The phages of halophilic vibrios and their use]

The range of the lytic activity of 46 phages of parahemolytic vibrios isolated from lysogenic strains, sea water samples, crabs and mussels has been studied. The phages are represented by virions belonging to morphological groups II, IV, V according to the phage classification currently used in Russia and to different serological groups. No relationship between the sensitivity of vibrio strains to the phages under study and the specificity of serotypes O and K has been established. The preparation of diagnostic phage [see text] suitable for the identification of 82% of strains of parahemolytic vibrios has been proposed.     

Isolation of parahemolytic vibrions from persons with acute gastrointestinal diseases]

Galophilic vibrios were for the first time in the Soviet Union isolated from the patients with gastrointestinal diseases in Novorossiisk during the summer-autumn period of 1973. The greatest number of strains of the parahemolytic vibrios was isolated in August and September, during intensive fishing season, from the patients in whom the disease usually developed 6 to 12 hours after eating the sea fish. The parahemolytic vibrios isolated from the patients were typical by all signs and produced a marked hemolysis on the Wagatsuma medium. The halophilic vibrios are inhabitants of the Black Sea. During the years of 1972-1973 there were isolated 109 strains of V. paraheamolyticus and 133 strains of V. alginolyticus from the sea water and various hydrobionts.     

Ecological relationship between Vibrio parahaemolyticus and agar-digesting vibrios as evidenced by bacteriophage susceptibility patterns.

Twenty bacteriophages active against Vibrio parahaemolyticus and agar-digesting vibrios, isolated from oysters (Crassostrea gigas) and Dungeness crab (Cancer magister) and by induction of a lysogenic agar digester, were tested as to their host range. These phages were specific for V. parahaemolyticus and various agar-digesting vibrios, and interspecies lysis occurred only between these two groups. V. alginolyticus, V. anguillarum and related species, V. cholerae, and a group of marine psychrophilic and psychrotrophic vibrios were not affected. No correlation was observed between the O and K serotypes of V. parahaemolyticus strains and bacteriophage susceptibility patterns, and 7 of 28 strains of V. parahaemolyticus were not lysed by any of the phages. Only two of the phage isolates were capable of lysing all susceptible V. parahaemolyticus strains. No correlation was observed between the inter-and intraspecies genetic relatedness (DNA homologies) of V. parahaemolyticus and agar-digesting vibrios and susceptibility patterns to different bacteriophages. Some of the phages were capable of plaque formation on V. parahaemolyticus as well as on some strains of agar-digesting vibrios that were separated by 70 to 80% differences in their DNA homologies. The possible ecological significance of these vibrio bacteriophages, particularly those having a wide host range, is discussed.     

Antibiotic sensitivity of Vibrio parahaemolyticus isolated in the Turkmene SSR

Sensitivity of 82 cultures of parahemolytic vibrios to 8 antibiotics was studied. It was shown that the majority of the strains were highly sensitive to levomycetin and gentamicin, sensitive to tetracycline, rifampicin, streptomycin, neomycin and kanamycin and resistant to ampicillin.     

Phage typing of cholera vibrios with different sets of phages]

A comparative study was made of two sets of Mukerjee and Drozhevkina-Arutyunova's bacteriophages in typing 514 strains of the El Tor vibrios and 45 strains of clasic biotype. It was shown that the Mukerjee or Drozhevkina-Arutyunova's phages could be used for the typing of cholera vibrios. The phages of the latter set prove to detect more phage types (18 against 11); they determine both the phage type and the biotype at the same time. The typing of cholera vibrios of both biotypes is possible, and the percentage of nontyping strains left is comparatively low (5.2 against 23.5 after Mukerjee). A table of the phage correspondence was made; it permits to obtain comparable data in using any set of the typing phages.     

