Promoter analysis and regulatory characteristics of vvhBA encoding cytolytic hemolysin of Vibrio vulnificus.
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Cytolytic hemolysin, a gene product of vvhA, is a putative virulence factor of the pathogenic bacterium Vibrio vulnificus. We have previously shown that hemolysin production is repressed by adding glucose to culture media and that production can be restored by adding cAMP. In this study, hemolysin activity and the level of vvh transcript were determined to reach a maximum in late exponential phase and were repressed when cells entered stationary phase. Northern blot and primer extension analyses revealed that vvhA is cotranscribed with a second gene, vvhB, located upstream of vvhA. Transcription of the vvhBA operon begins at a single site and is under the direction of a single promoter, P(vvh). A crp null mutation decreased hemolysin production and the level of vvhBA transcript by reducing the activity of P(vvh), indicating that the P(vvh) activity is under the positive control of cAMP receptor protein (CRP). A direct interaction between CRP and the regulatory region of the vvhBA operon was demonstrated by gel-mobility shift assays. The CRP binding site, centered at 59.5 bp upstream of the transcription start site, was mapped by deletion analysis of the vvhBA promoter region and confirmed by DNase I protection assays. These results demonstrate that the vvhBA expression is activated by CRP in a growth-dependent manner and that CRP exerts its effects by directly binding to DNA upstream of P(vvh). (+info)
Phage therapy of local and systemic disease caused by Vibrio vulnificus in iron-dextran-treated mice.
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Vibrio vulnificus is a gram-negative bacterium that contaminates filter-feeding shellfish such as oysters. After ingestion of contaminated oysters, predisposed people may experience highly lethal septicemia. Contamination of wounds with the bacteria can result in devastating necrotizing fasciitis, which can progress to septicemia. The extremely rapid progression of these diseases can render antibiotic treatment ineffective, and death is a frequent outcome. In this study, we examined the potential use of bacteriophages as therapeutic agents against V. vulnificus in an iron-dextran-treated mouse model of V. vulnificus infection. Mice were injected subcutaneously with 10 times the lethal dose of V. vulnificus and injected intravenously, either simultaneously or at various times after infection, with phages. Treatment of mice with phages could prevent death; systemic disease, as measured by CFU per gram of liver and body temperature; and local disease, as measured by CFU per gram of lesion material and histopathologic analysis. Two different phages were effective against three different V. vulnificus strains with various degrees of virulence, while a third phage that required the presence of seawater to lyse bacteria in vitro was ineffective at treating mice. Optimum protection required that the phages be administered within 3 h of bacterial inoculation at doses as high as 10(8) PFU. One of the protective phages had a half-life in blood of over 2 h. These results demonstrate that bacteriophages have therapeutic potential for both localized and systemic infections caused by V. vulnificus in animals. This model should be useful in answering basic questions regarding phage therapy. (+info)
In vitro and in vivo activities of newer fluoroquinolones against Vibrio vulnificus.
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The MICs of six fluoroquinolones as well as minocycline and cefotaxime for 46 clinical isolates of Vibrio vulnificus were determined by the agar dilution method. All the drugs tested had good activities against all isolates, with the MICs at which 90% of the isolates tested were inhibited (MIC(90)s) by five of the fluoroquinolones ranging between 0.03 and 0.06 micro g/ml. The MIC(90) of lomefloxacin, on the other hand, was 0.12 micro g/ml. Time-kill studies were conducted with these agents and a clinical strain of V. vulnificus, VV5823. When approximately 5 x 10(5) CFU of V. vulnificus/ml was incubated with any one of the above-mentioned six fluoroquinolones at concentrations of two times the MIC, there was an inhibitory effect on V. vulnificus that persisted for more than 48 h with no noted regrowth. The efficacies of the fluoroquinolones were further evaluated in vivo in the mouse model of experimental V. vulnificus infection and compared to the efficacy of a combination therapy using cefotaxime plus minocycline. With an inoculum of 1.5 x 10(7) CFU, 28 (87.5%) of 32 mice in the cefotaxime-minocycline-treated group survived and 29 (91%) of the 32 mice in the moxifloxacin-treated group survived while none of the 32 mice in the control group did. With an inoculum of 3.5 x 10(7) CFU, the difference in survival rates among groups of 15 mice treated with levofloxacin (13 of 15), moxifloxacin (10 of 15), gatifloxacin (10 of 15), sparfloxacin (11 of 15), ciprofloxacin (12 of 15), or lomefloxacin (10 of 15) was not statistically significant while none of the 15 mice treated with saline survived. We concluded that the newer fluoroquinolones as single agents are as effective as the cefotaxime-minocycline combination in inhibiting V. vulnificus both in vitro and in vivo. (+info)
Detection of cytotoxin-hemolysin mRNA in nonculturable populations of environmental and clinical Vibrio vulnificus strains in artificial seawater.
