Aims: Coral diseases have emerged over the last several decades, causing a loss of live coral cover in the Caribbean and Indo-Pacific reefs. Hence, microbiological and disease cultural techniques are commonly used to investigate their causative microbial agents. This is the first study to identify the potential of pathogenic Vibrio spp. isolated from apparently white syndrome (WS) coral disease in Tioman Island Marine Park using biochemical and molecular techniques. Methodology and results: The Vibrio colonies were isolated from 108 samples of WS infected corals (Acropora cytherea and Montipora aequituberculata) including seawater, sediment and algae found adjacent to infected coral colonies. A total of one hundred representative Vibrio isolates were characterized and most of them (n=50) were identified as V. vulnificus, V. alginolyticus and Photobacterium damselae following biochemical analysis. The molecular analysis revealed six Vibrio spp. (V. coralliilyticus, V. hepatarius, V. brasiliensis, V. tubiashi, V. campbellii, V. ishigakensis) and one Photobacterium rosenbergii. Vibrio coralliilyticus isolated from all infected coral samples may be highly responsible for the sign of WS disease. Conclusion, significance and impact of study The findings of this study provide baseline data and information on potential coral pathogens identified in the coastal waters of Tioman Island. Etiological disease study is suggested to validate their severity and virulence factors in the future.
Vibrio--pathogenicity ; Anthozoa

Vibrio cholerae ; classification ; pathogenicity

Genotype ; Humans ; Seafood ; microbiology ; Vibrio Infections ; epidemiology ; microbiology ; Vibrio parahaemolyticus ; drug effects ; genetics ; pathogenicity

Shewanella algae infections are rare in humans. Previously reported cases of S. algae have mainly been associated with direct contact with seawater. We report a case of primary S. algae bacteremia occurring after the ingestion of raw seafood in a patient with liver cirrhosis that presented a fulminent course of necrotizing fasciitis.
Bacteremia/*blood ; Fasciitis, Necrotizing/*microbiology ; Fatal Outcome ; Humans ; Korea ; Liver Cirrhosis/physiopathology ; Male ; Middle Aged ; Seafood/microbiology ; Sepsis/*microbiology ; Shewanella/*pathogenicity ; Vibrio/*pathogenicity ; Vibrio Infections/*blood

Vibrios are universal conditioned-pathogenic bacteria in marine culture environment, and the outbreak of vibrio disease resulted in a serious damage to aquaculture. Considering that vibrio disease in aquatic species, especially fishes, usually originated from mixed infection of different species (serotypes or subspecies) of vibrios, it is important to select the potential cross-protective protein antigens as candidates of polyvalent or combined vaccines. In present research, several strains of vibrios were isolated from infected large yellow croaker (Pseudosciaena crocea) and subsequently identified as six strains of V. harveyi, one V. parahaemolyticus and one V. alginolyticus by physiological, biochemical and molecular biological methods. Their outer membrane proteins (OMPs) were extracted and the SDS-PAGE and Western blotting results show that three immuno-blots with common molecular weight presented at approximate 45 kDa, 35 kDa and 22 kDa on their OMP electrophoretogram, indicating the existence of antigens with cross-protection in their OMPs. With the aids of combination of two-dimensional electrophoresis (2-D) and Western blotting and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), a deduced porin (GenBank Accession No. ZP_01260407) from V. alginolyticus and a maltoporin precursor (GenBank Accession No. NP_801154) from V. parahaemolyticus were able to react with polyclonal antibody to whole V. harveyi, suggesting these two proteins could act as the cross-protective antigens and the vaccines prepared with these porins would be probable to bring cross protection to three different vibrios.
Animals ; Antigens, Bacterial ; immunology ; Bacterial Outer Membrane Proteins ; immunology ; Cross Reactions ; Fish Diseases ; microbiology ; Perciformes ; microbiology ; Vibrio ; classification ; immunology ; isolation & purification ; pathogenicity ; Vibrio Infections ; microbiology

OBJECTIVETo explore the phenetic and genetic features of clinical Vibrio parahaemolyticus strains from 2007-2009 in China.

METHODSA total of 135 clinical Vibrio parahaemolyticus strains, isolated from Zhejiang, Jiangsu, Sichuan, Guangxi, Liaoning Provinces during 2007 to 2009, were selected for the research. The occurrence of virulence genes thermostable direct hemolysin (tdh) and TDH-related hemolysin (trh), species-specific genes thermolabile hemolysin (tlh), toxR, VPM and gyrB, the pandemic clone gene markers(GS-PCR, PGS-PCR, orf8 and HU-α) in 135 Vibrio parahaemolyticus strains was detected by PCR. The antimicrobial susceptibilities to eight antimicrobial agents of the experimental strains were determined by the broth microdilution method. All strains were serotyped and underwent the cluster analysis with pulsed-field gel electrophoreses.

