Identification and characterization of a conjugative blaVIM-1-bearing plasmid in Vibrio alginolyticus of food origin.

OBJECTIVES: To investigate the genetic features of the blaVIM-1 gene first detected in a cephalosporin-resistant Vibrio alginolyticus isolate, Vb1978. METHODS: The MICs of V. alginolyticus strain Vb1978 were determined, and the ß-lactamases produced were screened and analysed using conjugation, S1-PFGE and Southern blotting. The complete sequence of the blaVIM-1-encoding plasmid was also obtained using the Illumina and MinION sequencing platforms. RESULTS: V. alginolyticus strain Vb1978, isolated from a retail shrimp sample, was resistant to cephalosporins and exhibited reduced susceptibility to carbapenems. A novel blaVIM-1-carrying conjugative plasmid, designated pVb1978, was identified in this strain. Plasmid pVb1978 had 50 001 bp and comprised 59 predicted coding sequences (CDSs). The plasmid backbone of pVb1978 was homologous to those of IncP-type plasmids, while its replication region was structurally similar to non-IncP plasmids. The blaVIM-1 gene was found to be carried by the class 1 integron In70 and associated with a defective Tn402-like transposon. CONCLUSIONS: A novel blaVIM-1-carrying conjugative plasmid, pVb1978, was reported for the first time in V. alginolyticus, which warrants further investigation in view of its potential pathogenicity towards humans and widespread occurrence in the environment.

Marine bacterial DNase curtails virulence and disrupts biofilms of Candida albicans and non-albicans Candida species.

Candida is one of the most prevalent fungal pathogens in clinical settings which form antibiotic-resistant biofilms on biomedical devices. Hence, there is a need for non-antimicrobial alternatives to combat these infections. The present study investigates the anti-biofilm effect of marine bacterial DNase by targeting the eDNA present in the biofilms of Candida spp. A strain of Vibrio alginolyticus (AMSII) which showed enhanced DNase activity was isolated from marine sediment. Treatment of young and mature Candida biofilms with purified marine bacterial DNase (MBD) caused a 60-80% reduction in biofilm biomass, similar to treatment with DNase I from Bovine pancreas. Scanning electron microscopy showed that MBD significantly reduced the formation of biofilms on urinary catheters and more importantly prevented the virulent yeast to hyphae dimorphic switch in C. albicans. The present study identified a potential non-antibiotic alternative therapy to eradicate Candida biofilms and can be used to develop enzyme fabricated antifouling indwelling medical devices.

An experimental strategy validated to design cost-effective culture media based on response surface methodology.

For any fermentation process, the production cost depends on several factors, such as the genetics of the microorganism, the process condition, and the culture medium composition. In this work, a guideline for the design of cost-efficient culture media using a sequential approach based on response surface methodology is described. The procedure was applied to analyze and optimize a culture medium of registered trademark and a base culture medium obtained as a result of the screening analysis from different culture media used to grow the same strain according to the literature. During the experiments, the procedure quantitatively identified an appropriate array of micronutrients to obtain a significant yield and find a minimum number of culture medium ingredients without limiting the process efficiency. The resultant culture medium showed an efficiency that compares favorably with the registered trademark medium at a 95% lower cost as well as reduced the number of ingredients in the base culture medium by 60% without limiting the process efficiency. These results demonstrated that, aside from satisfying the qualitative requirements, an optimum quantity of each constituent is needed to obtain a cost-effective culture medium. Study process variables for optimized culture medium and scaling-up production for the optimal values are desirable.

Characterization of an IncA/C Multidrug Resistance Plasmid in Vibrio alginolyticus.

Cephalosporin-resistant Vibrio alginolyticus was first isolated from food products, with ß-lactamases encoded by blaPER-1, blaVEB-1, and blaCMY-2 being the major mechanisms mediating their cephalosporin resistance. The complete sequence of a multidrug resistance plasmid, pVAS3-1, harboring the blaCMY-2 and qnrVC4 genes was decoded in this study. Its backbone exhibited genetic homology to known IncA/C plasmids recoverable from members of the family Enterobacteriaceae, suggesting its possible origin in Enterobacteriaceae.

Collagenase From Vibrio alginolyticus Cultures: Experimental Study and Clinical Perspectives.

Purpose The purpose of this study is to examine the activity of collagenase from cultures of Vibrio alginolyticus as in vitro as in biological samples and to evaluate clinical perspectives of this product about the treatment of fibroproliferative diseases like Dupuytren's contracture. Methods The experimental part of the study has been divided in 2 stages. In the first stage, the collagenase has been produced in laboratory, assessing its purity, verifying the in vitro degradation of collagen by the enzyme and measuring the size of the fragments; in the second part, an experimental injection into samples of fibrous cord typical of Dupuytren's disease has been performed in vitro. For the injection we used only collagenase, or collagenase after having subjected them to 2 types of mechanical stress or a collagenase combined with ethylenediamine tetra-acetic acid. Considering that the human samples have been treated in vitro, our institution does not require a specific informed consent. Results It appeared evident that the collagenase obtained from Vibrio alginolyticus (nonpathogenic bacterium) is highly pure (>98%) and does not contain nonspecific protease. The collagenase from Vibrio alginolyticus therefore has an excellent degradative capacity against the collagen and this activity takes on a dose- and time-dependent behavior. The collagenase from Vibrio alginolyticus does not act negatively on cell survival and collagen peptides obtained may provide a better proliferative stimulus compared to controls. Conclusions The collagenase from Vibrio alginolyticus, given its obvious ability in vitro and biological samples, could be an option in the nonsurgical treatment of Dupuytren's disease. Level of evidence Level III, therapeutic.

