Characterization of Vibrio cholerae eltor in the city of Kazan in 2001

Information on V. cholerae eltor isolated in the focus of cholera in Kazan in 2001 at different periods of the outbreak is presented. The identity of strains isolated from patients, vibriocarriers and environmental objects, including their antibioticograms (sensitivity to cyprofloxacin and resistance to trimethoprim--sulfamethoxazole, streptomycin, furazolidone and nalidixic acid, which may be regarded as markers), is shown. Variable tandem repetitions in the DNA of 30 isolates strains of different origin have been determined. The results of this determination make it possible to classify all these strains as one genotype, which confirms the suggestion on the circulation of one subclone of the infective agent of cholera in the focus. As revealed in this investigation, the isolated strains are labile with respect to diagnostic phage eltor, while ctx+ strains are resistant to phage eltor ctx+.     

Sensitivity of Vibrio and Aeromonas to antibiotics]

The antibiotic sensitivity of 696 cultures belonging to the family Vibrionaceae (V. cholerae O1, V. cholerae non-O1, V. albensis, V. parahaemolyticus, V. alginolyticus and Aeromonas spp.) was studied and general regularities of the antibiotic sensitivity were shown: a high sensitivity to broad-spectrum antibiotics (tetracycline and chloramphenicol) and a low sensitivity to ++beta lactams (carbenicillin and ampicillin). The comparative examinations revealed similarity in the antibioticograms of V. cholerae O1 (el Tor++), V. cholerae non-O1 and V. albensis, especially the latter two groups, as well as the tested halophilic Vibrio cultures by the range of the MICs, Mo, Me and the nature of the antibiotic resistance. Cultures of V. cholerae and luminescent Vibrio tended to preserve a high sensitivity. High resistance levels were noted in the halophilic Vibrio and Aeromonas cultures. No significant differences in the sensitivity of the strains of various origin (from man and environmental objects) were detected. However, several more resistant strains were isolated from the environmental objects.     

Differences among marine and hospital strains of Vibrio cholerae during Peruvian epidemic

During a period of 18 months of an epidemic of Vibrio cholerae, cultures from 450 samples of fish, shellfish and seawater were isolated. The highest frequencies of occurrence observed were 5.2% in fish from inshore waters, 3.9% in marine snails, and 1.8% in mussels and crabs. No incidents were isolated from cultures of fish in the open seas or cultures from frozen shrimp. Cultures of marine origin were compared with cultures from hospitalized patients, and these revealed marked serological and toxigenic differences. Marine strains were mainly non-O1 V. cholerae, non toxigenic. We presume fishing off-shore not to be the cause of this outbreak. However, marine species from contaminated waters could contain toxigenic V. cholerae remaining viable and potentially pathogenic. Methods used were more sensitive and specific for detecting marine strains. In this paper the need to use more specific methods is 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.     

Isolation of Vibrio cholerae from aquatic birds in Colorado and Utah

Vibrio cholerae was isolated from cloacal swabs and freshly voided feces collected from 20 species of aquatic birds in Colorado and Utah during 1986 and 1987. About 17% (198 of 1,131) fecal specimens collected from July 1986 through August 1987 contained the organism. Both O1 and non-O1 V. cholerae strains were isolated from the fecal specimens. Isolates from eight birds (representing five species) agglutinated in O group 1 antiserum. Supernatants of broth cultures from three isolates which typed as V. cholerae O1 serotype Ogawa gave reactions typical of cholera toxin when tested on Y-1 mouse adrenal cell cultures. Several serovars of non-O1 V. cholerae were isolated from the fecal specimens; serovar 22 was the most prevalent type. All non-O1 isolates were cytotoxic to Y-1 mouse adrenal cells. Only non-O1 V. cholerae was detected in water samples collected from the habitat of the birds. The results of this study suggest that aquatic birds serve as carriers and disseminate V. cholerae over a wide area.     

Isolation of Vibrio cholerae from aquatic birds in Colorado and Utah.

Vibrio cholerae was isolated from cloacal swabs and freshly voided feces collected from 20 species of aquatic birds in Colorado and Utah during 1986 and 1987. About 17% (198 of 1,131) fecal specimens collected from July 1986 through August 1987 contained the organism. Both O1 and non-O1 V. cholerae strains were isolated from the fecal specimens. Isolates from eight birds (representing five species) agglutinated in O group 1 antiserum. Supernatants of broth cultures from three isolates which typed as V. cholerae O1 serotype Ogawa gave reactions typical of cholera toxin when tested on Y-1 mouse adrenal cell cultures. Several serovars of non-O1 V. cholerae were isolated from the fecal specimens; serovar 22 was the most prevalent type. All non-O1 isolates were cytotoxic to Y-1 mouse adrenal cells. Only non-O1 V. cholerae was detected in water samples collected from the habitat of the birds. The results of this study suggest that aquatic birds serve as carriers and disseminate V. cholerae over a wide area.     

