Antibiotic activity of lectins from marine algae against marine vibrios

Saline and aqueous ethanol extracts of marine algae and the lectins from two red algal species were assayed for their antibiotic activity against marine vibrios. Experimental studies were also carried out on the influence of environmental factors on such activity, using batch cultures. The results indicated that many of the saline extracts of the algal species were active and that the activity was selective against those vibrios assayed. The algal extracts were active against Vibrio pelagius and the fish pathogen V. vulnificus, but inactive against V. neresis. Algal lectins from Eucheuma serra (ESA) and Galaxaura marginata (GMA) strongly inhibited V. vulnificus but were inactive against the other two vibrios. The antibacterial activity of algal extracts was inhibited by pretreatment with various sugars and glycoprotein. Extracts of the two red algae, E. serra and Pterocladia capillacea, in saline and aqueous ethanol, inhibited markedly the growth rate of V. vulnificus at very low concentrations. Culture results indicated that metabolites active against V. vulnificus were invariably produced in P. capillacea over a wide range of temperature, light intensity, and nutritional conditions. Enhanced antibacterial activity occurred when P. capillacea was grown under higher irradiance, severe nutrient stress and moderate temperature (20 °C), reflecting the specific antibiotic characteristics of this alga. The strong antibiotic activity of lectins towards fish pathogenic bacteria reveals one of the important roles played by algal lectins, as well as the potential high economic value of those marine algae assayed for aquaculture and for biomedical purposes.     

Transformation ofVibrio vulnificus by electroporation

Vibrio vulnificus is a pathogenic, estuarine bacterium associated with primary septicemia and severe wound infections. Previous studies have reported limited success in the introduction of plasmids intoV. vulnificus by conjugation, and there have been no reports of successful electroporation procedures. We report here on the introduction of recombinant plasmids into four strains ofV. vulnificus by electroporation. In contrast to the complex medium that others have used for the electroporation ofVibrio cholerae, the efficiency of transformation ofV. vulnificus was greatly improved by the use of a defined growth medium containing glucose. The addition of glycine betaine further enhanced transformant yield.     

Isolation and characterization ofVibrio vulnificus inhabiting the marine environment of the southwestern area of Taiwan

Vibrio vulnificus, a marine bacterium, is of concern in Taiwan because it causes wound infections and sepsis with a high mortality rate every year. To examine forV. vulnificus, 13 samples of seawater or oysters were collected from nine sites in Yunlin, Chiayi, and Tainan. Seventy-seven strains ofV. vulnificus were isolated from 11 samples. Among these environmental isolates, 72 (91%) were indole-positive, a characteristic of biotype 1. The remaining five strains although indole-negative, a characteristic previously found exclusively in biotype 2 strains, were all ornithine decarboxylase- and mannitol-positive, which has never been reported for biotype 2 strains. Based on the overall biochemical reactions obtained using a commercial identification system, these indole-negative strains appeared to be more like biotype 1. Fifty-seven ribotypes were identified among these isolates, indicating the great genetic divergence in this species. Of the 30 environmental isolates tested, 17 (56.7%) exhibited virulence comparable to the clinical isolates in the mouse, implying that a high proportion of theV. vulnificus strains in the marine environments might be pathogenic to humans.     

Characterization and Virulence of Hemolysin III from <Emphasis Type="Italic">Vibrio vulnificus</Emphasis>

Vibrio vulnificus, a highly virulent marine bacterium, is the causative agent of both serious wound infections and fatal septicemia in many areas of the word. A gene (hlyIII) encoding a hemolysin was cloned and sequenced from V. vulnificus. Nucleotide sequence analysis predicted an open reading frame of 642 bp encoding a 214 amino acid polypeptide that showed 48% sequence identity to the hemolysin III of Bacillus cereus. When HlyIII of V. vulnificus was expressed in Escherichia coli, crude extracts exhibited hemolytic activity similar to that of hemolysin III from Bacillus cereus. A hlyIII isogenic mutant was constructed via insertional inactivation and showed an attenuated virulence compared with the wild-type strain when this mutant was administered intraperitoneally in mice.     

Genetic relatedness of clinical and environmental isolates of lactose-positiveVibrio vulnificus

Environmental isolates of lactose-positiveVibrio vulnificus from different geographic areas were compared with clinical strains ofV. vulnificus on the basis of their phenotypic traits, virulence, DNA base composition, and DNA-DNA reasociation. EnvironmentalV. vulnificus strains were phenotypically indistinguishable from clinical isolates. These strains had a DNA base composition of 47–48 mol% guanine + cytosine and 85% reassociation at stringent temperature with DNA from clinicalV. vulnificus strains. These result indicateV. vulnificus strains from widely separated regions of the marine environment are indistinguishable from strains that have been agents of septicemia associated with shellfish consumption and of wound infections associated with seawater exposure.     