Characterization of enteropathogenic Vibrio cholerae non O1/O139 isolated in Uzbekistan

The results of the serotyping of 244 V. cholerae non O1/O139 cultures isolated from patients in Uzbekistan in 2000 and 2001 are presented. All isolates were studied by the method of molecular probing and in the polymerase chain reaction for the presence of virulence genes and for sensitivity to phages ctx+, ctx- and hemolytic activity. The use of monoreceptor O-sera O2-O83 made it possible to determine vibrios of 32 serogroups with the dominating role in the etiology of acute enteric diseases belonging to serogroups O18, O62, O82, O37. Genes ctx AB were detected in none of the isolates, 5 of them contained gene tcp A. A group of cultures, sensitive to phage ctx+ and belonging mainly to enteropathogenic serogroups, was detected.     

Effect of sulfide water from natural springs on the properties of Vibrino cholerae

The authors studied the properties of E1 Tor cholera vibrios isolated from sulfide water of the natural sources. There was demonstrated under experimental and natural conditions the influence of ecological conditions of sulfide water on such vibrio properties as cholerogenicity, sensitivity to diagnostic and typing phages, hemolytic activity and the value of the hemolysin-destructive factor. A short-liver stay of cholera vibrios in sulfide water was accompanied by some reduction of their virulence.     

Halophilic Black Sea vibrios and their role in human pathology

During the period of 4 years 558 halophil vibrio strains (258 V. parahaemolyticus strains and 300 V. angiolyticus strains) were isolated from the water and from the representatives of the hydrobios of the Black Sea. The vibrios were isolated the whole year round, but during the winter and spring months their lowest concentration in water was observed, while in September-October it reached its maximum. During these 4 years 102 cases of gastroenteric diseases were registered, and in some of the patients halophil vibrios were isolated. In most cases the diseases developed after the consumption of sea fish and other sea products, as well as salted vegetables. The diseases had mainly a sporadic character. 90% of the strains isolated from humans belonged to serotypes O4 : K12 and O4 : K8; besides, the presence of serotypes O3 : K33, O3 : K57, O5 : K47, O6 : K46 and O10 : K52 was revealed.     

Cell surface characteristics of environmental and clinical isolates of Vibrio cholerae non-O1.

The cell surfaces of several toxigenic and nontoxigenic environmental and clinical isolates of Vibrio cholerae non-O1 have been examined. The environmental strains, irrespective of toxigenicity, are significantly more resistant to antibiotics and detergents than are V. cholerae O1 strains. The clinical isolates of non-O1 vibrios are as sensitive to a wide variety of chemicals as the O1 vibrios. The environmental non-O1 strains are also less susceptible to lysis when treated with protein denaturants or neutral and anionic detergents than are O1 vibrios and the clinical non-O1 strains. In contrast to O1 vibrios, the environmental non-O1 vibrios do not have exposed phospholipids in their outer membranes. These features of the cell surfaces of environmental non-O1 vibrios might have a role in the better survival of these organisms under environmental fluctuations.     

Cell surface characteristics of environmental and clinical isolates of Vibrio cholerae non-O1.

The cell surfaces of several toxigenic and nontoxigenic environmental and clinical isolates of Vibrio cholerae non-O1 have been examined. The environmental strains, irrespective of toxigenicity, are significantly more resistant to antibiotics and detergents than are V. cholerae O1 strains. The clinical isolates of non-O1 vibrios are as sensitive to a wide variety of chemicals as the O1 vibrios. The environmental non-O1 strains are also less susceptible to lysis when treated with protein denaturants or neutral and anionic detergents than are O1 vibrios and the clinical non-O1 strains. In contrast to O1 vibrios, the environmental non-O1 vibrios do not have exposed phospholipids in their outer membranes. These features of the cell surfaces of environmental non-O1 vibrios might have a role in the better survival of these organisms under environmental fluctuations.     

