A phenotypic and genetic study of sucrose nonfermenting strains ofVibrio mimicus andVibrio cholerae

Sixty-six strains unable to ferment sucrose and resemblingVibrio mimicus andV. cholerae were submitted to an extensive phenotypic characterization. DNA-DNA homology among selected strains and the type strain ofV. cholerae was studied by the S1 endonuclease method. Seven sucrose-negative strains were shown to have the phenotypic properties of and a high percentage DNA relatedness toV. cholerae and a low level of homology withV. mimicus. Eight luminescent strains phenotypically most closely resembledV. mimicus; however, two of these were shown to have a high level of DNA homology withV. cholerae and a low level of relatedness toV. mimicus. A single strain was found to be phenotypically and genetically unrelated to eitherV. cholerae orV. mimicus and may represent a new species. The remaining strains were phenotypically shown to beV. mimicus, and selected strains were shown to have a high percentage DNA homology withV. mimicus and a low level of homology withV. cholerae. Problems associated with the identification of these strains and differential traits are discussed.     

Two strains ofVibrio cholerae non-O1 possessing somatic (O) antigen factors in common withV. cholerae serogroup O139 synonym “Bengal”

Two strains (O22 reference strain, 169–68, and strain 490–93 isolated from a patient with diarrhea in Thailand) ofVibrio cholerae non-O1 possessing somatic (O) antigen factors in common withV. cholerae O139 synonym Bengal are described. The O antigens of these two strains were closely related to that ofV. cholerae O139 in an a,b-a,c type of relationship, but were not completely identical with serogroup O139. Therefore, both these strains are not classified into the O139 serogroup ofV. cholerae, because they have their own major antigens. As the strain 490–93 could not be placed into any of the 154 established O serogroups ofV. cholerae, this strain was assigned to a new serogroup, O155. For practical use, the diagnostic antiserum prepared against the O139 reference strain (MO45, ATCC 51394) ofV. cholerae must be absorbed with reference strains 169–68 and 490–93 representing serogroups O22 and O155 ofV. cholerae to remove cross-reacting agglutinins of the O22 and O155 strains, respectively.     

Extended serotyping scheme forVibrio cholerae

Fifty-seven new O serogroups have been added to the existing serotyping scheme ofVibrio cholerae to extend the scheme from O84 to O140. Prominent new additions were serogroups O139 and O140. The reference strain of O139 was isolated from a patient from an epidemic of cholera-like diarrhea in Madras, Southern India. Serogroup O140 was assigned to a group ofV. cholerae strains which were tentatively named as the Hakata serogroup and which possessed the C (Inaba) factor but not the B (Ogawa) nor the A (major specific antigen of O1 serogroup ofV. cholerae). As all antisera against reference strains ofV. cholerae contained some amount of antibody to the rough (R) antigen, all diagnostic O antisera must be absorbed with the reference rough strain, CA385.     

