Investigation of seven Vibrio virulence genes among Vibrio alginolyticus and Vibrio parahaemolyticus strains from the coastal mariculture systems in Guangdong, China

AIMS: To investigate the distribution of the virulence of two Vibrio species among different strains obtained from the mariculture systems on the coast of Guangdong in China and the correlation between the virulence strains and the virulence genes among Vibrio alginolyticus. METHODS: Besides three strains, 72 V. alginolyticus strains and seven Vibrio parahaemolyticus strains were examined by PCR or semi-nested PCR for the virulence genes (tlh, trh, tdh, toxR, toxRS, ctxA, VPI). Additionally, the virulence of 18 V. alginolyticus strains was tested. SIGNIFICANCE AND IMPACT OF THE STUDY: Virulence genes homologous to those in the V. parahaemolyticus and Vibrio cholerae are widely distributed among V. alginolyticus and V. parahaemolyticus in the coastal mariculture systems in Guangdong, China. Some of the V. alginolyticus strains are pathogenic to aquatic animals, and might have derived their virulence genes from V. parahaemolyticus or V. cholerae, representing a possible reservoir of these genes. However, there is no correlation between presence and absence of the virulence genes used to investigate V. alginolyticus and its virulent strains. In this report, we also show that tlh is distributed among V. alginolyticus.     

Adherence as a method of differentiating virulent and avirulent strains of Vibrio parahaemolyticus.

Usually only Kanagawa-positive strains of Vibrio parahaemolyticus are considered virulent; yet, a significant portion of V. parahaemolyticus food poisonings appear to be caused by Kanagawa-negative strains. Therefore, additional and more accurate measurements of a strain's food-poisoning potential are needed. Adherence of V. parahaemolyticus to human fetal intestinal (HFI) cells in vitro seems to offer this information. All strains of V. parahaemolyticus adhered to the HFI cells, but the degree of adherence was related to a number of factors. These included the age of the culture, the strain's Kanagawa reaction and source, the length of time the bacteria were exposed to the HFI cells, and the composition of the growth medium. Cells harvested during the late log phase of growth adhered more intensely than those harvested from the late stationary phase. Virulent strains, i.e., those involved in food poisoning, were observed to have a high adherence ability regardless of their Kanagawa reaction, whereas Kanagawa-negative strains isolated from seafood exhibited weak adherence intensities. Kanagawa-positive strains isolated from seafood adhered strongly to the HFI cells. The difference between the virulent and avirulent strains was quantitative in nature, and the greatest differences in adherence intensities were observed after short (10 to 15 min) exposure times. The presence of ferric iron in the growth medium was found to increase the adherence intensities of the virulent strains.     

Production of thermostable direct hemolysin by Vibrio parahaemolyticus enhanced by conjugated bile acids.

The effects of conjugated bile acids, glycocholic acid, and taurocholic acid (TC) on production of thermostable direct hemolysin (TDH) by Vibrio parahaemolyticus were determined by a reversed passive latex agglutination assay against TDH. The amount of TDH excreted in growth medium containing either glycocholic acid or taurocholic acid (5 mM/liter) was, on a per-cell basis, 4- to 16-fold greater than that excreted in medium without the bile acids. The amounts of TDH released from lysed cells grown with the bile acids (5 mM/liter) were 4- to 32-fold greater than those from lysed cells grown without, suggesting that the bile acids enhanced synthesis of TDH within bacterial cells. These data imply that the conjugated bile acids play a key role in the pathogenicity of V. parahaemolyticus.     

Remodelling of the Vibrio cholerae membrane by incorporation of exogenous fatty acids from host and aquatic environments

The Gram-negative bacteria Vibrio cholerae poses significant public health concerns by causing an acute intestinal infection afflicting millions of people each year. V. cholerae motility, as well as virulence factor expression and outer membrane protein production, has been shown to be affected by bile. The current study examines the effects of bile on V. cholerae phospholipids. Bile exposure caused significant alterations to the phospholipid profile of V. cholerae but not of other enteric pathogens. These changes consisted of a quantitative increase and migratory difference in cardiolipin, decreases in phosphatidylglycerol and phosphatidylethanolamine, and the dramatic appearance of an unknown phospholipid determined to be lyso-phosphatidylethanolamine. Major components of bile were not responsible for the observed changes, but long-chain polyunsaturated fatty acids, which are minor components of bile, were shown to be incorporated into phospholipids of V. cholerae. Although the bile-induced phospholipid profile was independent of the V. cholerae virulence cascade, we identified another relevant environment in which V. cholerae assimilates unique fatty acids into its membrane phospholipids - marine sediment. Our results suggest that Vibrio species possess unique machinery conferring the ability to take up a wider range of exogenous fatty acids than other enteric bacteria.     

