Morphological, Cultural, Biochemical, and Serological Comparison of Japanese Strains of Vibrio parahemolyticus with Related Cultures Isolated in the United States

Morphological, cultural, biochemical, and serological characteristics of 79 strains of Vibrio parahemolyticus isolated from patients suffering from gastroenteric disease in Japan were compared with 17 suspected V. parahemolyticus cultures isolated from wound infections and 14 nonpathogenic vibrios isolated from an estuarine environment in the United States. These groups were differentiated on the basis of several key reactions which included: the range of growth temperature and salt tolerance; the production of catalase and acetoin; the hydrolysis of starch; the fermentation and utilization as single carbon source of sucrose, cellobiose, and arabinose; and the ability to swarm on 1% agar. The separation of the groups on the basis of cultural and biochemical analyses was confirmed by means of slide agglutinations with specific anti-K antisera. The results of this study strongly suggest that the wound infection isolates are V. parahemolyticus species which are easily distinguished from the nonpathogenic estuarine vibrios.     

Genetic Characterization of Vibrio vulnificus Strains from Tilapia Aquaculture in Bangladesh

Outbreaks of Vibrio vulnificus wound infections in Israel were previously attributed to tilapia aquaculture. In this study, V. vulnificus was frequently isolated from coastal but not freshwater aquaculture in Bangladesh. Phylogenetic analyses showed that strains from Bangladesh differed remarkably from isolates commonly recovered elsewhere from fish or oysters and were more closely related to strains of clinical origin.Vibrio vulnificus causes severe wound infections and life-threatening septicemia (mortality, >50%), primarily in patients with underlying chronic diseases (10, 19, 23) and primarily from raw oyster consumption (21). This Gram-negative halophile is readily recovered from oysters (27, 35, 43) and fish (14) and was initially classified into two biotypes (BTs) based on growth characteristics and serology (5, 18, 39). Most human isolates are BT1, while BT2 is usually associated with diseased eels (1, 39). An outbreak of wound infections from aquacultured tilapia in Israel (6) revealed a new biotype (BT3). Phenotypic assays do not consistently distinguish biotypes (33), but genetic analyses have helped resolve relationships (20). A 10-locus multilocus sequence typing (MLST) scheme (8, 9) and a similar analysis of 6 loci (13) segregated V. vulnificus strains into two clusters. BT1 strains were in both clusters, while BT2 segregated into a single cluster and BT3 was a genetic mosaic of the two lineages. Significant associations were observed between MLST clusters and strain origin: most clinical strains (BT1) were in one cluster, and the other cluster was comprised mostly of environmental strains (some BT1 and all BT2). Clinical isolates were also associated with a unique genomic island (13).The relationship between genetic lineages and virulence has not been determined, and confirmed virulence genes are universally present in V. vulnificus strains from both clinical and environmental origins (19, 23). However, segregation of several polymorphic alleles agreed with the MLST analysis and correlated genotype with either clinical or environmental strain origin. Alleles include 16S rRNA loci (15, 26, 42), a virulence-correlated gene (vcg) locus (31, 41, 42), and repetitive sequence in the CPS operon (12). DiversiLab repetitive extrageneic palindromic (rep-PCR) analysis also confirmed these genetic distinctions and showed greater diversity among clinical strains (12).Wound infections associated with tilapia in Israel implicated aquaculture as a potential source of V. vulnificus in human disease (6, 40). Tilapia aquaculture is increasing rapidly, as shown by a 2.8-fold increase in tons produced from 1998 to 2007 (Food and Agriculture Organization; http://www.fao.org/fishery/statistics/en). Therefore, presence of V. vulnificus in tilapia aquaculture was examined in Bangladesh, a region that supports both coastal and freshwater sources of industrial-scale aquaculture. V. vulnificus strains were recovered from market fish, netted fish, and water samples, and the phylogenetic relationship among strains was examined relative to clinical and environmental reference strains collected elsewhere.     