Sugar Composition of Lipopolysaccharides of Family Vibrionaceae

A comparative study was carried out on the sugar composition of lipopolysaccharides (LPS) isolated from representative strains of members of the family Vibrionaceae including all of the constituting genera, i.e., Vibrio, Aeromonas, Photobacterium, Plesiomonas, and Lucibacterium. More than 100 strains were examined. It was found that, with the exception of Vibrio parahaemolyticus 06, 2-keto-3-deoxyoctonate (KDO), known generally as a component sugar in the core region of usual gram-negative bacterial LPS, is virtually absent from LPS of the Vibrionaceae strains so far examined. Furthermore, mannose was also lacking in LPS of Vibrionaceae strains with the exception of only one strain, A. anaerogenes (ATCC 15467). Instead, some KDO-like substances were found in LPS from Vibrio (“Beneckea”) nereida (ATCC 25917) and Plesiomonas shigelloides including the type strain (ATCC 14029), the same as those found in LPS from V. parahaemolyticus O7 and O12, and three strains of V. alginolyticus. These substances were strongly positive in the periodate-thiobarbituric acid test, yielding a color with maximum absorption at 549 nm. The spectra were identical to that of KDO, whereas substances differed from KDO at least in behavior in high-voltage paper electrophoresis and thin-layer chromatography. A particularly interesting feature from the chemotaxonomical point of view was found in the sugar composition of LPS isolated from V. cholerae. Fructose was present exclusively in LPS of V. cholerae (both O1 and non-O1 groups and classical and eltor biotypes) with the exception of one strain of Photobacterium phosphoreum (NCMB 844). In addition, a pair of rarely occurring amino sugars, perosamine and quinovosamine, was found in LPS from O1 group V. cholerae regardless of either the biotype (classical or eltor) or the serotype (Inaba or Ogawa), whereas this pair was not present in non-O1 group V. cholerae (the so-called NAG vibrios). This feature was confirmed with LPS from more than 30 additional strains of O1 group V. cholerae isolated from patients. The virtual absence of KDO in LPS of the family Vibrionaceae was demonstrated for the first time in this study. These results are compatible with the interpretation that the absence of KDO in LPS can be used as one of the taxonomical characteristics of Vibrionaceae in addition to (G+C) content, DNA (or RNA) homology, and numerical analysis data.     

Phenotypic characterization of clinical and environmental isolates ofVibrio cholerae from Australia

One hundred fifty-seven isolates possessing the biochemical traits associated withVibrio cholerae were submitted to an extensive phenotypic characterization. A numerical analysis of the results suggested that isolates presently assigned to the biotypescholerae, eltor, andalbensis ofV. cholerae do not possess consistent phenotypic differences supporting their separation into distinct biotypes. The results further indicated that clinical and environmental isolates of non-O1 serotypes ofV. cholerae are phenotypically indistinguishable from strains ofV. cholerae serotype O1. This study also confirmed the persistent presence ofV. cholerae in the Australian environment.     

Occurrence of <Emphasis Type="Italic">Vibrio cholerae</Emphasis> non-O1 in three wastewater treatment plants in Agadir (Morocco)

A total of 21 isolates of Vibrio cholerae non-O1 strains were isolated from three wastewater treatment plants in Agadir, Morocco. The isolates were analyzed by biochemical analysis, antibiogram, pulsed-field gel electrophoresis and the MALDI-TOF patterns of their protein masses were compared. Over 67% of isolates were susceptible to antimicrobial agents tested and 14% proved resistant to both trimethoprim-sulfamethoxazole and nalidixic acid. Typing by pulsed-field gel electrophoresis with NotI digestion revealed that the V. cholerae non-O1 strains from Agadir (Morocco) have a lower level of genetic homogeneity, the restriction patterns of whole-chromosomal DNA grouped the V. cholerae O1 and V. alginolyticus strains into a separate cluster from V. metschnikovii and V. cholerae non-O1 isolates. Furthermore, to gain additional analytical accuracy and reliability in the analysis we used dendrogram based on MALDI-TOF spectral patterns generated by the BioTyper 1.1™ software. All m/z signatures of all strains tested indicate that the mass spectral data contained sufficient information to distinguish between strains of V. cholerae.     

Detection of cholera (ctx) and zonula occludens (zot) toxin genes in Vibrio cholerae O1, O139 and non-O1 strains

Vibrio cholerae O1 and V. cholerae non-O1 strains isolated from environmental samples collected in São Paulo, Brazil, during cholera epidemics and pre-epidemic periods were examined for the presence of toxin genes. V. cholerae O1 strains isolated from clinical samples in Peru and Mexico, and V. cholerae O139 strains from India were also examined for the presence of ctx (cholera toxin gene) and zot (zonula occludens toxin gene) by polymerase chain reaction (PCR). A modified DNA-extraction method applied in this study yielded satisfactory recovery of genomic DNA from vibrios. Results showed that strains of V. cholerae O1 isolated during the preepidemic period were ctx ^-/zot ^- whereas strains isolated during the epidemic were ctx ⁺/zot ⁺. All V. cholerae non-O1 strains tested in the study were ctx ^-/zot ^-, whereas all V. cholerae O139 strains were ctx ⁺/zot ⁺. Rapid detection of the virulence genes (ctx and zot) can be achieved by PCR and this can serve as an important tool in the epidemiology and surveillance of V. cholerae.     