Vibrio parahaemolyticus infectious for both humans and edible mollusk abalone

The aims of this study are to report evidence of the first laboratory-acquired infection of Vibrio parahaemolyticus associated with handling experimentally infected abalones and to describe the virulence of the two bacterial strains tested in these animals. Two strains of V. parahaemolyticus, one from the stool of a patient with acute gastroenteritis (strain 880713) and the other from the hemolymph of a diseased small abalone Haliotis diversicolor supertexta (strain 880915), were identified and characterized. Both strains were lethal to small abalone, with similar LD(50) values (8.36-8.41 x 10(4) colony-forming units/g abalone). Laboratory-acquired infection resulted in one individual experiencing two episodes of acute gastroenteritis due to handling virulence tests during a 1-week interval. Our present results suggest that a V. parahaemolyticus strain isolated from the stool of a patient with gastroenteritis was infectious for small abalone, a major species of edible mollusk abalone cultured in Taiwan, while a similar strain isolated from hemolymph of a diseased small abalone was infectious for humans. This is the first report of V. parahaemolyticus virulent to small abalone as a zoonotic pathogen.     

Induction of viable but nonculturable state in Vibrio parahaemolyticus and its susceptibility to environmental stresses

AIMS: This work analysed factors that influence the induction of viable but nonculturable (VBNC) state in the common enteric pathogen, Vibrio parahaemolyticus. The susceptibility of the VBNC cells to environmental stresses was investigated. METHODS AND RESULTS: Bacterium was cultured in tryptic soy broth-3% NaCl medium, shifted to a nutrient-free Morita mineral salt-0.5% NaCl medium (pH 7.8) and further incubated at 4 degrees C in a static state to induce the VBNC state in 28-35 days. The culturability and viability of the cells were monitored by the plate count method and the Bac Light viable count method, respectively. Cells grown at the optimum growth temperature and in the exponential phase better induced the VBNC state than those grown at low temperature and in the stationary phase. Low salinity of the medium crucially and markedly shortened the induction period. The VBNC cells were highly resistant to thermal (42, 47 degrees C), low salinity (0% NaCl), or acid (pH 4.0) inactivation. CONCLUSIONS: Optimal conditions for inducing VBNC V. parahaemolyticus were reported. The increase in resistance of VBNC V. parahaemolyticus to thermal, low salinity and acidic inactivation verified that this state is entered as part of a survival strategy in an adverse environment. SIGNIFICANCE AND IMPACT OF THE STUDY: The methods for inducing VBNC V. parahaemolyticus in a markedly short time will facilitate further physiological and pathological study. The enhanced stress resistance of the VBNC cells should attract attention to the increased risk presented by this pathogen in food.     

Virulence factors in Vibrios and Aeromonads isolated from seafood

Thirty-one isolates from seafood, identified as Aeromonas hydrophila (7), Aeromonas caviae (11), Vibrio parahaemolyticus (3), Vibrio fluvialis (5), Vibrio alginolytictus (3), Vibrio metschnikovii (1) and Vibrio damsela (1), were tested for possible virulence factors including extracellular hydrolytic enzymes, haemolysins, cytotoxins (VERO and HEp-2 cells) and adherence ability (HEp-2 cells). All the A. hydrophila strains were beta-haemolytic and produced cytotoxins as well as one strain of V. fluvialis. A. hydrophila and A. caviae strains, frequently adhesive, showed both aggregative and diffusive patterns, while five Vibrio strains only (three V. fluvialis, one V. parahaemolyticus and one V. alginolyticus) were adhesive with an aggregative pattern.     

Effects of conjugated and unconjugated bile acids on the activity of the Vibrio cholerae porin OmpT

During infection, the enteric pathogen Vibrio cholerae encounters a bile-containing environment. Previous studies have shown that bile and/or bile acids exert several effects on the virulence and physiology of the bacterial cells. These observations have led to the suggestion that bile acids may play a signaling role in infection. We have previously reported that the bile component deoxycholic acid blocks the general diffusion porin OmpT in a dose-dependent manner, presumably as it transits through the pore. V. cholerae colonizes the distal jejunum and ileum, where a mixture of various conjugated and unconjugated bile acids are found. In this work, we have used patch clamp electrophysiology to investigate the effects of six bile acids on OmpT. Two bile acids (deoxycholic and chenodeoxycholic acids) were found to block OmpT at physiological concentrations below 1 mM, while glycodeoxycholic acid was mildly effective and cholic, lithocholic and taurodeoxycholic acids were ineffective in this range. The block was also voltage-dependent. These observations suggest the presence of a specific binding site inside the OmpT pore. Since deconjugation is due to the activity of the endogenous flora, the preferential uptake of some unconjugated bile acids by OmpT may signal the presence of a hospitable environment. The results are also discussed in terms of the possible molecular interactions between the penetrating bile acid molecule and the channel wall.     