RESULTSThe results of PCR methods claim that all experiment strains carry species-specific genes such as tlh, toxR, gyrB, VPM. Among clinical strains, 85.9% (116/135) carry tdh and/or trh. 85.2% (115/135) were positive for tdh, and 3.0% (4/135) were positive for trh; while 3 strains carried both.66.7% (90/135) , 80.7% (109/135) , 65.2% (88/135) , 66.7% (90/135) clinical strains carried the genes of GS-PCR, PGS-PCR, orf8, HU-α, respectively. The results of antibiotics susceptibility test showed that 8.1% (11/135) strains were resistant to at least one agent, including 9 strains were resistant to ampicillin, 2 strains were resistant to trimethoprim and sulphamethoxazole, and 1 strain were resistant to tetracycline. All clinical strains were sensitive to cefotaxime, ceftazidime, gentamicin, ciprofloxacin and chloromycetin.Serological analysis of the O and K antigens claimed that a total of 29 serotypes were identified for clinical strains, predominantly O3, O4 and O1 groups, accounting for 89.6% (121/135). O3: K6 was dominant serotype, accounting for 56.3% (76/135). The pandemic flora in China included O3: K6, O4: K68, O1: K36, O1: K25, O1: K5 and O3: K29 serotypes.Genomic DNAs of 135 clinical strains were digested with SfiI and NotI, the molecular size of PFGE restriction fragments used for analysis mainly ranged from 30-700 kb.When subjected to UPGMA clustering, 6 and 9 clusters were grouped by SfiI and NotI, and the minimal similarity was 52.6% and 58.7%, and pandemic flora were located in C groups and D group, respectively.

CONCLUSIONMost of Vibrio parahaemolyticus strains isolated from clinical sources in China were pathogenic. The pandemic clone, especially O3: K6 was prevalent. The GS-PCR and HU-α genes were reliable markers to identify the pandemic flora. The serotype by PFGE was reliable to distinguish the pandemic flora and the sporadic strains.

China ; epidemiology ; Drug Resistance, Bacterial ; Genes, Bacterial ; Humans ; Molecular Epidemiology ; Vibrio Infections ; epidemiology ; microbiology ; Vibrio parahaemolyticus ; genetics ; isolation & purification ; pathogenicity ; Virulence ; genetics

OBJECTIVETo determine the occurrence and distribution of specific clones of pathogenic Vibrio parahaemolyticus(VP)isolated in Shenzhen and to assess the relationship between serotype O3:K6 and the globally distributed pandemic clone.

METHODSA total of 1005 VPs isolated from diarrhea patients in 2002-2008 were sero-typed. Real-time PCR was used to detect the virulence genes tlh, toxR, tdh, trh and orf8 in 281 isolates from 68 different serotypes. The main serotypes were typed by pulsed field gel electrophoresis(PFGE). Strains with dominant serotypes and PFGE patterns were assayed by GS-PCR and toxRS sequencing for the identification of pandemic clone. Multilocus sequence typing(MLST)analysis was reserved for exemplary 41 O3 : K6 and O1 : K25 isolates.

RESULTSSeventy-nine serotypes were observed among the 1005 isolates, including O3 : K6(57.9%), O4 : K8(8.16%), O1 : KUT(5.87%), O1 : K25(5.27%), O4 : K68(1.39%), O1 : K56(1.39%) and O9 : K44(0.99%). Most of the strains(99.36%)showed PCR positive to tlh, toxR, and tdh but eleven strains were tdh negative. MLST showed that all the 36 O3 : K6 isolates belonged to ST3 and all the 5 O4 : K8 strains were ST189. These results matched the description of the pandemic VP clone.

CONCLUSIONA recognizable burden of diarrheal illness caused by VP had been seen in Shenzhen. Results from serotyping indicated that although there existing a large variety of diversities, the dominant serotype appeared to be O3 : K6. VP isolates identified in Shenzhen mainly showed as tdh positive but trh negative, in consistent with the current pandemic O3 : K6 clone. The pandemic O3 : K6 clone did appear to co-exist with other clones of O3 : K6, as well as O4 : K8,O1 : K25. Potential outbreak of VP could be monitored through the laboratory-based surveillance programs, suggesting that the strategies related to prevention and control of VP should be prioritized in Shenzhen.

China ; epidemiology ; Electrophoresis, Gel, Pulsed-Field ; Humans ; Multilocus Sequence Typing ; Real-Time Polymerase Chain Reaction ; Serotyping ; Vibrio Infections ; epidemiology ; microbiology ; Vibrio parahaemolyticus ; genetics ; isolation & purification ; pathogenicity

BACKGROUNDVibrio vulnificus (Vv) is an estuarine bacterium that can cause primary septicemia as well as serious wound infections. However, little is known about the mechanisms by which Vv infects dendritic cells (DCs) and its effects on cytoskeleton. In this study, we aimed to investigate the invasion, internalization, and the organelles damage of the cultured dendritic cells (a DC 2.4 strain) during Vv infection.

