Vibrio Hollisae Research Articles

Investigation of vibriosis caused by Vibrio anguillarum in rainbow trout (Oncorhynchus mykiss).

Identifying pathogenic microorganisms causing disease is important for epidemiological research, antimicrobial therapy, and control. The current study was carried out to use different methods for the identification of Vibrio anguillarum from diseased rainbow trout (Oncorhynchus mykiss) obtained from Türkiye (Muğla-Fethiye), the damage caused by the pathogenic microorganism in the tissues and organs, and the determination of the antibiotic effective against the pathogen. Hemorrhagic and ulcerative skin lesions and diffuse petechial hemorrhage in the internal organs were clinically detected in diseased fish obtained from the rainbow trout farm. Bacteria isolated from diseased fish were subjected to analysis using conventional bacteriological methods, a commercial bacterial identification test kit (API), an automated bacteria identification system known as Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS), BD Phoenix™, and 16S rRNA sequence analysis. All isolated bacteria were identified as V. anguillarum by API 20E and conventional bacteriological method. These results have been confirmed with 16S rRNA sequence analysis. However, the isolated bacteria were identified as Grimontia hollisae (syn. Vibrio holisae) with the BD Phoenix system. Histologically, tissue damage such as melano-macrophage centers and necrosis in the kidney and spleen, hyperemia and mononuclear cell infiltration in the liver, as well as mononuclear cell infiltration on muscles, talengectiasis in the gill tissue was observed. In addition, it has been determined that the most effective antibiotic against the pathogen was enrofloxacin. When comparing all identification methods used for this pathogen causing tissue damage, it was demonstrated that the MALDI-TOF MS provides better results than other methods in terms of cost and identification time, and it could be used as an alternative to the conventional method to the identification of V. anguillarum.

Rapid identification of enteric bacteria from whole genome sequences using average nucleotide identity metrics.

Identification of enteric bacteria species by whole genome sequence (WGS) analysis requires a rapid and an easily standardized approach. We leveraged the principles of average nucleotide identity using MUMmer (ANIm) software, which calculates the percent bases aligned between two bacterial genomes and their corresponding ANI values, to set threshold values for determining species consistent with the conventional identification methods of known species. The performance of species identification was evaluated using two datasets: the Reference Genome Dataset v2 (RGDv2), consisting of 43 enteric genome assemblies representing 32 species, and the Test Genome Dataset (TGDv1), comprising 454 genome assemblies which is designed to represent all species needed to query for identification, as well as rare and closely related species. The RGDv2 contains six Campylobacter spp., three Escherichia/Shigella spp., one Grimontia hollisae, six Listeria spp., one Photobacterium damselae, two Salmonella spp., and thirteen Vibrio spp., while the TGDv1 contains 454 enteric bacterial genomes representing 42 different species. The analysis showed that, when a standard minimum of 70% genome bases alignment existed, the ANI threshold values determined for these species were ≥95 for Escherichia/Shigella and Vibrio species, ≥93% for Salmonella species, and ≥92% for Campylobacter and Listeria species. Using these metrics, the RGDv2 accurately classified all validation strains in TGDv1 at the species level, which is consistent with the classification based on previous gold standard methods.

P.15: Usefulness of recombinant collagenase from Grimontia hollisae in islet isolation from chronic pancreatitis model

P.15: Usefulness of recombinant collagenase from Grimontia hollisae in islet isolation from chronic pancreatitis model

Insights into the catalytic mechanism of Grimontia hollisae collagenase through structural and mutational analyses.

Grimontia hollisae collagenase (Ghcol) consists of 767 amino acid residues with a single catalytic domain containing the zinc-binding motif H492 EYVH496 . The crystal structure of Ghcol in complex with its substrate (Gly-Pro-hydroxyproline-Gly-Pro-hydroxyproline) and site-directed mutagenesis of active-site Tyr residues revealed the catalytic mechanism: Glu493 functions as an acid and base catalyst while Tyr564 stabilizes the tetrahedral complex in the transition state.