Distribution of Virulence Genes in Clinical and Environmental Vibrio cholerae Strains in Bangladesh

Vibrio cholerae, an environmental organism, is a facultative human pathogen. Here, we report the virulence profiles, comprising 18 genetic markers, of 102 clinical and 692 environmental V. cholerae strains isolated in Bangladesh between March 2004 and January 2006, showing the variability of virulence determinants within the context of public health.     

Drug Resistance and R Plasmids in Escherichia coli Strains Isolated from Imported Pet Birds

Drug resistance in Escherichia coli strains isolated from pet birds (mynahs, macaws, finches, common bengals, parrots, and flamingos) imported into Japan from 10 foreign countries in 1977 and 1978 was investigated. Of the 309 strains isolated from 127 pet birds in the Animal Quarantine Service, 232 (75.1%) were drug resistant. Furthermore, strains resistant to oxytetracycline hydrochloride, dihydrostreptomycin, and sulfadimethoxine were relatively common. Resistance patterns varied from single to sextuple resistance, and 148 (63.8%) of the resistant strains had conjugative R plasmids. These results suggest that the high incidence of drug resistance and R plasmids in E. coli strains isolated from these pet birds may be a reflection of the prophylactic use of antibiotics for the prevention of diseases which increasingly occur with importation of the birds. Furthermore, the results suggest that the birds may be potential reservoirs of drug-resistant E. coli for families who raise and have intimate contact with such birds.     

Characterization of Vibrio anguillarum Strains Isolated from Diseased Fish in Finland

Characterization of V anguillarum strains (n=109) isolated from diseased salmonids was performed. Eight O serovars were found among the strains. Serovar Ol was predominant (90 %), while serovars O2, O3, O5, O8, O9, and a new serovar Va NT2, were represented by 1 or 2 strains. Two strains remained non-typeable. One of these was cross-reactive with several antisera, but had a LPS profile identical to that of serovar O8. All serovars showed specific LPS profiles. All but 1 of the Ol strains had a plasmid comparable in size to the pJMl virulence plasmid, while plasmids of different sizes were found in O2, Va NT2 and the non-typeable strains. Apart from a single strain resistant to tetracycline, all the strains were sensitive to oxolinic acid, tetracycline, and trimethoprim-sulfonamides. By their biochemical and antigenic properties strains causing vibriosis among salmonids in Finland closely resemble Scandinavian strains. Predominance of the serovars Ol and O2 suggests that commercial vaccines containing these serovars should afford sufficient protection against vibriosis in Finland.     

Molecular Diversity of Plasmids Bearing Genes That Encode Toluene and Xylene Metabolism in Pseudomonas Strains Isolated from Different Contaminated Sites in Belarus

Twenty different Pseudomonas strains utilizing m-toluate were isolated from oil-contaminated soil samples near Minsk, Belarus. Seventeen of these isolates carried plasmids ranging in size from 78 to about 200 kb (assigned pSVS plasmids) and encoding the meta cleavage pathway for toluene metabolism. Most plasmids were conjugative but of unknown incompatibility groups, except for one, which belonged to the IncP9 group. The organization of the genes for toluene catabolism was determined by restriction analysis and hybridization with xyl gene probes of pWW0. The majority of the plasmids carried xyl-type genes highly homologous to those of pWW53 and organized in a similar manner (M. T. Gallegos, P. A. Williams, and J. L. Ramos, J. Bacteriol. 179:5024–5029, 1997), with two distinguishable meta pathway operons, one upper pathway operon, and three xylS-homologous regions. All of these plasmids also possessed large areas of homologous DNA outside the catabolic genes, suggesting a common ancestry. Two other pSVS plasmids carried only one meta pathway operon, one upper pathway operon, and one copy each of xylS and xylR. The backbones of these two plasmids differed greatly from those of the others. Whereas these parts of the plasmids, carrying the xyl genes, were mostly conserved between plasmids of each group, the noncatabolic parts had undergone intensive DNA rearrangements. DNA sequencing of specific regions near and within the xylTE and xylA genes of the pSVS plasmids confirmed the strong homologies to the xyl genes of pWW53 and pWW0. However, several recombinations were discovered within the upper pathway operons of the pSVS plasmids and pWW0. The main genetic mechanisms which are thought to have resulted in the present-day configuration of the xyl operons are discussed in light of the diversity analysis carried out on the pSVS plasmids.     