Vibrio virulence genes in fishes collected from estuarine waters in Italy

Aims: The aim of this study was to investigate the presence of Vibrio vulnificus and potentially pathogenic strains of Vibrio parahaemolyticus in mullets collected from estuarine environment in Italy. Methods and Results: Two hundred and ninety‐five mullets were analysed by culture using the selective medium thiosulfate citrate bile salt sucrose agar, during a monitoring period of 2 years (2008–2009). Presumptive Vibrio colonies were initially identified by using biochemical tests, and strains identified as V. parahaemolyticus and V. vulnificus were subsequently examined by PCR for the presence of species‐specific and virulence genes (toxR, trh, tdh and vvh). V. parahaemolyticus was found in 55% (162/295) of fishes and V. vulnificus in 1% (3/295) with a higher presence in summer months. The trh+/tdh? strains were detected in 16% (47/295) of samples and only one strain resulted trh+/tdh+. One of the V. parahaemolyticus trh+ strains isolated belonged to the O1:KUT (K untypeable), a serotype recently associated to gastroenteritis in Italy. Conclusions: This is the first report demonstrating a high percentage of potential pathogenic V. parahaemolyticus trh+ strains in estuarine fishes of the Mediterranean area. Significance and Impact of the Study: These findings indicate the potential human health risk associated with the presence of pathogenic Vibrio spp. in wild fishes.     

Regulation systems of protease and hemolysin production in Vibrio vulnificus

Vibrio vulnificus, a gram‐negative halophilic estuarine bacterium, is an opportunistic human pathogen that causes rapidly progressive fatal septicemia and necrotizing wound infection. This species also causes hemorrhagic septicemia called vibriosis in cultured eels. It has been proposed that a range of virulence factors play roles in pathogenesis during human and/or eel infection. Among these factors, a metalloprotease (V. vulnificus protease [VVP]) and a cytolytic toxin (V. vulnificus hemolysin [VVH]) are of significant importance. VVP elicits the characteristic edematous and hemorrhagic skin damage, whereas VVH exhibits powerful hemolytic and cytolytic activities and contributes to bacterial invasion from the intestine to the blood stream. In addition, a few V. vulnificus strains isolated from diseased eels have recently been found to produce a serine protease designated as V. vulnificus serine protease (VvsA) instead of VVP. Similarly to VVP, VvsA may possess various toxic activities such as collagenolytic, cytotoxic and edema‐forming activity. In this review, regulation of V. vulnificus VVP, VVH and VvsA is clarified in terms of expression at the mRNA and protein levels. The explanation is given on the basis of the quorum sensing system, which is dependent on bacterial cell density. In addition, the roles of environmental factors and global regulators, such as histone‐like nucleoid structuring protein, cyclic adeno monophosphate receptor protein, RpoS, HlyU, Fur, ToxRS, AphB and LeuO, in this regulation are outlined. The cumulative impact of these regulatory systems on the pathogenicity of V. vulnificus is here delineated.     

Growth and hemolysin production by Haemophilus pleuropneumoniae cultivated in a chemically defined medium

A chemically defined medium (CDM) has been developed which supports both growth and hemolysin production by Haemophilus pleuropneumoniae. Although the growth rate in stationary cultures was substantially slower in CDM than in trypticase soy broth plus 0.6% yeast extract (TSBYE) and slightly slower than in heart infusion broth (HIB), extracellular hemolysin activity in CDM was slightly higher than in HIB and 16-fold greater than in TSBYE. Maximum hemolytic activity was produced in CDM in early to mid log phase of growth. Hemolytic activity in sterile, cell-free culture supernatant fluids persisted for over 10 days at 4 degrees C and 3-5 days at 37 degrees C, but was completely destroyed at 56 degrees C after 30 min. Total hemolysin inactivation was also achieved in the presence of trypsin or pronase (10 units/mL), but no decrease in hemolytic activity was noted in the presence of DNase or RNase. Iron had little effect on the hemolytic activity in the early stages of growth. However, in the later stages of growth, iron had a pronounced effect with hemolytic activity decreasing as the iron concentration increased from 1 to 500 microM. None of these iron concentrations had any effect on the hemolytic activity when added directly to prepared cell-free culture supernatant fluids. The extracellular hemolysin produced by H. pleuropneumoniae in CDM appears to be a heat-labile protein the activity of which is influenced by iron at certain phases of growth.     

