Ecology of Vibrio mimicus in aquatic environments.

An environmental study was done to examine the prevalence of Vibrio mimicus in some aquatic environments of Dhaka, Bangladesh, and of Okayama, Japan. Water samples from Dhaka environments and water and plankton samples from Okayama environments were quantitatively as well as qualitatively analyzed throughout the seasons for V. mimicus. The organism was isolated from Bangladesh environments throughout the year, whereas it was not isolated in Okayama when the water temperature fell below 10 degrees C. Samples with as many as 9.0 x 10(2) CFU of V. mimicus per 100 ml of water in Dhaka and 1.5 x 10(4) CFU of V. mimicus per 100 ml of water in Okayama were detected during the study period. V. mimicus was not found in any environment with an average salinity of 10% or more. Brackish environments with an average salinity of 4% were observed to be the optimal natural condition for the pathogen. Using the API 20E system with the conventional test methods, we observed variations in biochemical properties within the V. mimicus species. This study reveals the inefficacy of the API 20E system to identify a significant percentage of V. mimicus. Therefore, in addition to the API 20E system, a salt tolerance test and a string test are recommended for identification of this species. Susceptibility testing of strains isolated from Okayama environments showed higher resistance to ampicillin and susceptibility to trimethoprim-sulfamethoxazole when compared with environmental isolates of V. mimicus from Bangladesh.     

Ecology of Vibrio mimicus in Aquatic Environments

[This corrects the article on p. 2077 in vol. 55.].     

Ecology and seasonal distribution of Vibrio parahaemolyticus in aquatic environments of a temperate region

Abstract An environmental survey was done to study the ecology and distribution of Vibrio parahaemolyticus in 5 selected stations in Okayama Prefecture, which included fresh, brackish, and marine aquatic environments. Water and plankton samples were collected monthly for quantitative and qualitative analyses during the period October, 1987 to October, 1989 for V. parahaemolyticus . The pathogen was not detected from fresh water environments. A seasonality of the organism was observed in brackish and marine environments where average salinity ranged between 0.39 and 1.28%.Plankton samples yielded higher densities of V. parahaemolyticus compared with water samples. By applying several enrichment techniques, the pathogen was detected quite frequently during the winter months in the environments with temperatures ranging between 10 and 14°C. The identification following conventional tests, by the API 20E system and by serological methods reveal that the API 20E system is satisfactory to identify V. parahaemolyticus and further confirms that the serological method could be a simpler and more rapid procedure for V. parahaemolyticus identification.     

Expression of virulence-related properties by, and intestinal adhesiveness of, Vibrio mimicus strains isolated from aquatic environments.

A study of the major pathogenic characteristics of Vibrio mimicus was carried out with 77 strains isolated from aquatic environments in Okayama, Japan. Of the strains tested, 96% demonstrated in vitro adherence to the rabbit intestinal mucosa, of which 36, 20, and 43% belonged to the strongly, moderately, and weakly adhesive groups, respectively. Of the 27 strains which appeared to be enterotoxigenic in the experiments using rabbit ileal loops, 74% belonged to the strongly adhesive group. All strains of V. mimicus at early log phase showed cell-mediated hemagglutination, and 70% of strongly hemagglutinative strains belonged to the strongly adhesive group, implying a possible correlation between cell-mediated hemagglutination and bacterial adherence. However, no significant correlation could be detected in the production of putative exocellular pathogenic factors and bacterial adherence or enterotoxigenicity.     

Interactions of Vibrio phages and their hosts in aquatic environments

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Sources of Vibrio mimicus Contamination of Turtle Eggs

Vibrio mimicus contamination of sand increased significantly during the arrival of the olive ridley sea turtles (Lepidochelys olivacea) at Ostional anidation beach, Costa Rica. Statistical analysis supports that eggs are contaminated with V. mimicus by contact with the sand nest. V. mimicus was isolated from eggs of all nests tested, and ctxA⁺ strains were found in 31% of the nests, all of which were near the estuary.     