Vibrio hollisae isolated in a Hydropneumothorax

Introduction: Vibrio hollisae is a bacterium that is commonly responsible of digestive infections but in very rare cases, it can be isolated in other biological liquid. Case report: This case report is about a young man who suffered from an hydropneumothorax infected by Vibrio hollisae, following a chylous pleurisy drainage. The patient improved after surgical drainage and adequate antibiotherapy. Discussion and conclusion: Our case rises awareness of the exra-intersinal localisation of vibrio hollisae. The microbiologist should know that it may not grow in MacConkey plate agar even if it’s a gram-negative bacillus. The best growth is obtained with sheep blood agar plate.

Fungal and Bacterial Bioagents Efficiency on the Control of Potato Pest Phthorimaea operculella via Ingestion or Contact

Potatoes are one of the most important food products in the world and considered a main human nutrition sources source. Potato tuber moth (Phthorimaea operculella Zeller (PTM) (Lepidoptera: Gelechiidae)) causes remarkable economic losses to important crop, both in field and under storage conditions In this study, the insecticidal efficiency of the following bioagents: Brevibacillus brevis (FD-1), Bacillus atrophaeus (FD 17), Bacillus sphaericus (FD 49), Bacillus cereus (FD 63), Vibrio hollisae (FD 70), Bacillus thuringiensis subsp. kenyae (FDP 8) bacteria strains and Beauveria bassiana fungal isolate (ET 10), were evaluated on their efficacy to control P. operculella, under controlled conditions. In addition to insecticidal efficacy evaluations, analyses were also carried out to determine the differences between bioagents action mode: (1) uptake (ingestion as a gastric poison) and (2) contact. For (1), 20 larvae were fed on the tubers immersed in these suspensions to analyze efficacy by ingestion; for (2), suspensions of 1x108 CFU/ml of bacteria and 5.7x105 conidia/ml of fungus were prepared and sprayed to 20 larvae. FD-63 (91.67%) and FD-17 (88.33%) taken up by diet gave the most effective results against the pest.

Distribution, Prevalence, and Antibiotic Susceptibility Profiles of Infectious Noncholera Vibrio Species in Malaysia.

The noncholera Vibrio spp. which cause vibriosis are abundantly found in our water ecosystem. These bacteria could negatively affect both humans and animals. To date, there is a paucity of information available on the existence and pathogenicity of this particular noncholera Vibrio spp. in Malaysia in comparison to their counterpart, Vibrio cholera. In this study, we extracted retrospective data from Malaysian surveillance database. Analysis was carried out using WHONET software focusing noncholera Vibrio spp. including Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio fluvialis, Vibrio alginolyticus, Vibrio hollisae (Grimontia hollisae), Vibrio mimicus, Vibrio metschnikovii, and Vibrio furnissii. Here, we report the first distribution and prevalence of these species isolated in Malaysia together with the antibiotic sensitivity profile based on the species. We found that V. parahaemolyticus is the predominant species isolated in Malaysia. Noticeably, across the study period, V. fluvialis is becoming more prevalent, as compared to V. parahaemolyticus. In addition, this study also reports the first isolation of pathogenic V. furnissii from stool in Malaysia. These data represent an important step toward understanding the potential emergence of noncholera Vibrio spp. outbreaks.

Culture-dependent examination of the bacteriological quality of ready-to-eat African salads in Enugu metropolis, Nigeria and antibiotic resistance profile of associated bacteria