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.     

Lytic Activity of Vibrio Phages on Strains of Vibrio fetus Isolated from Man and Animals

Five phages isolated from lysogenic strains of Vibrio fetus var. venerealis and two from V. fetus var. intestinalis were tested for lytic activity on 95 V. fetus strains from various animal and human hosts. In addition, virion and plaque morphology of the seven phages were compared. Electron micrographs showed that all were the kite-tailed variety with minor variations in head and tail dimensions. Plaques of V45 and V2 were small, clear and irregular; those of V3, V8, and V19 were large, clear and regular at the edge; the plaques of V16 and V20 were intermediate in size, clear, and very irregular at the edge with satellite plaques. The number of strains lysed by one or more phages were as follows: 29 of 30 from cattle; 7 of 11 from sheep; 1 of 5 from pigs; 1 of 1 from a monkey; and 33 of 42 from human hosts. Four natural groups of phages were derived by statistical measures of percentage of similarity in lytic activity. Group III lysed more strains (46 of 95) than any of the others. Twenty-five strains were lysed by group IV, 23 strains by group I, and 19 strains by group II. Results of this study indicate that phage typing should be a practical supplement to other differential tests for V. fetus.     

Drug Resistance and R Plasmids in Vibrio anguillarum Isolated in Cultured Ayu (Plecoglossus altivelis)

Two hundred twenty-six strains of Vibrio anguillarum collected from cultured ayu (Plecoglossus altivelis) between 1978 and 1980 were studied for their sensitivities to 10 chemotherapeutics. In order to determine whether the drug-resistant strains possessed transferable R plasmids, they were conjugated with Escherichia coli. Almost all the strains isolated during the 3 years showed resistance to nalidixic acid (NA) and/or furazolidone (NF). NA and NF resistance were not transferred to Escherichia coli from any of the strains. Chloramphenicol-resistant strains were isolated in every year and almost all of them carried transferable R plasmids. Only one strain with tetracycline resistance was found among the strains tested. Strains resistant to sulfonamides, streptomycin, ampicillin (ABP), and trimethoprim (TMP) increased rapidly in 1980, and a large number of them carried transferable R plasmids. Transferable R plasmids encoded with resistance to ABP and TMP were detected for the first time in V. anguillarum strains. The R plasmids detected in the strains isolated in 1980 were classified into incompatibility groups E, A, and an untypable group. The R plasmid DNAs were cleaved by EcoRI to yield 11 to 13 fragments. The estimated molecular weights of the R plasmids from the five strains ranged from 97 to 104 M daltons.     

Characterization of Vibrio cholerae O139 Synonym Bengal Isolated from Patients with Cholera-Like Disease in Bangladesh

Vibrio cholerae O139 (synonym Bengal), a novel serovar of V. cholerae, is the causative agent of large outbreaks of cholera-like illness currently sweeping India and Bangladesh. Eight randomly selected V. cholerae O139 isolates were studied for their biological properties, which were compared with those of V. cholerae O1 and other V. cholerae non-O1. The V. cholerae O139 isolates were characterized by the production of large amount of cholera toxin, hemagglutination, weak hemolytic properties, resistance to polymyxin B, lysogeny with, and production of, kappa type phage (4/8 isolates only), and resistance to both classical and El Tor-specific phages. Thus, V. cholerae O139 isolates had an overall similarity with V. cholerae O1 El Tor.     