Morphological and physical characterization of the capsular layer of Vibrio cholerae O139

The morphological and physical characteristics of the capsule of Vibrio cholerae O139 were examined. An electron microscopic study using the freeze-substitution technique showed that all of the V. cholerae strains of the O139 serogroup examined have a very thin fibrous layer on the outside of the outer membrane. In contrast, the mutants of strain O139, strain MO10T4 (which lacks capsule synthesis), and strain Bengal-2R1 (which fails to synthesize both the capsule and the O-antigen of lipopolysaccharide) were all found to have lost the surface layer. In addition, the capsule layer could also not be observed on the surface of V. cholerae strain O1. To determine the biological characteristics of the capsule of strains of the O139 serogroup, we investigated the serum killing activity and bacterial phagocytosis by polymorphonuclear leukocytes. The O139 strains were more resistant to the serum killing activity than were the V. cholerae O1 strain and the O139 mutant strains, thus suggesting that the existence of the capsule gave a serum-resistant character to the O139 strains. The surface character of the O139 strains had the same hydrophobic character as did that of the O139 mutant strains and the O1 strain. In addition, all the V. cholerae O1 and O139 strains examined, including the mutant strains, were effectively ingested by the human polymorphonuclear leukocytes. The number of ingested bacteria was not significantly different among the strains, and the ingestion of the acapsular O139 mutants thus showed that the capsule does not play an antiphagocytic role. These data suggest that the capsule of V. cholerae O139 has a physiological function different from that of the ordinal hydrophilic capsule that is found in invasive bacteria such as Klebsiella pneumoniae. Received: 23 March 1998 / Accepted: 28 July 1998     

Molecular Epidemiology of Vibrio cholerae O1 Isolated from Sporadic Cholera Cases in Okinawa,Japan

In July 1994, 6 cholera cases due to Vibrio cholerae O1 El Tor Ogawa sporadically appeared in Okinawa. All 6 patients had no history of traveling abroad. In the period of this cholera outbreak, a strain of V. cholerae O1 El Tor Ogawa was detected from an imported fish at the Naha port quarantine station. The isolates were characterized to clarify whether or not, they belonged to a common clone. Phenotypes were identical except that one strain revealed cured Celebes and the others were original Celebes in kappa phage typing. The restriction fragment patterns of DNA of the isolates hybridized with an enzyme-labeled oligonucleotide probe for cholera toxin gene (ctx) were identical. Randomly amplified polymorphic DNA of the isolates were identical when a primer was used, but 2 patterns were seen when another primer was used. Pulsed-field gel electrophoresis of the chromosomal DNA digested with NotI restriction enzyme showed 3 patterns. The DNA fragment pattern of the strain isolated from the imported fish was different from the clinical isolates. These results suggested that there was no epidemiological relation among the strains of V. cholerae O1 isolated during this period.     

Cryptic Appearance of a New Clone of Vibrio cholerae Serogroup O1 Biotype El Tor in Calcutta,India

