Vibrio cholerae strain typing and phylogeny study based on simple sequence repeats

Vibrio cholerae is the etiological agent of cholera. Its natural reservoir is the aquatic environment. To date, practical typing of V. cholerae is mainly serological and requires about 200 antisera. Simple sequence repeats (SSR), also termed VNTR (for variable number of tandem repeats), provide a source of high genomic polymorphism used in bacterial typing. Here we describe an SSR-based typing method that combines the variation in highly mutable SSR loci, with that of shorter, relatively more stable mononucleotide repeat (MNR) loci, for accurate and rapid typing of V. cholerae. In silico screening of the V. cholerae genome revealed thousands of perfect SSR tracts with an average frequency of one SSR every 152 bp. A panel of 32 V. cholerae strains, representing both clinical and environmental isolates, was tested for polymorphism in SSR loci. Two strategies were applied to identify SSR variation: polymorphism of SSR tracts longer than 12 bp (L-SSR) assessed by capillary fragment-size analysis and MNR polymorphism assessed by sequencing. The nine L-SSR loci tested were all polymorphic, displaying 2 to 13 alleles per locus. Sequence analysis of eight MNR-containing loci (MNR-multilocus sequence typing [MLST]) provided information on both variations in the MNR tract itself, and single nucleotide polymorphism (SNP) in their flanking sequences. Phylogenetic analysis of the combined SSR data showed a clear discrimination between the clinical strains belonging to O1 and O139 serogroups, and the environmental isolates. Furthermore, discrimination between 27 strains of the 32 strains was achieved. SSR-based typing methods combining L-SSR and MNR-MLST were found to be efficient for V. cholerae typing.     

The Vibrio cholerae maneuver

Biofilms are communities of bacteria immersed in an extracellular matrix. Biofilms are considered a defensive strategy that protects bacteria from a hostile environment, including our immune system. Vidakovic et al. recently reported that Vibrio cholerae can build biofilms around immune cells and kill them, discovering an aggressive role for biofilms.     

Bacteriocin Typing of Vibrio cholerae

Bacteriocins of Vibrio cholerae have been demonstrated against enterobacterial and vibrio indicator organisms by conventional techniques. Abundant bacteriocin production took place on casein hydrolysate-yeast extract, tryptic soy, digest broth, proteose-peptone, and neopeptone agars. Essential factors were a citrate-phosphate buffer concentration of 0.5 to 0.7%, at pH 7.5 to 7.6, and cold shock. Thermal treatment of indicator organisms at 45 C for 12 min increased the percentage of typable strains. The bacteriocins of V. cholerae appeared to be powerful diffusible bactericidal agents. By using 8 indicator strains, 11 bacteriocin types have been recognized among 425 strains, of which 87% are typable at present.     

Der Stoffwechsel des Vibrio cholerae

Ohne Zusammenfassung     

Vibrio cholerae non-01 cellulitis

A 46-year-old man presented with pain and fever and a postphlebitic ulcer on his left leg. The wound was suppurative and open at the margins, but there was little underlying fasciitis and no apparent muscle or blood vessel involvement. Three separate wound cultures were obtained at two-day intervals, and all showed only Vibrio cholerae non-01. The patient was successfully treated with cefazolin sodium. This marks the second documented case of V cholerae non-01 type alone as a causative agent of cellulitis, and the first case where no saltwater origin could be demonstrated.     

Zur Biochemie des Vibrio cholerae

Ohne ZusammenfassungMit 3 Textabbildungen.     

Differential Medium for Vibrio cholerae

A differential medium designed for rapid presumptive identification of Vibrio cholerae was described and shown to be useful for enumeration of viable cholera vibrios in the presence of other intestinal bacteria.     

Action of some pancreatic enzymes on Vibrio cholerae

The action of pancreatic amylase, trypsin, lipase, and whole pancreatin was tested on five strains ofVibrio cholerae. Amylase did not act on any strain in concentrations to 10,000 IU/ml whereas trypsin increased vacuolization and lipase enhanced retraction of the protoplasm particularly in 2 of the 5 tested vibrio strains. Pancreatin caused damage hoth of the cell wall and the cytoplasm. It is suggested that these enzymes may play a role in the defense of the body against cholera vibrios.     

Evolutionary relationships of pathogenic clones of Vibrio cholerae by sequence analysis of four housekeeping genes

Studies of the Vibrio cholerae population, using molecular typing techniques, have shown the existence of several pathogenic clones, mainly sixth-pandemic, seventh-pandemic, and U.S. Gulf Coast clones. However, the relationship of the pathogenic clones to environmental V. cholerae isolates remains unclear. A previous study to determine the phylogeny of V. cholerae by sequencing the asd (aspartate semialdehyde dehydrogenase) gene of V. cholerae showed that the sixth-pandemic, seventh-pandemic, and U.S. Gulf Coast clones had very different asd sequences which fell into separate lineages in the V. cholerae population. As gene trees drawn from a single gene may not reflect the true topology of the population, we sequenced the mdh (malate dehydrogenase) and hlyA (hemolysin A) genes from representatives of environmental and clinical isolates of V. cholerae and found that the mdh and hlyA sequences from the three pathogenic clones were identical, except for the previously reported 11-bp deletion in hlyA in the sixth-pandemic clone. Identical sequences were obtained, despite average nucleotide differences in the mdh and hlyA genes of 1.52 and 3.25%, respectively, among all the isolates, suggesting that the three pathogenic clones are closely related. To extend these observations, segments of the recA and dnaE genes were sequenced from a selection of the pathogenic isolates, where the sequences were either identical or substantially different between the clones. The results show that the three pathogenic clones are very closely related and that there has been a high level of recombination in their evolution.     