Identification of the siderophores from Vibrio hollisae and Vibrio mimicus as aerobactin

Abstract Hydroxamate siderophores were purified from low-iron cultures of Vibrio hollisae ATCC 33564 and Vibrio mimicus ATCC 33653. By a combination of ¹H and ¹³C NMR spectroscopy, fast atom bombardment mass spectrometry, and compositional analysis, both of the siderophores were identified as aerobactin, a citrate-based dihydroxamate siderophore, which is highly prevalent in species of the family Enterobacteriaceae . Four other clinical strains belonging to these species also produced aerobactin. In response to iron limitation, all strains expressed two high molecular mass outer membrane proteins. The protein with an apparent molecular mass of 77 kDa, which was common to all strains examined, may be the ferric aerobactin receptor.     

Organization of the CTX prophage in environmental isolates of Vibrio mimicus

The organization of the CTX prophage in environmental strains of Vibrio mimicus was investigated. Sixteen hundred non-sucrose fermenting vibrios were examined for ctx gene by hybridization. Out of 1,600 isolates, 6 V. mimicus isolates contained ctxA gene. The organization of CTX prophage was determined by RFLP using ctxA probe. The CTX prophage integrated at a single site in V. mimicus genome which was present as a single copy flanked by at least a single RS element. Ribotype pattern revealed that a particular clone of V. mimicus acquired the CTXPhi in the aquatic environment. This study demonstrated that V. mimicus could act as a reservoir of CTXPhi in the aquatic environment.     

Analysis of the Structural Gene Encoding a Hemolysin in Vibrio mimicus

An environmental isolate of V. mimicus, strain E-33, has been reported to produce and secrete a hemolysin of 63 kDa. The hemolysin is enterotoxic in test animals. The nucleotide sequence of the structural gene of the hemolysin was determined. We found a 2,232 bp open reading frame, which codes a peptide of 744 amino acids, with a calculated molecular weight of 83,903 Da. The sequence for the structural gene was closely related to the V. cholerae el tor hlyA gene, coding an exocellular hemolysin. The amino terminal amino-acid sequence of the 63 kDa hemolysin, purified from V. mimicus, was determined by the Edman degradation method and found to be NH₂-S-V-S-A-N-N-V-T-N-N-N-E-T. This sequence is identical from S-152 to T-164 predicted from the nucleotide sequence. So, it seems that the mature hemolysin in V. mimicus is processed upon deleting the first 151 amino acids, and the molecular mass is 65,972 Da. Analyzing the deduced amino-acid sequence, we also found a potential signal sequence of 24 amino acids at the amino terminal. Our results suggest that, like V. cholerae hemolysin, two-step processing also exists in V. mimicus hemolysin.     

Purification and characterization of a heat-stable enterotoxin of Vibrio mimicus

A heat-stable enterotoxin produced by Vibrio mimicus (VM-ST) was studied. VM-ST was purified from a culture supernatant of V. mimicus strain AQ-0915 by ammonium sulfate fractionation, hydroxyapatite treatment, ethanol extraction, column chromatography on both SP-Sephadex C-50 and DEAE-Sephadex A-25, and HPLC, and the recovery rate was about 15%. Purified VM-ST was heat-stable. VM-ST activity was cross-neutralized by anti-STh antiserum. The amino acid composition of the purified VM-ST was determined 17 amino acid residues in the following sequence: Ile-Asp-Cys-Cys-Glu-Ile-Cys-Cys-Asn-Pro-Ala-Cys-Phe-Gly-Cys-Leu-Asn. This composition and sequence were identical to those of V. cholerae non-O1-ST. These results clearly demonstrate the production of a characteristic VM-ST by V. mimicus.     

The ability of two different Vibrio spp. bacteriophages to infect Vibrio harveyi, Vibrio cholerae and Vibrio mimicus