This study investigated the bacteriological quality of ready-to-eat (RTE) African salads in Enugu metropolis, Enugu, Nigeria. A total of 10 samples of African salad were purchased from 10 different vendors in Enugu metropolis. The samples were purchased from Agbani Road, Ogbete, Mayor, Uwani, Kenyatta, Achara Layout, Obiagu and Timber. Isolation and enumeration of bacterial isolates were done using Nutrient agar, Eosin Methylene Blue (EMB) agar, Thiosulphate-citrate-bile salts-sucrose (TCBS) agar, Salmonella-Shigella Agar (SSA) and MacConkey agar, following standard methods. Identification of the bacterial isolates were done through biochemical tests and the Analytical Profile Index (API 20E) test kit. The antibiotic sensitivity of the bacterial isolates was also done using the Kirby Bauer disc diffusion method. Total culturable heterotrophic count was above 300 colonies across the samples. The highest bacterial counts recorded on EMB, SSA and TCBS across the samples were 6.3 × 106 CFU/g, 7.4 × 106 CFU/g and 1.21 × 107 CFU/g respectively. The identities of the organisms were; Salmonella spp., Staphylococcus aureus, Escherichia coli, Vibrio mimicus, Vibrio fluvialis, Vibrio cholerae, Vibrio parahaemolyticus and Vibrio hollisae. The prevalent organism across the samples was Vibrio spp. The antibiotic sensitivity test suggested that Vibrio spp. was resistant to Ampiclox and Amoxycillin but sensitive to Erythromycin, Pefloxacin and Septrin. From this study, it was discovered that consumers of RTE African salad from majority of the vendors across Enugu metropolis are at risk of severe food poisoning.

Eco-friendly control method against invasive pest box tree moth, (Cydalima perspectalis (Walker) (Lepidoptera: Crambidae))

BackgroundBuxus sempervirens (Boxwood) is a type of plant that has economic and cultural significance, danger of extinction due to various factors. Cydalima perspectalis (Walker, 1859) (Lepidoptera: Crambidae), an invasive species, has an important role as a boxwood pest. The inadequacy of the pheromone trap methods recommended to control the pest or the negative effects of chemical insecticides on the environment have led to seeking alternative approaches in this regard. In this study, it was conducted to determine whether entomopathogens can be applied in pest control, the insecticidal effects of Bacillus thuringiensis subsp. kenyae (FDP-8), Bacillus cereus (FD-63), Brevibacillus brevis (FD-1) and Vibrio hollisae (FD-70) bacterial strains at the concentration of 1 × 108 CFU/ml and Beauveria bassiana (Bals.-Criv.) Vuill. (Cordycipitaceae) (ET 10) fungal isolate at the concentrations of 1 × 106, 1 × 107 and 1 × 108 conidia/ml were tested under controlled conditions.ResultsAs compared the results of the bacterial strains and fungal tested, it was determined that the best results were obtained from in the fungal isolate at the concentration of 1 × 108 conidia/ml. After 192 and 216 h. of observation, the 1 × 108 CFU/ml of bacterial strains: B. cereus FD-63, B. brevis FD-1 and V. hollisae FD-70; also, 1 × 108 conidia/ml concentration of ET 10 fungal isolate of B. bassiana control C. perspectalis caused mortality rate of 100% under laboratory conditions.ConclusionFuture studies of these microorganisms against this pest in the field, may be an important alternative method to control this pest.

Crystal structure of Grimontia hollisae collagenase provides insights into its novel substrate specificity toward collagen

Collagenase from the gram-negative bacterium Grimontia hollisae strain 1706B (Ghcol) degrades collagen more efficiently even than clostridial collagenase, the most widely used industrial collagenase. However, the structural determinants facilitating this efficiency are unclear. Here, we report the crystal structures of ligand-free and Gly-Pro-hydroxyproline (Hyp)-complexed Ghcol at 2.2 and 2.4 Å resolution, respectively. These structures revealed that the activator and peptidase domains in Ghcol form a saddle-shaped structure with one zinc ion and four calcium ions. In addition, the activator domain comprises two homologous subdomains, whereas zinc-bound water was observed in the ligand-free Ghcol. In the ligand-complexed Ghcol, we found two Gly-Pro-Hyp molecules, each bind at the active site and at two surfaces on the duplicate subdomains of the activator domain facing the active site, and the nucleophilic water is replaced by the carboxyl oxygen of Hyp at the P1 position. Furthermore, all Gly-Pro-Hyp molecules bound to Ghcol have almost the same conformation as Pro-Pro-Gly motif in model collagen (Pro-Pro-Gly)10, suggesting these three sites contribute to the unwinding of the collagen triple helix. A comparison of activities revealed that Ghcol exhibits broader substrate specificity than clostridial collagenase at the P2 and P2′ positions, which may be attributed to the larger space available for substrate binding at the S2 and S2′ sites in Ghcol. Analysis of variants of three active-site Tyr residues revealed that mutation of Tyr564 affected catalysis, whereas mutation of Tyr476 or Tyr555 affected substrate recognition. These results provide insights into the substrate specificity and mechanism of G. hollisae collagenase.