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The objective of this study was to develop a molecular detection method that better estimates the potential risk associated with the presence of Vibrio vulnificus. For that purpose, we applied seminested reverse transcription-PCR (RT-PCR) to viable but nonculturable (VBNC) populations of V. vulnificus and targeted the cytotoxin-hemolysin virulence gene vvhA. Three strains, two environmental, IF Vv10 and IF Vv18, and one clinical, C7184, were used in this study. Artificial seawater, inoculated with mid-log-phase cells, was maintained at 4 degrees C. VBNC cells resulted after 3, 6, and 14 days for C7184, IF Vv18, and IF Vv10, respectively. Our data indicate that seminested RT-PCR is sensitive for the detection of vvhA mRNA in artificial seawater when exclusively nonculturable bacteria are present. This is the first report of the expression of a toxin gene in VBNC V. vulnificus. Moreover, vvhA transcripts were shown to persist in nonculturable populations over a 4.5-month period, with a progressive decline of the signal over time. This result indicates that special attention should be given to the presence of potentially pathogenic VBNC cells in environmental samples when assessing public health risk. (+info)
Vibrio vulnificus induces macrophage apoptosis in vitro and in vivo.
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In this study, we compared the apoptotic activities of clinical and environmental isolates of Vibrio vulnificus toward macrophages in vitro and in vivo. The clinical isolates induced apoptosis in macrophage-like cells in vitro and in macrophages in vivo. This suggests that macrophage apoptosis may be important for the clinical virulence of V. vulnificus. (+info)
Vibrio vulnificus cytolysin induces hyperadhesiveness of pulmonary endothelial cells for neutrophils through endothelial P-selectin: a mechanism for pulmonary damage by Vibrio vulnificus cytolysin.
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Vibrio vulnificus cytolysin forms transmembrane pores that are permeable to calcium ions in pulmonary endothelial cells, and has been suggested as an important virulence factor that sequestrate neutrophils primarily in the lung. To elucidate the mechanism we investigated whether the cytolysin affect the expression of endothelial P-selectin and adhesiveness of pulmonary endothelial cells for neutrophils. The cytolysin increased the adhesiveness of CPAE cell, a pulmonary endothelial cell line, for neutrophils in a concentration- and time-dependent manner. The increase of adhesiveness occurred within several minutes after the cytolysin exposure, persisted up to 90 min, and was not affected by cycloheximide. Furthermore, flow cytometric analyses showed that cytolysin enhanced the level of P-selectin on CPAE cell surface. Therefore, these results suggest that the cytolysin-induced hyperadhesiveness of pulmonary endothelial cells for neutrophils is mediated by the mobilization of endothelial P-selectin to the cell surface. (+info)
Sequence polymorphism of the 16S rRNA gene of Vibrio vulnificus is a possible indicator of strain virulence.
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Vibrio vulnificus exhibits considerable strain-to-strain variation in virulence. Attempts to associate phenotypic or genotypic characteristics with strain virulence have been largely unsuccessful. Based on a 17-nucleotide difference throughout the sequence of the small subunit 16S rRNA gene, there are two major groups of V. vulnificus designated types A and B. In a survey of the 16S rRNA genotype in 67 V. vulnificus human clinical and nonclinical strains, we determined that the majority of nonclinical isolates are type A (31 of 33) and that there is a statistically significant association between the type B genotype and human clinical strains (26 of 34). (+info)
Identification of genetic loci required for capsular expression in Vibrio vulnificus.
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Transposon mutagenesis of an encapsulated, virulent strain of Vibrio vulnificus 1003(O) led to the identification of four genetic regions that are essential to capsular polysaccharide (CPS) expression and virulence. Of the four regions, three are believed to be part of a capsule gene locus comprised of biosynthesis, polymerization, and transport genes clustered on a single chromosomal fragment. Genes indicating a Wzy-dependent system of polymerization and transmembrane export are present, suggesting that the CPS of V. vulnificus is lipid linked. The fourth region, while it contains a gene essential for CPS expression, is characteristic of an integron-gene cassette region, similar to the super integron of V. cholerae. It is not believed to be part of a CPS gene locus and is located in a region of the chromosome separate from the putative CPS loci. It is comprised of open reading frames (ORFs) carrying genes of unknown function surrounded by direct repeats. This region also contains IS492, an insertion sequence located numerous times throughout a region of the genome, demonstrating a restriction fragment length polymorphism among an encapsulated and nonencapsulated morphotype of V. vulnificus. Collectively, 22 ORFs were recognized: 13 capsule synthesis genes, 4 insertion sequences, 1 truncated biosynthesis gene, and 4 genes of unknown function. This study has led to the identification of previously unrecognized genetic loci that may help to increase the understanding of capsular genetics and antigenic diversity among V. vulnificus strains. (+info)