Complete and Defective Bacteriophages of Classical Vibrio cholerae: Relationship to the Kappa Type Bacteriophage

The Classical Vibrio cholerae strain NIH 41 contains two temperate bacteriophages, designated VcA-1 and VcA-2, that are distinguished by immunity, plaque morphology, induction kinetics, and particle morphology. Both phage are serologically related to phage Kappa. However, only phage VcA-2 has the Kappa type host range and immunity. The induction kinetics and immunity patterns of Classical vibrios suggest that these strains may contain defective phage related to the phages isolated from NIH 41. Classical strain 569B releases phage-tail structures upon induction that are morphologically and serologically related to both phages VcA-1 and VcA-2. The possible reason for the defectiveness of these phages in 569B is discussed. It is concluded that complete or defective bacteriophages of the Kappa type morphology and serology are extremely prevalent in V. cholerae, regardless of biotype.     

Method for detecting small numbers of Vibrio cholerae in very polluted substrates.

A method is presented for the indirect detection of Vibrio cholerae by the multiplication of two specific bacteriophages: phiH74/64 for El-Tor vibrios, and phage group IV (Mukerjee) for classical vibrios. The product to be examined is seeded in alkaline tryptone water for enrichment, as in the classical method, and is then incubated for 6 h at 37 C. Thereafter, a loopful is transferred to each of two nutrient broth (pH 9) tubes. One of these receives a drop of phage phiH74/64; the other receives a drop of phage group IV. The stock phages are diluted so as to contain about 3,800 plaque-forming units in one drop; this is the maximum amount which, when added to 10 ml of broth, will not be detected in a loopful of 1 mm diameter. The tubes containing phage phiH74/64 are incubated at 42 C; those with phage group IV are incubated at 37 C. After 18 h the cultures are killed by agitation with chloroform, and a 1-mm loopful is deposited on a layer seeded with the detector strains: Makassar 757 for El-Tor phage and V. cholerae 154 for classical cholera phage. After 4 to 5 h at 37 C, lysis appears on the spot areas if there has been phage multiplication in the respective broth tubes. With experimentally contaminated sewage water, vegetables, or stools, 1 to 10 cholera vibrios were detected in every sample. In rare cases, false-positive results were obtained by multiplication of the phage on non-cholera vibrios.     

Studies on halophilic vibrios causing a food poisoning outbreak in the city of Vladivostok

V. parahaemolyticus and V. alginolyticus strains isolated from patients during an outbreak of an acute enteric disease in Vladivostok in 1997 were studied. All strains were found to possess typical taxonomic signs. V. parahaemolyticus isolated from humans had direct heat stable haemolysin exotoxin. The overwhelming majority of these strains belonged to serovar O3K6. Among the cultures under study 7 phage types were determined: phage types 1, 2, 7, 10 in 8 V. parahaemolyticus strains and phage types 2, 4. 5. 7 in 5 V. alginolyticus strains. The diagnostic halophilic phage lyzed vibrios in 30.2% of strains. The cultures under study were found to be highly sensitive to chloramphenicol, cefotaxime, nalidixic acid and cyprofloxacin. The study proved that the outbreak of alimentary toxicoinfection was caused by vibrios of serogroup O3:K6.     

Predatory Bacteria as Natural Modulators of Vibrio parahaemolyticus and Vibrio vulnificus in Seawater and Oysters