Seasonal variation of microbiota in Shank Acanthopagrus latus living in both brackish and fresh water

The present study was conducted to monitor the microbiota in the posterior intestine of Shank fish living in the river (fresh water) and Al-Razzaza lake (salt or brackish water) in Kerbala, Iraq. Cultivable bacteria were calculated and identified from specimens obtained from the posterior intestine of mucosa (PM) and digesta (PD) during summer and winter seasons. The total culturable bacteria (TCB) of bacteria isolated from both intestinal regions from fresh water fish during summer time were higher than counterparts in winter time. In contrast, the TVC of bacteria isolated from the PM from salt water fish during winter time were higher than those reported in summer time for the same region. Up to 13 species of bacteria were identified from the posterior intestine. Aeromonas hydrophila was the predominant microbe isolated in high percentages from all samples (e.g PD, PM, summer, winter, river and lake). Grimontia hollisae, Pasteurella multocida, Enterobacter cloacae and Pseudomonas fluorescens were isolated from the PM of fresh water fish during summer time. Brucella spp. were found only in PD of fish from salt water during summer time. The mean log of lactic acid bacteria (LAB) in the PD varied between 5.4 ± 0.5 and 4.5 ± 0.1 CFU g-1 during summer and winter in the river location. The mean log of LAB populations in the PM ranged from 4.3 ± 0.9 to 5.9 ± 0.1 CFU g-1 during the seasons of summer and winter in the river location. The LAB population in the both regions of intestine in Shank living in the lake during both seasons were completely absent. In conclusion, seasonal variation in river and lacustrine temperature and salinity influenced the TCB and LAB population and distribution of bacterial species during the study.

Outbreak of seafood-related food poisoning from undetectable Vibrio parahaemolyticus-like pathogen, Chiang Mai Province, Thailand, December 2020.

On 1 December 2020, the Department of Disease Control of Thailand was notified of a cluster of food poisoning cases among participants at a church festival in Mae Ai district, Chiang Mai province. We conducted an outbreak investigation to confirm diagnosis, describe the epidemiological characteristics of the outbreak, identify possible sources of the outbreak and provide appropriate control measures. We reviewed medical records of the food poisoning cases from the health care centres. Active case finding was conducted among participants who had consumed food and water at the festival. An environmental survey was done in the village where the festival was held. A case-control study was conducted to identify the source of the outbreak. Samples for laboratory analysis included rectal swabs and fresh stool specimens from the cases and food handlers, surface swabs of cooking equipment, food, water and ice samples. Among 436 participants surveyed, 368 (84.4%) cases of food poisoning were identified. The most common clinical manifestation was abdominal pain (89.7%), followed by watery diarrhoea (45.7%), nausea (43.5%), vomiting (38.9%), fever (18.5%) and bloody diarrhoea (4.6%). None died in this outbreak. The case-control study showed that mixed spicy seafood salad served in the festival was significantly associated with the disease by both univariable and multivariable analyses. However, the causative agent could not be identified. The environmental investigation suggested this seafood might have been undercooked. Clinical manifestations of the cases, incubation period and the suspected seafood salad suggested seafood-related food poisoning. Grimontia hollisae, the organism causing illness similar to Vibrio parahaemolyticus and commonly undetectable in the laboratory with routine testing, might be the pathogen that caused this outbreak. G.hollisae should be in differential diagnosis and identified in seafood-associated outbreaks.