Bile acids induce a cationic current, depolarizing pancreatic acinar cells and increasing the intracellular Na+ concentration

Biliary disease is a major cause of acute pancreatitis. In this study we investigated the electrophysiological effects of bile acids on pancreatic acinar cells. In perforated patch clamp experiments we found that taurolithocholic acid 3-sulfate depolarized pancreatic acinar cells. At low bile acid concentrations this occurred without rise in the cytosolic calcium concentration. Measurements of the intracellular Na(+) concentration with the fluorescent probe Sodium Green revealed a substantial increase upon application of the bile acid. We found that bile acids induce Ca(2+)-dependent and Ca(2+)-independent components of the Na(+) concentration increase. The Ca(2+)-independent component was resolved in conditions when the cytosolic Ca(2+) level was buffered with a high concentration of the calcium chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). The Ca(2+)-dependent component of intracellular Na(+) increase was clearly seen during stimulation with the calcium-releasing agonist acetylcholine. During acetylcholine-induced Ca(2+) oscillations the recovery of cytosolic Na(+) was much slower than the recovery of Ca(2+), creating a possibility for the summation of Na(+) transients. The bile-induced Ca(2+)-independent current was found to be carried primarily by Na(+) and K(+), with only small Ca(2+) and Cl(-) contributions. Measurable activation of such a cationic current could be produced by a very low concentration of taurolithocholic acid 3-sulfate (10 microm). This bile acid induced a cationic current even when applied in sodium- and bicarbonate-free solution. Other bile acids, taurochenodeoxycholic acid, taurocholic acid, and bile itself also induced cationic currents. Bile-induced depolarization of acinar cells should have a profound effect on acinar fluid secretion and, consequently, on transport of secreted zymogens.     

Electrostatic mechanism of survival of virulent Aeromonas salmonicida strains in river water.

The ecological mechanism of survival of Aeromonas salmonicida, the bacterial pathogen of fish furunculosis, in river water was investigated by laboratory-based experiments with two virulent strains (which were autoagglutinating) and two virulent strains (which were nonagglutinating). A difference in net electrical charge of A. salmonicida cells was detected by electrophoresis; cells of the virulent strains were negative, whereas cells of the avirulent strains were positive. Despite the loss of viable cells within a week in distilled water and physiological saline (0.85% sodium chloride), the cells of the virulent strains survived for more than 15 weeks in the presence of diluted humic acid (10 micrograms/ml), tryptone (10 micrograms/ml), and cleaned river sand (100 g/100 ml of medium), but loss of viable cells occurred within 5 weeks in the absence of sand. The cells of the avirulent strains lost viability within 2 weeks with no relation to the presence of sand. Using ion-exchange columns, humic acid and the amino acids of tryptone were found to be anionic and cationic in water (pH 7.0), respectively. Sand particles had a high capacity to adsorb humic acid alone and amino acid-humic acid complexes. Thirty to fifty times the environmental concentration of amino acids (10 micrograms/ml) were accumulated on the surface of sand particles, thereby permitting only bacterial cells carrying net negative electrical charges (virulent cells) to survive for a long period on the surface of the sand particles. These electrostatic interrelationships among river sand, humic acid, and bacterial cells are closely implicated in the mechanism of long-term survival of virulent A. salmonicida in river sediments.     

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.     

Effects of conjugated and unconjugated bile acids on the activity of the Vibrio cholerae porin OmpT

Abstract

During infection, the enteric pathogen Vibrio cholerae encounters a bile-containing environment. Previous studies have shown that bile and/or bile acids exert several effects on the virulence and physiology of the bacterial cells. These observations have led to the suggestion that bile acids may play a signaling role in infection. We have previously reported that the bile component deoxycholic acid blocks the general diffusion porin OmpT in a dose-dependent manner, presumably as it transits through the pore. V. cholerae colonizes the distal jejunum and ileum, where a mixture of various conjugated and unconjugated bile acids are found. In this work, we have used patch clamp electrophysiology to investigate the effects of six bile acids on OmpT. Two bile acids (deoxycholic and chenodeoxycholic acids) were found to block OmpT at physiological concentrations below 1 mM, while glycodeoxycholic acid was mildly effective and cholic, lithocholic and taurodeoxycholic acids were ineffective in this range. The block was also voltage-dependent. These observations suggest the presence of a specific binding site inside the OmpT pore. Since deconjugation is due to the activity of the endogenous flora, the preferential uptake of some unconjugated bile acids by OmpT may signal the presence of a hospitable environment. The results are also discussed in terms of the possible molecular interactions between the penetrating bile acid molecule and the channel wall.     