This study shows that naturally occurring Vibrio predatory bacteria (VPB) exert a major role in controlling pathogenic vibrios in seawater and shellfish. The growth and persistence of Vibrio parahaemolyticus and Vibrio vulnificus were assessed in natural seawater and in the Eastern oyster, Crassostrea virginica. The pathogens examined were V. vulnificus strain VV1003, V. parahaemolyticus O1:KUT (KUT stands for K untypeable), and V. parahaemolyticus O3:K6 and corresponding O3:K6 mutants deficient in the toxRS virulence regulatory gene or the rpoS alternative stress response sigma factor gene. Vibrios were selected for streptomycin resistance, which facilitated their enumeration. In natural seawater, oysters bioconcentrated each Vibrio strain for 24 h at 22°C; however, counts rapidly declined to near negligible levels by 72 h. In natural seawater with or without oysters, vibrios decreased more than 3 log units to near negligible levels within 72 h. Neither toxRS nor rpoS had a significant effect on Vibrio levels. In autoclaved seawater, V. parahaemolyticus O3:K6 counts increased 1,000-fold over 72 h. Failure of the vibrios to persist in natural seawater and oysters led to screening of the water samples for VPB on lawns of V. parahaemolyticus O3:K6 host cells. Many VPB, including Bdellovibrio and like organisms (BALOs; Bdellovibrio bacteriovorus and Bacteriovorax stolpii) and Micavibrio aeruginosavorus-like predators, were detected by plaque assay and electron microscopic analysis of plaque-purified isolates from Atlantic, Gulf Coast, and Hawaiian seawater. When V. parahaemolyticus O3:K6 was added to natural seawater containing trace amounts of VPB, Vibrio counts diminished 3 log units to nondetectable levels, while VPB increased 3 log units within 48 h. We propose a new paradigm that VPB are important modulators of pathogenic vibrios in seawater and oysters.     

Isolation of Escherichia coli bacteriophages from the stool of pediatric diarrhea patients in Bangladesh

A 3-week coliphage survey was conducted in stool samples from 140 Bangladeshi children hospitalized with severe diarrhea. On the Escherichia coli indicator strain K803, all but one phage isolate had 170-kb genomes and the morphology of T4 phage. In spot tests, the individual T4-like phages infected up to 27 out of 40 diarrhea-associated E. coli, representing 22 O serotypes and various virulence factors; only five of them were not infected by any of these new phages. A combination of diagnostic PCR based on g32 (DNA binding) and g23 (major capsid protein) and Southern hybridization revealed that half were T-even phages sensu strictu, while the other half were pseudo-T-even or even more distantly related T4-like phages that failed to cross-hybridize with T4 or between each other. Nineteen percent of the acute stool samples yielded T4-like phages, and the prevalence was lower in convalescent stool samples. T4-like phages were also isolated from environmental and sewage water, but with low frequency and low titers. On the enteropathogenic E. coli strain O127:K63, 14% of the patients yielded phage, all of which were members of the phage family Siphoviridae with 50-kb genomes, showing the morphology of Jersey- and beta-4 like phages and narrow lytic patterns on E. coli O serotypes. Three siphovirus types could be differentiated by lack of cross-hybridization. Only a few stool samples were positive on both indicator strains. Phages with closely related restriction patterns and, in the case of T4-like phages, identical g23 gene sequences were isolated from different patients, suggesting epidemiological links between the patients.     

The expression of the pathogenic properties of the Vibrio cholerae O139 serogroup in vitro

Cholera toxin, hemolysin, dermonecrotic and proteolytic factors have been detected and identified in V. cholerae O139. The production of these substances has been found to depend on the conditions of the cultivation of vibrios, and the types of proteases have been determined.     

三种消毒剂对大黄鱼致病性弧菌的体外杀灭作用研究

为了解不同消毒剂对大黄鱼中致病性弧菌的抑菌效果。采用纸片扩散法比较了聚维酮碘(PVP-I)、聚六亚甲基双胍(PHMB)和过氧化氢(H2O2)对溶藻弧菌和副溶血性弧菌的抑菌效果,再以肉汤稀释法测定了三种消毒剂的最低抑菌浓度(MIC)、最低杀菌浓度(MBC)及MBC/MIC比值,在此基础上,获得了抑菌效果相对较优的H2O2、PHMB对溶藻弧菌、副溶血性弧菌及其混菌的杀菌动力学曲线。结果表明,H2O2(3%)对两种弧菌具有最大的抑菌圈直径(19~21mm),PHMB(30mg/mL)次之(约为15mm),PVP-I(10%)的抑菌效果最弱(小于10mm)。3种消毒剂的MIC值由小到大依次为PHMB相似文献   