In this study, pulsed-field gel electrophoresis (PFGE) was applied to determine if the Vibrio cholerae O1 strains which reappeared after being temporarily displaced in Calcutta by the O139 serogroup were different from those isolated before the advent of the O139 serogroup. NotI digestion generated a total of 11 different patterns among the 24 strains of V. cholerae randomly selected to represent different time frames. Among the V. cholerae O1 strains isolated after July 1993, 4 PFGE banding patterns designated as H through K were observed with pattern H dominating. Pattern H was distinctly different from all other patterns encountered in this study including patterns A, B and C of V. cholerae O1 El Tor, which dominated before November 1992, and pattern F, which was the dominant V. cholerae O139 pattern. Further, pattern H was also different from the NotI banding patterns of the representative strains of the 4 toxigenic clonal groups of V. cholerae O1 El Tor currently prevailing in different parts of the world. NotI fragments of the new clone of V. cholerae O1 did not hybridize with an O139 specific DNA probe, indicating that there was no O139 genetic material in the new clone of V. cholerae O1. Hybridization data with an O1-specific DNA probe again differentiated between the clones of V. cholerae O1 existing before the genesis of the O139 serogroup and the O1 strains currently prevalent.     

Pili of a Vibrio cholerae O139

The pili of a strain of Vibrio cholerae O139 were purified and characterized. They were morphologically, electrophoretically and immunologically indistinguishable from the pili with 16 kDa subunit protein of V. cholerae O1. All 22 strains of V. cholerae O139 examined possessed the pili. The pili were different in hemagglutination inhibition pattern from V. cholerae O1 16K pili.     

The Gene Encoding the Heat-Stable Enterotoxin of Vibrio cholerae Is Flanked by 123-Base Pair Direct Repeats

The heat-stable enterotoxin (O1-ST) gene (sto) was cloned from chromosome of the strain GP156 of Vibrio cholerae O1 (Inaba, El Tor) in Escherichia coli K-12, and its nucleotide seqence was determined. The nucleotide sequence of sto was very similar to that of NAG-ST gene (stn) of V. cholerae non-O1. Both sto and stn were flanked by 123-base pair direct repeats which had at least 93% homology to one another and included some inverted repeats. All the strains of V. cholerae, V. mimicus, V. metschnikovii, V. hollisae and Yersinia enterocolitica examined by colony hybridization had the direct repeat sequence regardless of ST-gene possession.     

Ecology ofVibrio cholerae in the freshwater environs of Calcutta,India

Seasonal incidence ofVibrio cholerae was monitored for a year in a man-made freshwater lake, an open sewage canal, and a pond composed of rainwater accumulations, located in Calcutta.V. cholerae was found in all sites. It exhibited a distinct bimodal seasonal cycle in the lake with a primary peak in August–September and a secondary peak in May–June. Correlation with environmental parameters revealed that temperature and, to a certain extent, pH were the important factors governing the densities ofV. cholerae. In the lake, sediment samples harbored high densities ofV. cholerae immediately after months when peak counts were observed in plankton, suggesting a cycle of cells between sediment and water. At the other sampling areas, no defined seasonality was observed. Instead, high counts ofV. cholerae were observed at these severely polluted sites throughout the study period, including the winter months. All the 15 water samples passed via the ligated loop of rabbits yielded pure cultures ofV. cholerae, indicating that the rabbit intestine selects outV. cholerae from a mixed flora. Uniformly high isolation rates ofV. cholerae were observed from brackish water and freshwater species of export quality prawns.V. cholerae was found to be abundant and was represented by 32 individual Louisiana State University (LSU) serovars, including two new serovars. The 01 serovar could not be isolated from any of the samples examined in this study. It was concluded thatV. cholerae non-01 is common in the freshwater environs of Calcutta.     

Detection and molecular characterization of Vibrio cholerae O1 Inaba biotype El Tor strain in Kerala, S. India

The resurgence of enteric pathogen Vibrio cholerae, the causative organism of epidemic cholera, remains a major health problem in many developing countries. The outbreaks of cholera follow a seasonal pattern in regions of endemicity. The southern Indian state of Kerala is endemic to cholera. A V. cholerae strain isolated from the stool sample of a patient in Piravam, Kerala, South India, was analysed. However, this case occurred at a time not associated with cholera outbreaks, leading to concern among the State health officials. We compared the virulence potential of the isolate with that of the standard or reference strains, that have been widely used as positive control. The isolate was identified as V. cholerae O1 biotype El Tor serotype Inaba. The resistance pattern of the isolate to common antibiotics was examined and it was found to be multi-drug resistant in nature. The strain was analysed for the presence of the CTX genetic element, which encodes genes for cholera toxin and other important regulatory genes. It was found to be positive for all the genes tested. In Kerala, most of the cholera outbreaks have been reported to be caused by V. cholerae O1 El Tor belonging to Ogawa serotype. Interestingly, the V. cholerae strain isolated from this case has been found to be of Inaba serotype, which is rarely reported.     