An outbreak of cholera (El Tor) in Samarkand Province, Uzbekistan in 1990]

A group outbreak of cholera caused by Vibrio eltor in the Samarkand Province++ in 1990 is analyzed. The retrospective analysis of the isolation of V. cholerae from water of surface reservoirs has been made. The study points out that since the cholera epidemic of 1985 V. cholerae avirulent strains, serogroup O1, have been periodically isolated in the Samarkand Province. The conclusion has been made that the isolation of even avirulent strains only from environmental objects is indicative of the unfavorable epidemiological situation in this area and of the necessity to take measures for improving epidemiological surveillance.     

Dynamics of the isolation and biological properties of Vibrio cholerae cultures,serogroups 01 and 0139, isolated from water reservoirs and sewage water on the territory of the city of Rostov-on-Don

The dynamics of the isolation of V. cholerae cultures from various water objects on the territory of Rostov-on-Don during the period of 1994-2001 was analyzed and biological properties of 14 such cultures were studied. In the absence of epidemic complications during the above-mentioned period, a growth in the amount of V. cholerae isolates, serogroups 01 and 0139, including toxigenic V. cholerae 01, was registered. The microbiological and epidemiological aspects of the monitoring of surface reservoirs and sewage were considered and the expediency of the profound and systematic study of its results for epidemiological surveillance on cholera was emphasized.     

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.     

Role of cyanobacteria in the persistence of Vibrio cholerae O139 in saline microcosms

Recently, a new strain of cholera, Vibrio cholerae O139, has emerged as an epidemic strain, but there is little information about its environmental reservoir. The present investigation was aimed to determine the role of cyanobacteria in the persistence of V. cholerae O139 in microcosms. An environmental isolate of V. cholerae O139 and three cyanobacteria (Anabaena sp., Nostoc sp., and Hapalosiphon sp.) were used in this study. Survival of culturable V. cholerae O139 in microcosms was monitored using taurocholate-tellurite gelatin agar medium. Viable but nonculturable V. cholerae O139 were detected using a fluorescent antibody technique. Vibrio cholerae O139 could be isolated for up to 12 days in a culturable form in association with cyanobacteria but could not be isolated in the culturable form after 2 days from control water without cyanobacteria. The viable but nonculturable V. cholerae O139 could be detected in association with cyanobacteria for up to 15 months. These results, therefore, suggest that cyanobacteria can act as a long-term reservoir of V. cholerae O139 in an aquatic environment.     

Hemolytic Vibrio cholerae O1 that is sensitive to Mukerjee's cholera phage IV and the phage produced by the hemolytic vibrio lysogenized with the infection of Mukerjee's cholera phage IV

Strains of hemolytic Vibrio cholerae O1 (El Tor vibrio) which are sensitive to Mukerjee's cholera phage group IV were isolated from cholera patients in North-East Thailand in 1986. Plaques of the phage on these hemolytic V. cholerae O1 were usually translucent but almost transparent on some strains, just like the plaques on non-hemolytic V. cholerae O1 (classical vibrio). These hemolytic V. cholerae O1 were lysogenized with the infection of cholera phage IV, and the lysogenized strains produced phage different from cholera phage IV. These hemolytic strains were classified into Cured type in prophage typing of V. cholerae O1, El Tor, because they were also lysogenized with Kappa phage and were hemolytic. When Cured-type V. cholerae O1, El Tor previously isolated in various countries were examined for the sensitivity to cholera phage IV, some of the isolates were sensitive.     

Genetic diversity of Vibrio cholerae in Chesapeake Bay determined by amplified fragment length polymorphism fingerprinting