A potential bacterial biocontrol agent,strain S2V2 against pathogenic marine <Emphasis Type="Italic">Vibrio</Emphasis> in aquaculture

We isolated a marine bacterium strain S2V2 which inhibited the growth of pathogenic marine Vibrio spp. The aims of this research were to identify a new antibiotic-producing marine bacterium strain S2V2, and evaluate its spectrum activity and pathogenic property. Analysis of 16S rDNA sequence placed strain S2V2 in the genus Pseudoalteromonas, but the sequence similarity was low (95.46%) implying the strain might be a new species in this genus. Strain S2V2 inhibited the growth of 67.9% of 28 Vibrio strains tested. This strain inhibited V. alginolyticus, V. anguillarum, V. fluvialis, V. harveyi, V. metschnikovii, V. splendidus, V. ordalii, V. parahaemolyticus, and V. vulnificus, but inactive against V. campbellii, Aeromonas hydrophyla and Staphylococcus aureus. Strain S2V2 produced extracellular non proteinaceous antibacterial substances. The highest antibacterial activity was found when strain S2V2 was cultured for 96 h in ZoBell broth medium. An artificial infection to post larvae of Lithopenaeus vanname indicated that strain S2V2 was a non pathogenic bacterium. Non pathogenic property and specific antibacterial activity against a broad range of fish pathogenic marine Vibrio of strain S2V2 suggest that this strain is a prospective source of unique antibiotic and a potential biocontrol agent in marine aquaculture.     

Development and characterization of a catalytically inactive cysteine protease domain of RtxA1/MARTXVv as a potential vaccine for Vibrio vulnificus

Recent studies have defined several virulence factors as vaccine candidates against Vibrio vulnificus. However, most of these factors have the potential to cause pathogenic effects in the vaccinees or induce incomplete protection. To overcome these drawbacks, a catalytically inactive form, CPD_Vv(C3725S), of the well‐conserved cysteine protease domain (CPD) of V. vulnificus multifunctional autoprocessing repeats‐in‐toxin (MARTX_Vv/RtxA1) was recombinantly generated and characterized. Notably, active and passive immunization with CPD_Vv(C3725S) conferred protective immunity against V. vulnificus strains. These results may provide a novel framework for developing safe and efficient subunit vaccines and/or therapeutics against V. vulnificus that target the CPD of MARTX toxins.     

Evaluation of Two Assay Kits for Thermostable Direct Hemolysin (TDH) as an Indicator of TDH-Related Hemolysin (TRH) Produced by Vibrio parahaemolyticus

Reversed passive latex agglutination (RPLA) or enzyme-linked immunosorbent assay kits with beads (Bead-ELISA) are commercially available in Japan to detect the thermostable direct hemolysin (TDH) produced by Vibrio parahaemolyticus isolates. We evaluated whether these kits can be used to assay the pathogenic toxin, TDH-related hemolysin (TRH), produced by some so-called Kanagawa phenomenon-negative V. parahaemolyticus strains isolated from patients with diarrhea. Our results showed that the two kits, RPLA and Bead-ELISA, can detect TRH, although they were originally developed for detection of TDH. This may be due to the use of polyclonal anti-TDH antisera that cross react with TRH. Although the sensitivity for TDH detection by RPLA and Bead-ELISA differed tenfold, that for TRH detection was essentially equal. The minimum concentration of TRH required for detection by the two assay kits was about 10 ng/ml.     