Genetic organization and functional analysis of the otn DNA essential for cell-wall polysaccharide synthesis in Vibrio cholerae O139

In 1992 a new Vibrio cholerae strain, designated V. cholerae O139 Bengal, emerged which has been responsible for large outbreaks of cholera in India and Bangladesh. Previously, we have shown that this strain arose from a V. cholerae O1 strain by the acquisition of novel DNA. Sequence analysis revealed that the novel DNA is flanked by two genes, rfaD and rfbQRS, which are also found in O1 strains. The mosaic structure of rfaD_vco139 indicated that it was one of the regions involved in recombination between donor and acceptor DNA. However, sequence divergence between the O1 and O139 rfbQRS genes indicated that the second recombination site between donor and O1-acceptor DNA is probably located downstream of rfbD_vco139. The DNA region between rfaD_vco139 and rfbQRS_vco139, designated otn, contained seven open reading frames (ORFs). Two ORFs, otnA and otnB, showed homology with genes involved in cell-wall polysaccharide synthesis. Mutations in otnA and otnB indicated that they are required for capsule synthesis but not lipopolysaccharide synthesis. The otn DNA is also found inV. cholerae O69 and O141 strains, and the organization of this DNA was essentially identical to that in the O139 strain. However, sequence divergence of the otnAB genes indicated that the O139 otn DNA region was not derived from the O69 or O141 strains. No antigenic relationship was found between the different V. cholerae serotypes carrying otn DNA, so the genes determining the antigenic specificity of the O antigen or capsule must be located outside the otn DNA. The O139 otn DNA contained a JUMPstart sequence, which is associated with polysaccharide-synthetic genes in several bacterial species. Furthermore, a repeat motif was observed in extragenic regions. A number of observations suggest that these sequences may facilitate gene flow between V. cholerae strains and the assembly of clusters of functionally related genes.     

Geographical variation in the distribution of phage types of Vibrio cholerae O1 and non-O1

A Vibrio cholerae O1 phage-typing scheme, developed in the USSR, has been used to type 120 strains of V. cholerae O1 isolated in Africa and Asia, and 56 non-O1 V. cholerae isolated in England and Wales from infections contracted abroad. 90.9% of V. cholerae O1 were typable. Phage type 13 predominated among African strains whereas types 11 and 15 were more common among strains from Asia. Only 14.3% of non-O1 V. cholerae reacted with the phages.     

Vibrio ordalii sp. nov.: A causative agent of vibriosis in fish

Vibrio ordalii sp. nov. is the name proposed for the bacterium previously designated asV. anguillarum biotype 2. The change in the classification of this fish pathogen is based on differences between the classicalV. anguillarum andV. ordalii in cultural and biochemical characteristics, and in deoxyribonucleic acid (DNA) sequence relatedness. Phenotypically,V. ordalii was distinguishable fromV. anguillarum based on: negative Voges-Proskauer reaction; negative reaction with arginine in Moeller's medium; negative Simmons' and Christensen's citrate test; negative ONPG test; failure to hydrolyze starch; failure to show lipase activity; inability to grow at 37°C; and failure to ferment cellobiose, glycerol, sorbitol, and trehalose. Genotypically, strain ofV. ordalii formed a highly conserved DNA homology group which showed 83 to 100% within-group homology and only 58 to 69% relatedness toV. anguillarum. In contrast, theV. anguillarum strains tested showed greater than 70% withingroup homology and 53 to 67% relatedness toV. ordalii. NeitherV. ordalii norV. anguillarum were related toV. parahaemolyticus orV. alginolyticus. The proposed type strain (holotype) ofV. ordalii is ATCC 33509 (=DF₃K=Dom F₃ kid).     