霍乱弧菌毒力表达调控基因toxR缺失株的构建及其功能的研究

目的　通过构建霍乱弧菌toxR基因缺失株来研究toxR基因对霍乱弧菌减毒菌株IEM101和高产毒株569B毒力表达的调控作用。方法　采用自杀性质粒和接合转移技术,将2个中间含有四环素基因的toxR基因分别与霍乱弧菌减毒株IEM101和高产毒株569B染色体toxR基因重组,从而获得toxR基因缺失株IEM101-4和569B-43,并对2个toxR基因缺失株和其原出发菌株的霍乱肠毒素的产率和主要外膜蛋白图谱进行比较。结果　采用GM1-ELISA检测受测菌CT基因表达,toxR基因缺失株569B-43的P/N值为1.82,而其原出发菌株569B的P/N为4.52,而IEM101和其toxR基因缺失株的P/N值均低于2。采用SDS-PAGE对受试菌外膜蛋白进行分析,toxR基因缺失株569B和IEM101的外膜蛋白图谱相比,均多出2条相对分子质量（Mr）为40×103和43×103外膜蛋白区带。结论　toxR蛋白是霍乱肠毒素基因ctx表达的正调控因子,是霍乱弧菌主要外膜蛋白（Mr为40×103和43×103)编码基因的负调控因子。     

The cytolysin gene of Vibrio vulnificus: sequence and relationship to the Vibrio cholerae E1 Tor hemolysin gene.

A cytolysin of ca. 56 kilodaltons has been suggested as a possible virulence factor in Vibrio vulnificus infections. We sequenced the DNA encoding cytolytic activity and found that the sequence contained two open reading frames, vvhA and vvhB. vvhA encoded the structural gene for the cytolysin and contained the N-terminal amino acid sequence previously reported for the protein. Regions of the vvhA gene showed homology to the structural gene for the Vibrio cholerae E1 Tor hemolysin.     

Immunological properties of complex conjugates based on Vibrio cholerae O1 Ogawa lipopolysaccharide antigen

Host protection by humoral immunity against Vibrio cholerae O1 confers lipopolysaccharide (LPS)-specific vibriocidal antibodies. Levels of relevant specific antibodies are closely related to complement-mediated inactivation of the vibrios inoculum, especially on the mucosal surface of intestine. We have tested complex V. cholerae O1 Ogawa-detoxified lipopolysaccharide (dLPS) conjugates. The first conjugate contained glucan both as the immunomodulator and the matrix; the second conjugate contained immunologically inert amylose as matrix. Both d-LPS conjugates contain multiply attached dLPS antigen. These conjugates elicited a statistically significant increase of antigen-specific IgG levels in mice (P<0.001 and P<0.05, respectively). The specific anti-conjugate IgG and IgA response after the second (booster) dose were significantly higher compared to pre-immune and whole-cell response. The most effective vibriocidal activity was observed in the case of conjugate, with glucan as the matrix. The highest correlation was found between vibriocidal activity and specific IgG2b (r=0.765) and IgA (r=0.887) sera levels. The determination of specific IgG subclasses and IgG2a + 2b/IgG1 ratio revealed a dominant T(H)1 cell response crucial for effective vaccine candidate.     

General Transduction in Vibrio cholerae.

Evidence was obtained for general transduction in Vibrio cholerae. Transduction of three amino acid markers and three antibiotic resistance characters was demonstrated using strains of biotype eltor and biotype cholerae. Some of the genetic characters were transduced from a biotype eltor donor (and its mutant derivatives) to biotype cholerae and eltor recipients. For the genetic traits examined, the frequencies of transduction ranged between 10(-5) and 10(-8). Maximal frequencies were obtained with transducing phage lysates that were irradiated with ultraviolet light. The development of a system of general transduction will now aid in fine structure analysis and detailed mapping of the chromosome of V. cholerae.     

Phage beta interaction with Vibrio cholerae

The role of temperate phage beta in determining the serology and eltor-lytic phage sensitivity in Vibrio cholerae was investigated. The only serological change found in six host strains was a change to roughness. This was accompanied by failure to adsorb several of the lytic phages. Various phage-sensitivity changes were induced by phage beta in two hosts at the post-adsorption level. In strain HP47, three types of progeny were obtained of which one was universally resistant to lytic phages. These untypable lysogens were culturally stable but gave rise to segregants of the rare phage-type 6 on single colony selection.     

2 hemocultures positive for Vibrio cholerae

The authors report two cases of Vibrio cholerae isolation in hemocultures even though the copro-cultures where negative. In both cases, post-operated patients are concerned. The first one had supported a renal transplantation and was under immuno-depressor. The second one, after an aorto-aneurysm surgical treatment suffered from a mesenteric infarct. During the disease the vibrio was found in the hemoculture.     

Siderophore production by Vibrio cholerae.

Vibrio cholerae produces a phenolate-type siderophore that stimulates growth of the organism in low-iron medium. This compound is similar, but not identical, to enterochelin, the siderophore produced by Salmonella and Escherichia coli.     

Adsorption and growth of Vibrio cholerae on chitin.

Incubation of Vibrio cholerae of O-group serotype 1 with chitin particles resulted in adsorption of vibrios onto chitin; chitin-adsorbed V. cholerae survived exposure to acid better than nonadsorbed vibrios. V. cholerae multiplied in dialyzed chitin suspended in 4.2% NaCl, suggesting that adherence to ingested chitin of crustacea might be of epidemiological significance by providing a substrate for vibrio multiplication as well as protection from gastric acid during stomach transit.