Vibrio cholerae is indigenous to the aquatic environment, and serotype non-O1 strains are readily isolated from coastal waters. However, in comparison with intensive studies of the O1 group, relatively little effort has been made to analyze the population structure and molecular evolution of non-O1 V. cholerae. In this study, high-resolution genomic DNA fingerprinting, amplified fragment length polymorphism (AFLP), was used to characterize the temporal and spatial genetic diversity of 67 V. cholerae strains isolated from Chesapeake Bay during April through July 1998, at four different sampling sites. Isolation of V. cholerae during the winter months (January through March) was unsuccessful, as observed in earlier studies (J. H. L. Kaper, R. R. Colwell, and S. W. Joseph, Appl. Environ. Microbiol. 37:91-103, 1979). AFLP fingerprints subjected to similarity analysis yielded a grouping of isolates into three large clusters, reflecting time of the year when the strains were isolated. April and May isolates were closely related, while July isolates were genetically diverse and did not cluster with the isolates obtained earlier in the year. The results suggest that the population structure of V. cholerae undergoes a shift in genotype that is linked to changes in environmental conditions. From January to July, the water temperature increased from 3 degrees C to 27.5 degrees C, bacterial direct counts increased nearly an order of magnitude, and the chlorophyll a concentration tripled (or even quadrupled at some sites). No correlation was observed between genetic similarity among isolates and geographical source of isolation, since isolates found at a single sampling site were genetically diverse and genetically identical isolates were found at several of the sampling sites. Thus, V. cholerae populations may be transported by surface currents throughout the entire Bay, or, more likely, similar environmental conditions may be selected for a specific genotype. The dynamic nature of the population structure of this bacterial species in Chesapeake Bay provides new insight into the ecology and molecular evolution of V. cholerae in the natural environment.     

Difference of Phenotype and Genotype Between Human and Environmental: Isolated Vibrio cholerae in Surabaya,Indonesia

Cholera due to Vibrio cholerae has been spreading worldwide, although the reports focusing on Indonesian V. cholerae are few. In this study, in order to investigate how V. cholerae transmitted to human from environment. We extended an epidemiological report that had investigated the genotype of V. cholerae isolated from human pediatric samples and environmental samples. We examined 44 strains of V. cholerae isolated from pediatric diarrhea patients and the environment such as shrimps or oysters collected in three adjacent towns in Surabaya, Indonesia. Susceptibilities were examined for 11 antibiotics. Serotype O1 or O139 genes and pathogenic genes including cholera toxin were detected. Multi-locus sequence typing (MLST) and enterobacterial repetitive intergenic consensus (ERIC)-PCR were also performed to determine genetic diversity of those isolates. Serotype O1 was seen in 17 strains (38.6%) with all pathogenic genes among 44 isolates. Other isolates were non-O1/non-O139 V. cholerae. Regarding antibiotic susceptibilities, those isolates from environmental samples showed resistance to ampicillin (11.4%), streptomycin (9.1%) and nalidixic acid (2.3%) but those isolates from pediatric stools showed no resistance to those 3 kinds of antibiotics. MLST revealed sequence type (ST) 69 in 17 strains (38.6%), ST198 in 3 strains (6.8%) and non-types in 24 strains (54.5%). All the ST69 strains were classified to O1 type with more than 95% similarity by ERIC-PCR, including all 6 (13.6%) isolates from environmental samples with resistance to streptomycin. In conclusion, V. cholerae O1 ST69 strains has been clonally spreading in Surabaya, exhibiting pathogenic factors and antibiotic resistance to streptomycin, especially in the isolates from environment.     

Vibrio cholerae 01, variant eltor,in the environment

The possibilities of the autochthonous existence of V. cholerae in open water reservoirs, depending on the combined effect of different biotic and abiotic factors are considered. The role of adaptive variability of V. cholerae O1, biovar eltor for its preservation in the environment is emphasized. The data on the duration of the V. cholerae O1 isolation from different environmental objects in some regions of Ukraine are presented.     

Evaluation of polysaccharide antigens of Vibrio cholerae O139 of different origin by monoclonal antibodies

The epitope composition of O-polysaccharides in the lipopolysaccharide (LPS) of V. cholerae, serogroup O139, isolated from clinical material and water of surface reservoirs was analyzed with the use of monoclonal antibodies. The analysis demonstrated that these O-polysaccharides were similar in their structure and chemical composition. In LPS of V. cholerae O139 clinical strains O-polysaccharide determinants occurred more often. Among V. cholerae isolated from water strains on whose surface individual epitopes of O-polysaccharide occurred less frequently or were absent appeared to be more numerous. A decrease in the concentration of microbial cells in the process of their testing by immunological methods led to increased percent of negative reactions with specific antibodies. Some V. cholerae O139 strains isolated from water were similar in the epitope composition of their O-polysaccharide and binding activity to cultures isolated from humans. As indicated by the results of these studies, cholera vibrios Bengal and vibrios isolated from river water on the territory of Russia had quantitative differences due to a higher level of the production of O-polysaccharide determinants and their occurrence in V. cholerae of serogroup O139.     