High-Frequency Rugose Exopolysaccharide Production by Vibrio cholerae Strains Isolated in Haiti

In October, 2010, epidemic cholera was reported for the first time in Haiti in over 100 years. Establishment of cholera endemicity in Haiti will be dependent in large part on the continued presence of toxigenic V. cholerae O1 in aquatic reservoirs. The rugose phenotype of V. cholerae, characterized by exopolysaccharide production that confers resistance to environmental stress, is a potential contributor to environmental persistence. Using a microbiologic medium promoting high-frequency conversion of smooth to rugose (S–R) phenotype, 80 (46.5%) of 172 V. cholerae strains isolated from clinical and environmental sources in Haiti were able to convert to a rugose phenotype. Toxigenic V. cholerae O1 strains isolated at the beginning of the epidemic (2010) were significantly less likely to shift to a rugose phenotype than clinical strains isolated in 2012/2013, or environmental strains. Frequency of rugose conversion was influenced by incubation temperature and time. Appearance of the biofilm produced by a Haitian clinical rugose strain (altered biotype El Tor HC16R) differed from that of a typical El Tor rugose strain (N16961R) by confocal microscopy. On whole-genome SNP analysis, there was no phylogenetic clustering of strains showing an ability to shift to a rugose phenotype. Our data confirm the ability of Haitian clinical (and environmental) strains to shift to a protective rugose phenotype, and suggest that factors such as temperature influence the frequency of transition to this phenotype.     

High genetic diversity of Vibrio cholerae in the European lake Neusiedler See is associated with intensive recombination in the reed habitat and the long‐distance transfer of strains

Coastal marine Vibrio cholerae populations usually exhibit high genetic diversity. To assess the genetic diversity of abundant V. cholerae non‐O1/non‐O139 populations in the Central European lake Neusiedler See, we performed a phylogenetic analysis based on recA, toxR, gyrB and pyrH loci sequenced for 472 strains. The strains were isolated from three ecologically different habitats in a lake that is a hot‐spot of migrating birds and an important bathing water. We also analyzed 76 environmental and human V. cholerae non‐O1/non‐O139 isolates from Austria and other European countries and added sequences of seven genome‐sequenced strains. Phylogenetic analysis showed that the lake supports a unique endemic diversity of V. cholerae that is particularly rich in the reed stand. Phylogenetic trees revealed that many V. cholerae isolates from European countries were genetically related to the strains present in the lake belonging to statistically supported monophyletic clades. We hypothesize that the observed phenomena can be explained by the high degree of genetic recombination that is particularly intensive in the reed stand, acting along with the long distance transfer of strains most probably via birds and/or humans. Thus, the Neusiedler See may serve as a bioreactor for the appearance of new strains with new (pathogenic) properties.     

Comparison of different cationic probes for electron microscopic visualization of bacterial polyanionic capsular material

We have isolated from the ovine rumen eight bacterial strains belonging to the speciesButyrivibrio fibrisolvens. DNA hybridization studies showed that the eight strains could be divided into four homology groups, of which none was closely related to the type strain ATCC 19171. Measurement of cross-hybridization between selected pairs of bacterial strains showed that DNA types which produced low, but significant, cross-hybridization on dot-blots were able to form heteroduplexes with between 8.4% and 32.9% of the efficiency of homoduplex formation. Thermal denaturation of the same heteroduplexes resulted in Tm values 6.4–7.5°C lower than those of the homologous duplexes formed under the same conditions. In some cases, hybridization between strains was below the level of reliable measurement. Similar experiments with ten recently isolated strains ofBacteroides ruminicola sub-sp.brevis revealed a similar degree of genetic divergence between isolates.