AIMS: To determine the host range of the Vibrio harveyi myovirus-like bacteriophage (VHML) and the cholera toxin conversion bacteriophage (CTX Phi) within a range of Vibrio cholerae and V. mimicus and V. harveyi, V. cholerae and V. mimicus isolates respectively. METHODS AND RESULTS: Three V. harveyi, eight V. cholerae and five V. mimicus isolates were incubated with VHML and CTX Phi. Polymerase chain reaction (PCR) was used to determine the presence of VHML and CTX Phi in infected isolates. We demonstrated that it was possible to infect one isolate of V. cholerae (isolate ACM #2773/ATCC #14035) with VHML. This isolate successfully incorporated VHML into its genome as evident by positive PCR amplification of the sequence coding part of the tail sheath of VHML. Attempts to infect all other V. cholerae and V. mimicus isolates with VHML were unsuccessful. Attempts to infect V. cholerae non-01, V. harveyi and V. mimicus isolates with CTX Phi were unsuccessful. CONCLUSIONS: Bacteriophage infection is limited by bacteriophage-exclusion systems operating within bacterial strains and these systems appear to be highly selective. One system may allow the co-existence of one bacteriophage while excluding another. VHML appears to have a narrow host range which may be related to a common receptor protein in such strains. The lack of the vibrio pathogenicity island bacteriophage (VPI Phi) in the isolates used in this study may explain why infections with CTX Phi were unsuccessful. SIGNIFICANCE AND IMPACT OF THE STUDY: The current study has demonstrated that Vibrio spp. bacteriophages may infect other Vibrio spp.     

Vibrio mimicus diarrhea following ingestion of raw turtle eggs.

Clinical and epidemiological characteristics of diarrhea associated with Vibrio mimicus were identified in 33 hospitalized patients referred to the Costa Rican National Diagnostic Laboratory Network between 1991 and 1994. The relevant symptoms presented by patients included abundant watery diarrhea, vomiting, and severe dehydration that required intravenous Dhaka solution in 83% of patients but not fever. Seroconversion against V. mimicus was demonstrated in four patients, from whom acute- and convalescent-phase sera were obtained. Those sera did not show cross-reaction when tested against Vibrio cholerae O1 strain VC-12. All the V. mimicus isolates from these cases produced cholera toxin (CT) and were susceptible to commonly used antibiotics. Attempts to isolate this bacterium from stool samples of 127 healthy persons were not successful. Consumption of raw turtle eggs was recalled by 11 of the 19 (58%) individuals interviewed. All but two V. mimicus diarrheal cases were sporadic. These two had a history of a common source of turtle (Lepidochelys olivacea) eggs for consumption, and V. mimicus was isolated from eggs from the same source (a local market). Among the strains, variations in the antimicrobial susceptibility pattern were observed. None of the strains recovered from market turtle eggs nor the four isolates from river water showed CT production. Further efforts to demonstrate the presence of CT-producing V. mimicus strains in turtle eggs were made. Successful results were obtained when nest eggs were tested. In this case, it was possible to isolate CT- and non-CT-producing strains, even from the same egg. For CT detection we used PCR, enzyme-linked immunosorbent assay (ELISA), and Y-1 cell assay, obtaining a 100% correlation between ELISA and PCR results. Primers Col-1 and Col-2, originally described as specific for the V. cholerae O1 ctxA gene, also amplified a 302-bp segment with an identical restriction map from V. mimicus. These results have important implications for epidemiological surveillance in tropical countries where turtle eggs are used for human consumption, serving as potential sources of cholera-like diarrhea.     

Sugar composition of the polysaccharide portion of lipopolysaccharides of Vibrio fluvialis, Vibrio vulnificus, and Vibrio mimicus

A chemotaxonomic study was carried out on Vibrio fluvialis and V. vulnificus on the basis of the sugar composition of the polysaccharide portion of their lipopolysaccharides (LPS). A previously developed rapid method of preparing samples for compositional sugar analysis was employed. Nineteen O-serogroups of V. fluvialis were divided into 14 chemotypes while seven O-serogroups of V. vulnifucus were divided also into seven chemotypes since the polysaccharide portion of LPS of each serogroup has a different sugar composition from that of the other serogroups. Close similarities in the sugar composition of the same portion were demonstrated between serologically cross-reacting non-O1 group V. cholerae and V. fluvialis, and non-O1 V. cholerae and V. mimicus.     

Existence of a Novel Hemagglutinin Having No Protease Activity in Vibrio mimicus

The protease elaborated by Vibrio mimicus is known to possess hemagglutinating ability to chicken erythrocytes, the well-known HA/protease. A non-protease hemagglutinin (HA) with strong agglutinating ability towards rabbit erythrocytes was obtained from 32 hr culture supernatant of a pathogenic environmental strain of V. mimicus. This HA (V. mimicus HA: VMHA) appeared stable at relatively higher temperature and agglutinated the erythrocytes from rabbit, guinea pig and mouse but not the erythrocytes from chicken, bovine, horse and sheep. Simple sugars, metal ions and chelating agents failed to inhibit the activity of VMHA. The activity of VMHA was found to be sensitive to digestion by proteolytic enzymes including HA/protease. These results provide evidence for the existence of novel HA other than HA/protease in V. mimicus.     