English

Heart disease is a leading cause of morbidity and mortality in the United States with diet being a major contributor. As solution, fish rich in omega-3 fatty acids is recommended. In this study, the impact of brackish water on fish quality, was done by sampling canal water in Edinburg, Texas. Canal water was membrane filtered and cultured on bacteriological media. Pure cultures were phenotypically identified, and isolates confirmed on a BD Phoenix analyzer. The BD Phoenix analyzer, confirmed the following isolates: Grimontia hollisae, Pseudomonas oryzihabitans, Pseudomonas pseudoalcaligenes, Tatumella ptyseos, Pseudomonas fluorescens, Klebsiella pneumonia subsp. ozaenae, Staphylococcus pettenkoferi, Pasteurella pneumotropica, Achromobacter species, Pasteurella aerogenes, Moraxella species and Pseudomonas species. Fish consumption is good for maintaining a healthy heart, but consumers risk exposure to foodborne pathogens that can cause watery diarrhea, severe abdominal cramps, nausea, vomiting, fever, bacteremia, urinary tract infections and even dead. Key words: Brackish, Microbiome, α-Linolenic acid, Linoleic acid, 2,5-diketo-D-gluconic acid, Tatumellaptyseos, Grimontia hollisae, Staphylococcus pettenkoferi, Esoteric, Nosocomial.

The roles of histidine and tyrosine residues in the active site of collagenase in Grimontia hollisae.

Collagenase from the Grimontia hollisae strain 1706B (Ghcol) is a zinc metalloproteinase with the zinc-binding motif H492EXXH496. It exhibits higher collagen-degrading activity than the collagenase from Clostridium histolyticum, which is widely used in industry. We previously examined the pH and temperature dependencies of Ghcol activity; Glu493 was thought to contribute acidic pKa (pKe1), while no residue was assigned to contribute alkaline pKa (pKe2). In this study, we introduced nine single mutations at the His or Tyr residues in and near the active site. Our results showed that H412A, H485A, Y497A, H578A and H737A retained the activities to hydrolyze collagen and gelatin, while H426A, H492A, H496A and Y568A lacked them. Purification of active variants H412A, H485A, H578A and H737A, along with inactive variants H492A and H496A, were successful. H412A preferred (7-methoxycoumarin-4-yl)acetyl-L-Lys-L-Pro-L-Leu-Gly-L-Leu-[N3-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl]-L-Ala-L-Arg-NH2 to collagen, while H485A preferred collagen to the peptide, suggesting that His412 and His485 are important for substrate specificity. Purification of the active variant Y497A and inactive variants H426A and Y568A were unsuccessful, suggesting that these three residues were important for stability. Based on the reported crystal structure of clostridial collagenase, Tyr568 of Ghcol is suggested to be involved in catalysis and may be the ionizable residue for pKe2.

Recombinant collagenase from Grimontia hollisae as a tissue dissociation enzyme for isolating primary cells

Collagenase products are crucial to isolate primary cells in basic research and clinical therapies, where their stability in collagenolytic activity is required. However, currently standard collagenase products from Clostridium histolyticum lack such stability. Previously, we produced a recombinant 74-kDa collagenase from Grimontia hollisae, which spontaneously became truncated to ~60 kDa and possessed no stability. In this study, to generate G. hollisae collagenase useful as a collagenase product, we designed recombinant 62-kDa collagenase consisting only of the catalytic domain, which exhibits high production efficiency. We demonstrated that this recombinant collagenase is stable and active under physiological conditions. Moreover, it possesses higher specific activity against collagen and cleaves a wider variety of collagens than a standard collagenase product from C. histolyticum. Furthermore, it dissociated murine pancreata by digesting the collagens within the pancreata in a dose-dependent manner, and this dissociation facilitated isolation of pancreatic islets with masses and numbers comparable to those isolated using the standard collagenase from C. histolyticum. Implantation of these isolated islets into five diabetic mice led to normalisation of the blood glucose concentrations of all the recipients. These findings suggest that recombinant 62-kDa collagenase from G. hollisae can be used as a collagenase product to isolate primary cells.