Conventional and molecular methods to detect bacterial pathogens in mussels

Aim: To detect Aeromonas spp., Salmonella spp., Vibrio cholerae, Vibrio parahaemolyticus and Vibrio vulnificus in mussels and water samples from a farming area, conventional and molecular methods were applied to enrichment cultures. Methods and Results: The aerolysin gene (aero) of Aeromonas spp., the invasion plasmid antigen B (ipaB) gene of Salmonella spp., the enterotoxin secretion protein (epsM) gene of V. cholerae, the species‐specific region of 16S rRNA gene of V. vulnificus, the 16S–23S rDNA (IGS) gene of V. parahaemolyticus and the pR72H fragment of V. parahaemolyticus were amplified by multiplex polymerase chain reaction (PCR) assays on DNA extracted from enrichment cultures. The haemolysin gene (tdh) of pathogenic V. parahaemolyticus was also amplified. Conventional culture method allowed the isolation of V. parahaemolyticus and V. vulnificus from water and mussels. The genes aero, epsM and 16S rRNA of V. vulnificus were occasionally detected in the enrichment cultures. In mussels, the ipaB and IGS genes were detected from June to September and from April to November, respectively. All genes, except aero, were amplified from mussels collected in September, when pathogenic V. parahaemolyticus (tdh+) strains were also isolated. Conclusions: Multiplex‐PCR assays were more sensitive and faster than conventional procedures. Significance and Impact of the Study: The results emphasize the need of an accurate and rapid detection of bacterial pathogens in mussels to protect human health.     

Simple Purification Method for a Vibrio vulnificus Hemolysin by a Hydrophobic Column Chromatography in the Presence of a Detergent

A Vibrio vulnificus hemolysin (VVH) was purified by two steps of hydrophobic column chromatography on Phenyl-Sepharose HP. The first chromatography was carried out at pH 6.0. In this pH condition, VVH efficiently bound to the column, but the hemolysin fraction eluted was accompanied with colored substance(s). To eliminate this colored substance, the second chromatography was carried out at pH 9.8 in the presence of 1% 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), a zwitterionic detergent. Homogeneity of the hemolysin thus obtained was shown by polyacrylamide gel electrophoresis. The specific activity increased 33, 600 times and the yield was 35%. The method is simple and useful to supply enough VVH for study of the role of the hemolysin in the infection by V. vulnificus or on the mechanism of action of the hemolysin.     

Isolation of bacteriophage infectious for Vibrio vulnificus

Nine phage isolates infectious for Vibrio vulnificus and falling into four morphological groups were isolated from estuarine waters collected in Louisiana. Of the 60 V. vulnificus strains tested, 87% were susceptible to one or more of the isolates. With the exception of V. fluvialis, Vibrio species other than vulnificus were resistant to infection. A spectrum of enteric bacterial strains were similarly resistant. Susceptibility differences were seen between opaque (virulent) V. vulnificus strains and those with translucent (nonvirulent) colony types, with the former being more susceptible. Susceptibility patterns to infection by the nine phage isolates among the V. vulnificus test strains suggest that the latter may fall into several groups. Other aspects relating to the phage isolates are presented.     

NaCl concentration determines the outcome of competition experiments betweenVibrio pelagius andVibrio cholerae in chemostat cultures

The effect of NaCl concentration gradients on the growth of a marine species,Vibrio pelagius, and two freshwater and estuarine species,Vibrio cholerae andAeromonas hydrophila, was determined in chemostat cultures containing a mineral medium withd-glucose as the sole source of carbon and energy. Complementary cell density profiles were obtained with the marine and the freshwater-estuarine species at the extreme ends of the NaCl concentration gradients. At the lower NaCl concentrations,V. cholerae andA. hydrophila attained maximal cell densities, while no growth or submaximal cell yields were characteristic ofV. pelagius; the reverse was true at the higher NaCl concentrations. At the intermediate concentrations (100–400 mM) all three organisms were able to grow with cell densities ranging from 80%–100% of the maximum. Competition experiments betweenV. cholerae andV. pelagius within this region (100, 150, and 300 mM) indicated that the lower NaCl concentrations favoredV. cholerae while the higher concentrations favoredV. pelagius. The results suggest that the responses ofV. pelagius, V. cholerae, andA. hydrophila to different concentrations of NaCl are important in determining the environmental distribution of these species.     

Ecology of Vibrio parahaemolyticus and Vibrio vulnificus in the Coastal and Estuarine Waters of Louisiana,Maryland, Mississippi,and Washington (United States)

Vibrio parahaemolyticus and Vibrio vulnificus, which are native to estuaries globally, are agents of seafood-borne or wound infections, both potentially fatal. Like all vibrios autochthonous to coastal regions, their abundance varies with changes in environmental parameters. Sea surface temperature (SST), sea surface height (SSH), and chlorophyll have been shown to be predictors of zooplankton and thus factors linked to vibrio populations. The contribution of salinity, conductivity, turbidity, and dissolved organic carbon to the incidence and distribution of Vibrio spp. has also been reported. Here, a multicoastal, 21-month study was conducted to determine relationships between environmental parameters and V. parahaemolyticus and V. vulnificus populations in water, oysters, and sediment in three coastal areas of the United States. Because ecologically unique sites were included in the study, it was possible to analyze individual parameters over wide ranges. Molecular methods were used to detect genes for thermolabile hemolysin (tlh), thermostable direct hemolysin (tdh), and tdh-related hemolysin (trh) as indicators of V. parahaemolyticus and the hemolysin gene vvhA for V. vulnificus. SST and suspended particulate matter were found to be strong predictors of total and potentially pathogenic V. parahaemolyticus and V. vulnificus. Other predictors included chlorophyll a, salinity, and dissolved organic carbon. For the ecologically unique sites included in the study, SST was confirmed as an effective predictor of annual variation in vibrio abundance, with other parameters explaining a portion of the variation not attributable to SST.     