Medium-dependent production of extracellular enterotoxins by non-O-1 Vibrio cholerae, Vibrio mimicus, and Vibrio fluvialis.

Fluid accumulation at 4 h in the intestines of suckling mice enabled us to distinguish non-O-1 Vibrio cholerae, V. mimicus, and V. fluvialis clinical isolates from environmental isolates. Enterotoxin production was culture medium dependent. Filtrates of cultures grown in tryptic soy broth without glucose but with added 0.5% NaCl did not exhibit marked enterotoxin activity in the assay. Culture filtrates of all clinical strains grown in brain heart infusion broth supplemented with 0.5% NaCl induced large amounts of fluid accumulation in mouse intestines. However, most environmental strains grown in brain heart infusion broth amended as described above were unable to induce fluid accumulation. The enterotoxin present in culture filtrates lost activity at 56 degrees C and appeared to be distinct from previously described virulence factors, including the well-described cholera toxin. The new enterotoxin could represent an important virulence mechanism common to all three species.     

RNA Colony Blot Hybridization Method for Enumeration of Culturable Vibrio cholerae and Vibrio mimicus Bacteria

A species-specific RNA colony blot hybridization protocol was developed for enumeration of culturable Vibrio cholerae and Vibrio mimicus bacteria in environmental water samples. Bacterial colonies on selective or nonselective plates were lysed by sodium dodecyl sulfate, and the lysates were immobilized on nylon membranes. A fluorescently labeled oligonucleotide probe targeting a phylogenetic signature sequence of 16S rRNA of V. cholerae and V. mimicus was hybridized to rRNA molecules immobilized on the nylon colony lift blots. The protocol produced strong positive signals for all colonies of the 15 diverse V. cholerae-V. mimicus strains tested, indicating 100% sensitivity of the probe for the targeted species. For visible colonies of 10 nontarget species, the specificity of the probe was calculated to be 90% because of a weak positive signal produced by Grimontia (Vibrio) hollisae, a marine bacterium. When both the sensitivity and specificity of the assay were evaluated using lake water samples amended with a bioluminescent V. cholerae strain, no false-negative or false-positive results were found, indicating 100% sensitivity and specificity for culturable bacterial populations in freshwater samples when G. hollisae was not present. When the protocol was applied to laboratory microcosms containing V. cholerae attached to live copepods, copepods were found to carry approximately 10,000 to 50,000 CFU of V. cholerae per copepod. The protocol was also used to analyze pond water samples collected in an area of cholera endemicity in Bangladesh over a 9-month period. Water samples collected from six ponds demonstrated a peak in abundance of total culturable V. cholerae bacteria 1 to 2 months prior to observed increases in pathogenic V. cholerae and in clinical cases recorded by the area health clinic. The method provides a highly specific and sensitive tool for monitoring the dynamics of V. cholerae in the environment. The RNA blot hybridization protocol can also be applied to detection of other gram-negative bacteria for taxon-specific enumeration.Vibrio cholerae is autochthonous to the aquatic environment, but some strains produce enterotoxins and are capable of causing epidemics of the human disease cholera. Strains of V. cholerae are classified by their O antigen, with over 210 serogroups recognized to date. Seven cholera pandemics have occurred since 1832: while microbiologic data on the earlier pandemics are not available, the last two are known to have been caused by strains within serogroup O1, with the major pathogenic factor being production of cholera toxin. The genes encoding cholera toxin and other pathogenic factors have been shown to reside in a mobile genetic element of phage origin, designated CTXΦ (20).Standard microbiologic methods for isolation of V. cholerae present in natural waters rely primarily on a method originally developed for clinical diagnosis, namely, enrichment in alkaline peptone water, followed by subculture on selective media and confirmation using selected biochemical and immunological tests (7). The alkaline nature of the enrichment broth allows differential multiplication of Vibrio species but renders this method inappropriate for enumeration. PCR methods and oligonucleotide hybridization have been used to detect and enumerate toxigenic V. cholerae bacteria (3, 11, 12, 14, 15, 21). These methods typically rely on amplification of or hybridization to pathogenic markers, such as O1/O139 wbe, tcpA, and ctxA DNA sequences.However, occasional localized outbreaks of cholera have been caused by non-O1, non-O139 V. cholerae, which may be toxigenic or nontoxigenic. Conversely, many environmental V. cholerae O1 strains isolated from areas of endemicity do not harbor ctx genes (9). It has also been shown that CTXΦ is capable of lysogenic conversion of strains that are CTXΦ negative (20). Additionally, the cholera toxin (CTX) prophage has also been detected in clinical strains of V. mimicus, and V. mimicus has been proposed as a natural reservoir for CTXΦ (2). Furthermore, ecological studies of V. cholerae are often hampered by the fact that toxigenic strains represent only a small percentage of the total V. cholerae population in the environment, especially in areas where cholera is not endemic. These facts underline the need for a method of detection of the total number of V. cholerae bacteria present in environmental samples.The many copies of 16S rRNA molecules in each V. cholerae cell offer appropriate targets for species-specific enumeration. In this study, the probe Vchomim1276, previously described by Heidelberg et al. (4-6), was employed in an RNA colony blot hybridization protocol. The specificity and sensitivity of the probe were tested using type strains and environmental and clinical isolates. The method was evaluated using laboratory microcosms to which cells of V. cholerae were added, and the protocol was used to enumerate V. cholerae bacteria in samples collected from ponds in a region of cholera endemicity in Bangladesh.     