Occurrence of Vibrio spp. along the Algerian Mediterranean coast in wild and farmed Sparus aurata and Dicentrarchus labrax

Background and Aim:Vibrio bacteria are autochthonous inhabitants of aquatic and marine environments. Certain strains are responsible for important seafood-borne outbreaks in developed nations. The aim of this study was to investigate the occurrence of Vibrio spp. along the Algerian Mediterranean coast in several samples of two prominent wild and farmed marine fishes, Sparus aurata and Dicentrarchus labrax.Materials and Methods:A total of 350 S. aurata (n=280 farmed and n=70 wild) and 340 D. labrax (n=250 farmed and n=90 wild) were sampled among three different locations along the Algerian Mediterranean coastal area. The samples were processed for Vibrio research according to the ISO methods. Isolated colonies were identified utilizing biochemical tests and consecutively confirmed with matrix-assisted laser desorption ionization-time-of-flight mass spectrometry, combined with polymerase chain reaction (PCR) analysis when appropriate, or confirmed with PCR analysis alone.Results:A total of 42 Vibrio spp. were detected only among the farmed fishes. Taking into account, all 690 fishes sampled, the incidence of Vibrio spp. was 6.08% (with peaks up to 7.92%) among the total number of farmed fishes. Overall, 25 strains were isolated from S. aurata and 17 strains were isolated from D. labrax. The isolated strains belonged to four different species and were represented as follows: Vibrio alginolyticus (n=20), Vibrio cholerae (n=15), Vibrio fluvialis (n=5), and Vibrio hollisae (n=2). The incidence of Vibrio was higher in places characterized by greater levels of anthropogenic contamination of seawater.Conclusion:Considering the growing production and consequent rising consumption of farmed fish in Algeria, the reported incidence of Vibrio and the presence of potentially pathogenic strains of Vibrio such as V. cholerae cause particular concern for food safety matters. Even if innovative and natural techniques are desired in aquaculture, proper hygiene and manufacturing practices are essential for the correct management of Vibrio infection risk in farmed fishes at both industrial and domestic levels.

Multiple Pleomorphic Tetramers of Thermostable Direct Hemolysin from Grimontia hollisae in Exerting Hemolysis and Membrane Binding

Oligomerization of protein into specific quaternary structures plays important biological functions, including regulation of gene expression, enzymes activity, and cell–cell interactions. Here, we report the determination of two crystal structures of the Grimontia hollisae (formally described as Vibrio hollisae) thermostable direct hemolysin (Gh–TDH), a pore-forming toxin. The toxin crystalized in the same space group of P21212, but with two different crystal packing patterns, each revealing three consistent tetrameric oligomerization forms called Oligomer–I, –II, and –III. A central pore with comparable depth of ~50 Å but differing in shape and size was observed in all determined toxin tetrameric oligomers. A common motif of a toxin dimer was found in all determined structures, suggesting a plausible minimum functional unit within the tetrameric structure in cell membrane binding and possible hemolytic activity. Our results show that bacterial toxins may form a single or highly symmetric oligomerization state when exerting their biological functions. The dynamic nature of multiple symmetric oligomers formed upon release of the toxin may open a niche for bacteria survival in harsh living environments.

The C-terminal segment of collagenase in Grimontiahollisae binds collagen to enhance collagenolysis.

The collagenase secreted by Grimontia hollisae strain 1706B is a 74 kDa protein that consists of two parts: the catalytic module and a C‐terminal segment that includes the bacterial pre‐peptidase C‐terminal domain. Here, we produced a recombinant C‐terminal segment protein and examined its ability to bind collagen and other characteristics as compared with collagen‐binding domains (CBDs) derived from Hathewaya histolytica (Clostridium histolyticum) collagenases; these CBDs are the only ones thus far identified in bacterial collagenases. We found that the C‐terminal segment binds to collagen only when the collagen is in its triple‐helical conformation. Moreover, the C‐terminal segment and the CBDs from H. histolytica have comparable characteristics, including binding affinity to type I collagen, substrate spectrum, and binding conditions with respect to salt concentration and pH. However, the C‐terminal segment has a completely different primary structure from those of the CBDs from H. histolytica. As regards secondary structure, in silico prediction indicates that the C‐terminal segment may be homologous to those in CBDs from H. histolytica. Furthermore, we performed collagenase assays using fluorescein isothiocyanate‐labeled type I collagen to show that the C‐terminal segment positively contributes to the collagenolytic activity of the 74 kDa collagenase from G. hollisae.