METHODSThe study model was the cultured DCs infected by a Vv 1.758 strain. Electron microscopy was used to observe the localization of bacteria at the different time points of infection, cell morphology, and the process of organelles changes. The cytoskeleton structure including the microfilaments and the microtubules rearrangement was examined under a fluorescence microscope.

RESULTSThe Vv were pinocytosised into the DC cells through double-sides, and localized at 1 - 2 mm of the inner side membrane. It took 1.3, 1.9, and 3.4 hours to reach the infection ratio of 25%, 50%, and 75%, respectively. Using electron microscopy, the DCs had been observed to have developed chromatin aggregation within 4.0 hours, and significant cytoskeleton structure disruption was noted within 6.0 hours.

CONCLUSIONThe high lethality of Vv infection may be associated with the direct disruption of the DCs cytoskeleton structure.

Animals ; Apoptosis ; physiology ; Cells, Cultured ; Cytoskeleton ; metabolism ; ultrastructure ; DNA Fragmentation ; Dendritic Cells ; metabolism ; microbiology ; ultrastructure ; Mice ; Microscopy, Electron ; Microscopy, Electron, Transmission ; Vibrio Infections ; metabolism ; Vibrio vulnificus ; pathogenicity

Lipoprotein plays a role in the host defense against bacterial infection, and its serum level has been demonstrated to be an important prognosis factor of survival. We have previously demonstrated that LDL directly inactivates the hemolytic activity of Vibrio vulnificus cytolysin (VVC) in vitro. The object of this study was therefore to examine whether the LDL-mediated inactivation of VVC leads to protection against lethal infection of V. vulnificus in vivo, using wild and VVC-deficient V. vulnificus strains. Unexpectedly, we found that LDL protects mouse lethality induced by VVC-deficient as well as wild V. vulnificus strain. We also demonstrated that LDL blocks V. vulnificus LPS-induced lethality in mice. These results suggest that LDL preferentially act on endotoxin rather than exotoxin in the protection against V. vulnificus-induced mice lethality.
Animals ; Disease Models, Animal ; Female ; Humans ; Lipopolysaccharides/*antagonists & inhibitors ; Lipoproteins, LDL/*pharmacology ; Mice ; Mice, Inbred ICR ; Perforin/antagonists & inhibitors/genetics ; Vibrio Infections/prevention & control ; Vibrio vulnificus/*drug effects/genetics/*pathogenicity ; Virulence/drug effects/genetics/physiology

OBJECTIVETo study the serotypes, antimicrobial resistance, virulence genes and molecular characteristics of Vibrio parahaemolyticus isolated from outbreaks and sporadic cases in Guangdong, 2013.

METHODS36 Vibrio parahaemolyticus strains isolated from outbreaks and 43 strains from sporadic cases were sero-typed and tested for antimicrobial resistance. PCR was used to detect for tdh(thermostable direct hemolysin gene), trh (tdh(-) related hemolysin gene), GS-PCR and orf8 genes. All the samples were analyzed by pulsed-field gel electrophoresis (PFGE).

RESULTS36 isolates from outbreaks were all identified as O3 : K6, and among the 43 sporadic isolates, O3 : K6 (23, 53.49%) was the dominant serotype. Vibrio parahaemolyticus isolates showed high resistance rate to ampicillin (96.20%) and cefalotin (40.50%), but were high sensitive to cotrimoxazole (100%) and chloramphenicol (100%). 83.33% (30/36) outbreak isolates were resistant to multi-drugs but only 37.21% (16/43) of the sporadic isolates showed so. Results from virulence gene detection suggested that all the 36 outbreak isolates belonged to tdh(+) trh(-) strains, while 86.05% (37/43) of the sporadic isolates were tdh(+)trh(-) and 11.63% (5/43)were tdh(-)trh(+) . Only one tdh(+)trh(+) strain was found. All the outbreak isolates contained GS-PCR and/or orf8 genes, whereas among the sporadic isolates only 51.16% (22/43) of them carrying the similar genes. Results from PFGE analysis suggested that 79 isolates were discriminated into 3 clusters and 32 different PFGE patterns with the similarity value between 59.8% and 100.0%. Outbreak isolates seemed to gather in the same cluster, while the sporadic isolates spreading in all the three clusters.

CONCLUSIONO3 : K6 was the dominant serotype in Vibrio parahaemolyticus strains isolated in Guangdong, 2013. These strains showed high sensitivity to most antibiotics, but with multi-drug resistance. Positive rate of tdh gene was high, and most O3 : K6 strains contained GS-PCR and/or orf8 genes. PFGE analysis revealed genetic diversity was within the Vibrio parahaemolyticus strains in Guangdong.

Bacterial Toxins ; China ; epidemiology ; Disease Outbreaks ; Drug Resistance, Bacterial ; Electrophoresis, Gel, Pulsed-Field ; Genetic Variation ; Hemolysin Proteins ; Humans ; Polymerase Chain Reaction ; Serotyping ; Vibrio Infections ; epidemiology ; Vibrio parahaemolyticus ; genetics ; pathogenicity ; Virulence