Hydrographic Status of Sundarbans Mangrove, Bangladesh with Special Reference to Post-larvae and Juveniles Fish and Shrimp Abundance

Environmental factors, including water parameters were correlated with assemblages of shell and finfish post-larvae and juveniles in the 5 rivers of Sundarbans mangrove for 2 years, located in the south-west of Bangladesh. Fifteen species of shellfish belonging to 6 families and 37 species of finfish belonging to 27 families were recorded. Four species of shrimp (Metapenaeus monoceros, M. brevicornis, Macrobrachium villosimanus and Acetes spp.) and megalopa of crabs (79.3–96.83%), and 4 species of finfish (Panchax melastigma, Liza parsia, L. tade and Gobidae) (40–76%) were the most abundant species. Significant (p<0.05) inter-month variations in temperature, transparency and chlorophyll-a; inter-month and inter-river variations in salinity, conductivity and TDS were observed in the Sundarbans water. Ordination on correlation coefficients between temperature and salinity with the abundance of dominant species showed that Acetes spp. and Gobidae were abundant in high salinity and high temperature areas; Stolephorus tri in low salinity and high temperature areas; Macrobrachiun villosimanus, Leiognathus spp. and Liza tade in low salinity and low temperature areas, however, other species showed variations among 2 years of study. Out of 7 dominant shellfishes, 4 species showed positive correlation to temperature, pH and TDS; 3 species to salinity, transparency, conductivity and chlorophyll-a in the river water. Among 8 dominant fish species, 4 species were found to positively correlate with temperature and pH; 3 with salinity, conductivity and chlorophyll-a; 6 with transparency and 5 species showed positive correlation to TDS in Sundarbans water. Abundance of Penaeus monodon also showed positive correlation to observed water parameters except chlorophyll-a.     

Genetic and Biophysical Study of R Plasmids Conferring Sulfonamide Resistance in Shigella Strains Isolated in 1952 and 1956

The conjugative plasmids determining sulfonamide resistance in five Shigella strains, each isolated from a different patient, have been characterized. One S. flexneri 2a strain, isolated in 1952, harbored an fi(+) plasmid of molecular weight 53 x 10(6), which specified synthesis of F-like pili and bore determinants for sulfonamide resistance (Su) and bacteriocinogeny (Col). This plasmid was compatible with plasmids of groups F(I), F(II), I(alpha), and P. A second S. flexneri 2a strain isolated in 1952 harbored an fi(-) plasmid of molecular weight 59 x 10(6), bearing the Su determinant and compatible with all plasmids tested. This strain also harbored an fi(+) group-F(II) plasmid of molecular weight 42 x 10(6), which bore the Col determinant and specified synthesis of F-like pili. Three S. dysenteriae 2 strains isolated in 1956 carried apparently identical fi(-) plasmids of molecular weight 58 x 10(6), which bore the Su determinant, could form transconjugants in Pseudomonas but not in Proteus, and were incompatible with the P-group plasmid RP4.     

Bacterial Strains Isolated from Different Niches Can Exhibit Different Patterns of Adhesion to Substrata

Various mechanisms have been demonstrated to be operative in bacterial adhesion to surfaces, but whether bacterial adhesion to surfaces can ever be captured in one generally valid mechanism is open to question. Although many papers in the literature make an attempt to generalize their conclusions, the majority of studies of bacterial adhesion comprise only two or fewer strains. Here we demonstrate that three strains isolated from a medical environment have a decreasing affinity for substrata with increasing surface free energy, whereas three strains from a marine environment have an increasing affinity for substrata with increasing surface free energy. Furthermore, adhesion of the marine strains related positively with substratum elasticity, but such a relation was absent in the strains from the medical environment. This study makes it clear that strains isolated from a given niche, whether medical or marine, utilize different mechanisms in adherence, which hampers the development of a generalized theory for bacterial adhesion to surfaces.