Effect of physiological conditions on the autolysis ofStaphylococcus aureus strains

The effect of physiological conditions on autolysis and autolytic activity in various strains ofStaphylococcus aureus was determined. The rate of whole cell autolysis ofS. aureus was growth phase dependent and a maximum rate was observed in early stationary phase cultures. However, the autolysins extracted by the freeze-thaw method (cell-wall bound autolytic activity) did not show any significant increase in activity. The addition of NaCl to the growth medium enhanced the rate of autolysis with the highest rate being displayed by cultures grown in 1.5 M NaCl. However, lower autolytic activity was found in the freeze-thaw extracts of cultures grown at higher concentrations of NaCl. The rate of autolysis of cultures grown at 30°C was higher than cultures grown at 37 or 43°C. Thus, the rate of autolysis seems to be independent of the bacterial growth rate. Cultures grown in slightly acidic conditions showed a faster rate of autolysis compared to cultures grown under alkaline conditions. SDS-polyacrylamide gel containing 0.2% crude cell-wall ofS. aureus did not show any obvious correlation with the appearance of any particular lytic band in the zymogram to autolytic activity or rate of autolysis of cultures grown under various environmental conditions. A nonhemolytic phenotype, mutations in the accessory gene regulator, and lysogeny (phages ø11, ø12, ø13) had no obvious effect either on the rate of autolysis or on the pattern of lytic bands in the zymograms.     

Protective effect of polygoni cuspidati radix and emodin on <Emphasis Type="Italic">Vibrio vulnificus</Emphasis> cytotoxicity and infection

Vibrio vulnificus, a good model organism of bacterial septicemia, causes fatal septicemia manifesting a fulminating course and a high mortality rate within days. In order to identify new natural substances preventing V. vulnificus infection, a plant library was screened for inhibiting cytotoxicity to host cells by using Trypan blue staining and LDH assay. We found that Polygoni Cuspidati Radix potently suppressed the acute death of HeLa and RAW264.7 cells in a dose dependent manner. Further studies revealed that Polygoni Cuspidati Radix inhibited V. vulnificus growth and survival in HI broth and seawater, respectively. We confirmed that Polygoni Cuspidati Radix contained high level of emodin by thin layer chromatography (TLC). Emodin showed direct antibacterial activity against V. vulnificus. In addition, emodin prevented the morphologic damages and acute death of HeLa cells caused from V. vulnificus. The safety of Polygoni Cuspidati Radix and emodin to host cells was confirmed by MTT assay. Polygoni Cuspidati Radix and emodin protected mice from V. vulnificus infection.     

牛源致病性蜡样芽孢杆菌溶血素BL亚基的原核表达及其生物学活性分析

蜡样芽孢杆菌属于革兰氏阳性菌,分布广泛,具有一定的致病性。不同的蜡样芽孢杆菌携带有不同的毒力因子,这直接决定了蜡样芽孢杆菌株致病性的差异。研究和探讨毒力因子分布以及具体毒素的生物活性有助于对蜡样芽孢杆菌病采取更科学的防控。本研究通过原核表达系统将溶血素BL三亚基重组表达,并对表达蛋白进行了纯化和部分生物学活性的检测。研究结果表明,牛源致病性蜡样芽孢杆菌溶血素BL可以在原核表达系统中成功表达和纯化,牛源致病性蜡样芽孢杆菌溶血素BL拥有溶血性、细胞毒性、很好的免疫原性以及对小鼠具有一定的免疫保护性。本研究表达了溶血素BL三亚基,并探究了牛源致病性蜡样芽孢杆溶血素BL的生物学活性。本研究为进一步揭示蜡样芽孢杆菌溶血素BL的致病作用机制和建立针对牛源致病性蜡样芽孢杆菌病的检测方法奠定了理论基础。