Enterotoxin production by Vibrio cholerae and Vibrio mimicus grown in continuous culture with microbial cell recycle

We have examined the effect of complete cell recycle on the production of cholera toxin (CT) by Vibrio cholerae and CT-like toxin by Vibrio mimicus in continuous culture fermentations. Complete cell recycle was obtained by filtering culture fluids through Amicon hollow fibers with an exclusion limit of 100,000 daltons (H1P100-20) and returning the concentrated cell slurry to the fermentor. A single 1-liter laboratory fermentor system modified with this recycle loop was capable of producing over 20 liters of cell-free culture filtrate per day. Toxin production in this system was compared with yields obtained in traditional continuous cultures and in shake flask cultures. Yields of CT from V. cholerae 569B in the recycle fermentor were highest at the highest dilution rate employed (1.0 vol/vol per h). The use of complete cell recycle dramatically increased yields over those obtained in continuous culture and equaled those obtained in shake flasks. The concentration of CT in the filtrate was slightly less than half of that measured in culture fluids sampled at the same time. Similarly, V. mimicus 61892 grown in the presence of 50 micrograms of lincomycin per ml produced 280 ng of CT per ml in the recycle fermentor, compared with 210 ng/ml in shake flasks under optimal conditions. The sterile filtrate from this fermentation contained 110 ng/ml.     

Iron-binding compounds and related outer membrane proteins in Vibrio cholerae non-O1 strains from aquatic environments

A total of 156 strains of Vibrio cholerae non-O1 from aquatic origins were examined for the presence of iron uptake mechanisms and compared with O1 strains and other Vibrio species. All non-O1 strains were able to grow in iron-limiting conditions, with MICs of ethylenediaminedi (O-hydroxyphenylacetic acid) ranging from 20 microM to 2 mM. The production of siderophores was demonstrated by growth in chrome azurol S agar and cross-feeding assays. All strains produced phenolate-type compounds, as assessed by the chemical tests and by bioassays with Salmonella typhimurium enb-7. Some of the strains also promoted the growth of S. typhimurium enb-1 (which can use only enterobactin as a siderophore) as well as some strains of Vibrio anguillarum deficient in the anguibactin-mediated system. The chromatographic analyses and absorption spectra of siderophores extracted from culture supernatants suggest that vibriobactin may be produced by the strains examined. Interestingly, some strains also produced hydroxamate-type compounds, as determined by chemical tests, and were able to promote the growth of an aerobactin-deficient strain of Escherichia coli. These results were confirmed by the absorption spectra and chromatographic analyses of the culture extracts. The synthesis of iron-regulated outer membrane proteins in representative strains was also examined. The molecular sizes of the main induced proteins ranged from 70 to 78 kilodaltons. These results indicate that several iron uptake mechanisms which could be involved in environmental survival and pathogenicity are present in environmental V. cholerae non-O1 strains.