP) and PCR-SSCP analysis of groEL-I on chromosome 1 and groEL-II on chromosome 2, V. cholerae isolates gave distinct profiles compared with V. mimicus isolates. In addition, PCR-SSCP analysis of groEL-I and groEL-II could differentiate between V. cholerae epidemic and nonepidemic isolates. Interestingly, the relationships among strains based on groEL-I from chromosome 1 and groEL-II from chromosome 2 were congruent with each other, highlighting the conserved evolutionary history of both chromosomes in this species. CONCLUSIONS: PCR-SSCP is a powerful typing technique, which has the ability to differentiate V. cholerae and V. mimicus isolates. The epidemic V. cholerae O1/O139 serogroup isolates represent a clonal complex distinct from non-O1/non-O139 isolates that can be identified by PCR-SSCP analysis. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlights the effectiveness of using reliable molecular typing methods and in particular PCR-SSCP, to identify genetic variation among V. cholerae and V. mimicus isolates.     

Detection by multiplex polymerase chain reaction and typing of Chlamydia trachomatis isolates

Abstract The multiplex polymerase chain reaction (PCR) was applied for the detection of the Chlamydia trachomatis chromosome and plasmid. The multiplex PCR demonstrated a sensitivity of 0.8 fg of chlamydial DNA, corresponding to the detection of about 5 copies of the plasmid. Analysis of 195 genital specimens collected randomly from a female population, showed that the multiplex PCR is more sensitive and rapid than culturing for detecting Chlamydia trachomatis . Moreover, sequencing of the II variable domain of the ompl gene, directly from DNA of the clinical specimens, appears to be a simple and rapid method for determining serovar isolates.     

Comparison of different primers for rapid detection of Vibrio parahaemolyticus using the polymerase chain reaction

AIMS: The aim of this study was to compare different primers for rapid and effective detection of Vibrio parahaemolyticus by polymerase chain reaction (PCR). METHODS AND RESULTS: A total of four pairs of primers, three previously published and one based on a newly developed V. parahaemolyticus metalloprotease (vpm) gene, have been assayed for PCR detection of V. parahaemolyticus. They have been tested for specificity and sensitivity on a total of 101 strains including reference and environment isolates belonging to V. parahaemolyticus and other species in Vibrio. Of the four sets of primers tested, the one designed on the basis of the metalloprotease gene (675 bp) gave optimal results with bacterial strains examined as they only amplified the specific fragment in strains that had been genetically and biochemically assessed as V. parahaemolyticus and the limit of detection was 4 pg of purified target DNA. CONCLUSIONS: The primers designed on the metalloprotease gene gave optimal results for specific, sensitive and rapid detection of V. parahaemolyticus by PCR. SIGNIFICANCE AND IMPACT OF THE STUDY: PCR amplification with the optimal primer set VPM1/VPM2 could facilitate the rapid diagnosis and surveillance of potentially pathogenic strains of V. parahaemolyticus and reduce food-borne illness.