DNA sequence of the tail fiber genes 37, encoding the receptor recognizing part of the fiber, of bacteriophages T2 and K3

The DNA sequences of genes 37 of bacteriophages T2 and K3 are presented and compared with that of phage T4. The corresponding proteins constitute, as dimers, the part of the long tail fibers that recognizes the bacterial receptor. The CO2H termini of the polypeptides are located at the free ends of the fibers. Morphologically, the three phages are essentially identical, but they use different receptors. The genes from phages T4, T2 and K3 encode proteins consisting of 1026, 1341 and 1243 amino acid residues, respectively. DNA-DNA hybridizations had shown earlier that genes 37, in contrast to the gene for the major capsid protein, of a number of T-even type phages are highly polymorphic. The deduced amino acid sequences now show that this polymorphism extends to the protein primary structures. About 50 NH2-terminal residues are conserved and are probably required for binding to the adjacent protein 36. This area is followed by more or less irregularly spaced regions of non-homology, partial homology or complete homology. The heterogeneity is most prominent in a region encompassing about 600 CO2H-terminal residues of the T2 or K3 proteins. Nevertheless, the amino acid compositions of the three proteins are very similar and all are rich in glycine. It has been found that the receptor specificities of phages K3 and T2 are determined by protein 38, a polypeptide required for the efficient dimerization of protein 37 of phage T4. Proteins 38 of phages K3 and T2 are functionally interchangeable, those of T4 and T2 or K3 are not. Proteins 37 of phages K3 and T2 possess a conserved sequence of 160 CO2H-terminal residues. This area is missing in the T4 protein. This region may serve as a binding site for polypeptides 38 of phages K3 and T2. The overall picture of the protein primary structures of the three phages strongly suggests that the evolution of genes 37, which was most likely driven by selection for variations in receptor recognition specificities, has not been a steady process but has involved loss and gain of segments of DNA.     

A method for studies of an El Tor-associated antigen of Vibrio cholerae O1

A method for studying the biotype El Tor associated mannose-sensitive haemagglutinin (MSHA) of V. cholerae O1 has been developed. By using crude MSHA adsorbed to chicken erythrocytes as solid phase antigen in an enzyme-linked immunosorbent assay (ELISA), antisera against V. cholerae of the El Tor biotype reacted in high titre with the MSHA-coated cells, whereas antisera against vibrios of the classical biotype did not bind significantly, i.e. in higher titre than pre-immune sera. The binding of anti-MSHA serum, or a monoclonal antibody against MSHA, to the MSHA-coated erythrocytes could be efficiently inhibited by crude MSHA as well as by El Tor vibrios whereas neither V. cholerae lipopolysaccharide nor different strains of classical vibrios had any inhibitory effect. These results support the existence of an El Tor-associated immunogen. They also suggest a possibility of determining antibodies against different haemagglutinins in ELISA without having access to purified antigens.     

The genome of bacteriophage K1F, a T7-like phage that has acquired the ability to replicate on K1 strains of Escherichia coli

Bacteriophage K1F specifically infects Escherichia coli strains that produce the K1 polysaccharide capsule. Like several other K1 capsule-specific phages, K1F encodes an endo-neuraminidase (endosialidase) that is part of the tail structure which allows the phage to recognize and degrade the polysaccharide capsule. The complete nucleotide sequence of the K1F genome reveals that it is closely related to bacteriophage T7 in both genome organization and sequence similarity. The most striking difference between the two phages is that K1F encodes the endosialidase in the analogous position to the T7 tail fiber gene. This is in contrast with bacteriophage K1-5, another K1-specific phage, which encodes a very similar endosialidase which is part of a tail gene "module" at the end of the phage genome. It appears that diverse phages have acquired endosialidase genes by horizontal gene transfer and that these genes or gene products have adapted to different genome and virion architectures.