Detection and identification of Vibrio parahaemolyticus by multiplex PCR and DNA-DNA hybridization on a microarray

In this paper,we developed a rapid and accurate method for the detection of Vibrio parahaemolyticus strains,using multiplex PCR and DNA-DNA hybridization.Multiplex PCR was used to simultaneously amplify three diagnostic genes(tlh,tdh and fia)that serve as molecular markers of V.parahaemolyticus.Biotinylated PCR products were hybridized to primers immobilized on a microarray,and detected by chemiluminesce with avidin-conjugated alkaline phosphatase.With this method,forty-five samples were tested.Eight known virulent strains (tlh+/tdh+/fia+)and four known avirulent strains(tlh+/tdh-/fla+)of the V.parahaemolyttcus were successtuny aetectea,ana no non-spectnc hybridization and cross-hybridization reaction were found from fifteen closely-related strains(tin-/tdh-/fta+)or the Vibrio spp.In addition,all the other eighteen strains of non-Vibrio bacteria(tlh-/tdh-/fla-)gave negative results.The DNA microarray successfully distinguished V.parahaemolyticus from other Vibrio spp.The results demonstrated that this was an efficient and robust method for identifying virulent strains of V.parahaemolyticus.     

多重实时PCR检测产毒素性霍乱弧菌和副溶血弧菌

设计引物和探针,优化多重实时PCR条件,以同时检测霍乱弧菌霍乱毒素基因ctxA、副溶血弧菌种特异性基因gyrB和耐热肠毒素基因tdh。该多重实时PCR方法检测产毒素性的O1群(3株)和O139群(44株)霍乱弧菌菌株、不产毒素的O1群(12株)和O139群(6株)及非O1非O139群(7株)霍乱弧菌菌株的ctxA,阳性和阴性结果与普通PCR检测结果100%符合;检测副溶血弧菌种特异性gyrB,116株副溶血弧菌均阳性,而9株其它细菌和72株霍乱弧菌均阴性;检测tdh的阳性和阴性结果也与普通PCR结果完全一致。另外还建立了检测副溶血弧菌菌株trh1和trh2的单重实时PCR方法。     

Genome sequence of the clinical O4:K12 serotype Vibrio parahaemolyticus strain 10329

Vibrio parahaemolyticus is the leading cause of food-borne illnesses worldwide. Here, we report a draft genome of V. parahaemolyticus strain 10329 of the O4:K12 serotype. It belongs to the main U.S. West Coast clonal complex of V. parahaemolyticus (sequence type 36 [ST36]) causing oyster-associated human illness. It contains the virulence determinants tdh and trh but appears to infect at much lower doses than V. parahaemolyticus strains with these same determinants from other areas, such as the U.S. Gulf and Atlantic coasts.     

副溶血弧菌毒力评价小鼠模型的建立与应用

目的建立用于评价副溶血弧菌毒力的小鼠模型,为研究副溶血弧菌的致病机制奠定基础。方法将适宜浓度的菌液经腹腔感染4～5周龄雌性BALB/c小鼠,观察小鼠的症状及死亡数。结果高盐(2%NaCl)条件下培养的强毒株RIMD2210633,经腹腔感染107CFU的菌量,小鼠存活率为20%～30%,而环境无毒株S251的小鼠存活率为100%。结论建立了评价副溶血弧菌毒力的实验小鼠模型,并应用于不同盐分浓度培养的强毒株与环境无毒株的毒力比较实验。     

Serologic and molecular characterization of Vibrio parahaemolyticus strains isolated from seawater and fish products of the Gulf of Mexico

The thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) are the main virulence factors of Vibrio parahaemolyticus. We isolated V. parahaemolyticus from seawater, fish, and oysters obtained from the Pueblo Viejo Lagoon in Veracruz, determined the serogroups, phenotypically and genotypically characterized TDH and TRH, and investigated the presence of the toxR gene. A total of 46 V. parahaemolyticus strains were isolated, and all of them amplified the 368-bp toxR gene fragment. The trh gene was not identified in any of the strains; 4 of the 46 strains were Kanagawa phenomenon (KP) positive and amplified the 251-bp tdh gene fragment. The most frequent serogroup was serogroup O3. This is the first report of the presence of KP-positive tdh-positive environmental V. parahaemolyticus strains in Mexico.