Toxigenic Vibrio cholerae in the aquatic environment of Mathbaria, Bangladesh

Toxigenic Vibrio cholerae, rarely isolated from the aquatic environment between cholera epidemics, can be detected in what is now understood to be a dormant stage, i.e., viable but nonculturable when standard bacteriological methods are used. In the research reported here, biofilms have proved to be a source of culturable V. cholerae, even in nonepidemic periods. Biweekly environmental surveillance for V. cholerae was carried out in Mathbaria, an area of cholera endemicity adjacent to the Bay of Bengal, with the focus on V. cholerae O1 and O139 Bengal. A total of 297 samples of water, phytoplankton, and zooplankton were collected between March and December 2004, yielding eight V. cholerae O1 and four O139 Bengal isolates. A combination of culture methods, multiplex-PCR, and direct fluorescent antibody (DFA) counting revealed the Mathbaria aquatic environment to be a reservoir for V. cholerae O1 and O139 Bengal. DFA results showed significant clumping of the bacteria during the interepidemic period for cholera, and the fluorescent micrographs revealed large numbers of V. cholerae O1 in thin films of exopolysaccharides (biofilm). A similar clumping of V. cholerae O1 was also observed in samples collected from Matlab, Bangladesh, where cholera also is endemic. Thus, the results of the study provided in situ evidence for V. cholerae O1 and O139 in the aquatic environment, predominantly as viable but nonculturable cells and culturable cells in biofilm consortia. The biofilm community is concluded to be an additional reservoir of cholera bacteria in the aquatic environment between seasonal epidemics of cholera in Bangladesh.     

Characterization of a Vibrio cholerae phage isolated from the coastal water of Peru

A Vibrio cholerae bacteriophage, family Myoviridae, was isolated from seawater collected from the coastal water of Lima, Peru. Genome size was estimated to be 29 kbp. The temperate phage was specific to V. cholerae and infected 12/13 V. cholerae O1 strains and half of the four non-O1/non-O139 strains tested in this study. Vibrio cholerae O139 strains were resistant to infection and highest infection rates were obtained in low nutrient media amended with NaCl or prepared using seawater as diluent.     

Lecithinase activity of Vibrio cholerae and of vibrios not agglutinated by O-cholera serum

The lecithinase activities of 187 V. cholerae strains differing in their virulence and 70 cultures of NAG vibrios of different origin varied in all experimental groups. The level of lecithinase activity in the groups of V. cholerae strains having low virulence, or no virulence at all, was considerably higher than in virulent strains. NAG vibrios isolated from diarrhea patients and from samples of lake and pond water did not differ in the activity of this enzyme.     

Antibiotic sensitivity of Vibrio cholerae 01 isolated from environmental objects]

The sensitivity of 252 Vibrio cholerae-O1 strains isolated from environmental objects to antibiotics of various groups was assayed by the method of serial dilutions on solid media. The biological characteristics of the isolates are presented. The Vibrio cholerae isolates with serological variation were the most frequent (36.6 per cent), so are the cultures detected by their sensitivity to the specific phages (87.5 per cent). It was found that changes in some biological properties of the strains did not coincide with the changes in the antibiotic sensitivity. The isolates were highly sensitive to tetracycline, chloramphenicol, gentamicin, erythromycin and rifampicin and less sensitive to novobiocin and the other aminoglycosides. The sensitivity to the beta-lactams was the lowest. The resistance determinants were detected in single strains (6.3 per cent), the kanamycin and novobiocin resistance determinants being detected in 15 out of the 16 strains tested. The study showed that the cultures of Vibrio cholerae-O1 isolated from the environmental objects generally preserved their sensitivity to the diverse group antibiotics.     

Biofilm acts as a microenvironment for plankton-associated Vibrio cholerae in the aquatic environment of Bangladesh

The role of biofilm as a microenvironment of plankton-associated Vibrio cholerae was investigated using plexiglass as a bait. A total of 72 biofilm samples were tested using culture, direct fluorescent antibody (DFA) and molecular techniques following standard procedures. Culturable V. cholerae (smooth and rugose variants) were isolated from 33% of the samples. V. cholerae O1 were detected by FA technique throughout the year except April and June. All V. cholerae O1 isolates were positive for tcpA, ctxA and ace genes while V. cholerae non-O1, non-O139 isolates lacked these genes. V. cholerae O1 (both Inaba and Ogawa) strains had identical ribotype pattern (R1), but V. cholerae non-O1, non-O139 had different ribotype patterns. All V. cholerae O1 strains were resistant to vibrio-static compound (O/129). All V. cholerae O1 except one were resistant to trimethoprime-sulphamethoxazole, streptomycin, nalidixic acid and furazolidone but sensitive to ciprofloxacin, and tetracycline. This study indicates that plexiglass can act as a bait to form biofilm, a microenvironment that provides shelter for plankton containing V. cholerae in the aquatic environment of Bangladesh.