Shellfish as reservoirs of bacterial pathogens

Shellfish comprises a variety of animals including bivalves – oysters, clams, quahogs, scallops, mussels, and others including crustaceans – lobsters, crabs, and shrimps. Shellfish naturally found in Grenada, an island nation in the Caribbean, include: blue land crabs (Cardisoma guanhumi), clams (Mercenaria mercenaria), oysters (Isognomon isognomon), queen conchs, also called “lambi” (Strombus gigas), and whelks (West Indian top shell) (Cittarium pica), all of which are used for human consumption in this country. Bacteria are ubiquitous in the marine environment. Shellfish can be a source of commensal bacteria that can be pathogenic to shellfish, as well as those pathogenic to humans. Raw and partially cooked molluscan shellfish (clams, oysters, and mussels) have a long history as vectors of infectious agents[1]. According to Potasman et al.[2], the earliest reports of shellfish-transmitted diseases were documented in the late 19th and early 20th century. Apart from typhoid fever, the most notable examples include Vibrio species, which account for 20% of all outbreaks of shellfishassociated human disease. Other bacteria are Salmonella species, Shigella species, Plesiomonas shigelloides, and Listeria species[2]. Several Vibrio species native to both marine and estuarine environments, have been identified as the causative agents of shellfish-associated human illnesses. These include Vibrio parahaemolyticus (V. parahaemolyticus), Vibrio vulnificus (V. vulnificus), Vibrio cholerae non-O1, Vibrio cholerae, Vibrio cholerae O1, Vibrio fluvialis (V. fluvialis), Vibrio hollisae, Vibrio mimicus, Vibrio alginolyticus (V. alginolyticus), and unidentified or miscellaneous Vibrio species. Among these, all except V. vulnificus, are associated with gastroenteritis of varying severity[1]. Oysterassociated V. vulnificus septicemia and death were first reported in 1975[3]. Another bacterium of public health concern is Salmonella. Compared to vibrios, there have been much less studies on Salmonella in various shellfish. Setti et al.[4] studied the dynamics of Salmonella contamination in the coastal areas of Agadir, a populous region of Southern Morocco where shellfish production is important to the local economy. Salmonella enterica (S. enterica) serovar Kentucky was found almost exclusively in mussels, whereas Salmonella blockley was found in mussels and sediment samples. Identification of Salmonella to serovar level is required ARTICLE INFO ABSTRACT

Hydrogen peroxide causes Vibrio vulnificus bacteriolysis accelerated by sulfonyl fluoride compounds.

Induction of bacteriolysis of Vibrio vulnificus cells by 10 mM hydrogen peroxide (H(2)O(2)) was analyzed. All Vibrio species examined, except for Vibrio hollisae, were lysed by 10 mM H(2)O(2). Bacteriophage induction was not the cause of H(2)O(2)-induced bacteriolysis. Autolysis is also known to cause bacteriolysis. VvpS protein is a serine protease of V. vulnificus essential for autolysis. vvpS mutant underwent H(2)O(2)-induced bacteriolysis in the same manner as the wild type. Protease inhibitors including serine protease inhibitors did not inhibit H(2)O(2)-induced bacteriolysis, which means that bacteriolysis is not due to autolysis. Unexpectedly, H(2)O(2)-induced bacteriolysis was accelerated by adding 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) and phenylmethylsulfonyl fluoride which are serine protease inhibitors. The hydroxyl radical was generated by H(2)O(2)-AEBSF interaction. It was considered that H(2)O(2)-induced bacteriolysis was caused by the hydroxyl radical which was generated by Fenton reaction, and possibly mediated by AEBSF. Deferoxamine, an agent chelating ferric ion and Fenton reaction inhibitor, suppressed both H(2)O(2)-induced bacteriolysis and its acceleration by AEBSF. This suggests that both phenomena were Fenton reaction dependent, and hydroxyl radical generated by Fenton reaction caused bacteriolysis of V. vulnificus though the reason for high susceptibility of Vibrio species to hydroxyl radical is not known.