[Virulence-related genes of Vibrio alginolyticus and its virulence in mice].

OBJECTIVE: We determinated the virulence factors of Vibrio alginolyticus strains isolated from the environment by multiplex PCRs and animal experiments, in order to compare the differences between the highly virulent strain and attenuated virulent strain, and to explore the virulent mechanism of V. alginolyticus in mammals. METHODS: The virulence-related genes of V. alginolyticus were investigated by multiplex PCRs. Hemolysin and pathogenic proteins were detected using Kanagawa phenomenon tests and enzyme activity tests. In vivo pathogenetic tests of V. alginolyticus were done through orogastric and intraperitoneal Kunming mouse. RESULTS: Amylase and lecithinase activities were observed in 100% of the strains, whereas lipase and gelatinase activities were found in only 70% and urease activity was not detected. In Kanagawa phenomenon tests 60% of the strains gave positive results. The related virulence genes such as toxR, Collagenase, tlh, FlaA, ompW, AspA and fur were distributed among 10 strains of V. alginolyticus collected, with the exception of toxS, trh, tdh and UreR. Among those 10 strains, VA009 has shown a strong pathogenesis to the mouse, which caused fluid accumulation and led the mortality rate as high as 80% within 7 days by intraperitoneal infection. CONCLUSION: This study indicates that there is a great difference in pathogenicity among V. alginolyticus strains to mouse. The cell toxicity of V. alginolyticus made more contribution than extracellular secretion, while the extracellular secretion of V. parahaemolyticus played a major role in its toxicity. The virulence gene profiles were consistent between the highly virulent and attenuated virulent strains, indicating that V. alginolyticus might have a different virulence system and different pathogenic mechanism compared with V. parahaemolyticus.

A new association between hyaluronic acid and collagenase in wound repair: an open study.

BACKGROUND: Most ulcers occur with slough and need debridement treatment. Surgical treatment is usually performed but many patients need an additional chemical therapy to promote healing process. This type of wound bed preparation is slower than surgical one, but it is essential in those patients who are not eligible for surgery because of systemic diseases. A collagenase derived from the bacterium Clostridium hisolyticum is the most used. AIM: To evaluate the safety and the effectiveness of a new collagenase produced by Vibrio algynoliticum. MATERIALS AND METHODS: Forty patients were enrolled and daily treated for five weeks with Bionect Start® ointment. Parameters took into account were fibrin degradation and pain relief. The same team carried out treatment during the study period of five weeks. RESULTS: 32 patients achieved a relevant reduction of the ulcer size, an improvement of the wound bed and a reduction of fibrin and exudates. Among of 32, 14 patients presented with a complete healing. Eight patients had mild or no improvements. The patients referred a remarkable pain reduction, 20 out of 40 patients reported a decrease in pain during treatment, 16 patients referred no pain at all and four patients had no significant changes. CONCLUSIONS: The application of this product on the lesions promotes vascularized granulation tissue and reduces formation of fibrin and exudate. Furthermore, a macroscopical margins proliferation is highlighted after a short time and an improvement of periwound skin is observed during the treatment period. Bionect Start® allowed easy removal of dressing, less pain for patient, reduction of dressing time.

Insight into the binding of the wild type and mutated alginate lyase (AlyVI) with its substrate: a computational and experimental study.

The homology model of the wild type alginate lyase (AlyVI) marine bacterium Vibrio sp. protein, was built using the crystal structure of the Family 7 alginate lyase from Sphingomonas sp. A1. To rationalize the observed structure-affinity relationships of aliginate lyase alyVI with its (GGG) substrate, molecular docking, MD imulations and binding free energy calculations followed by site-directed mutagenesis and alyVI activity assays were carried out. Per-residue decomposition of the (GGG) binding energy revealed that the most important contributions were from polar and charged residues, such as Asn138, Arg143, Asn217, and Lys308, while van der Waals interactions were responsible for binding with the catalytic His200 and Tyr312 residues. The mutants H200A, K308A, Y312A, Y312F, and W165A were found to be inactive or almost inactive. However, the catalytic efficiency (k(cat)/K(m)) of the double mutant L224V/D226G increased by two-fold compared to the wild type enzyme. This first structural model with its substrate binding mode and the agreement with experimental results provide a suitable base for the future rational design of new mutated alyVI structures with improved catalytic activity.

Crystal structure and mutational analysis of aminoacylhistidine dipeptidase from Vibrio alginolyticus reveal a new architecture of M20 metallopeptidases.

Aminoacylhistidine dipeptidases (PepD, EC 3.4.13.3) belong to the family of M20 metallopeptidases from the metallopeptidase H clan that catalyze a broad range of dipeptide and tripeptide substrates, including L-carnosine and L-homocarnosine. Homocarnosine has been suggested as a precursor for the neurotransmitter γ-aminobutyric acid (GABA) and may mediate the antiseizure effects of GABAergic therapies. Here, we report the crystal structure of PepD from Vibrio alginolyticus and the results of mutational analysis of substrate-binding residues in the C-terminal as well as substrate specificity of the PepD catalytic domain-alone truncated protein PepD(CAT). The structure of PepD was found to exist as a homodimer, in which each monomer comprises a catalytic domain containing two zinc ions at the active site center for its hydrolytic function and a lid domain utilizing hydrogen bonds between helices to form the dimer interface. Although the PepD is structurally similar to PepV, which exists as a monomer, putative substrate-binding residues reside in different topological regions of the polypeptide chain. In addition, the lid domain of the PepD contains an "extra" domain not observed in related M20 family metallopeptidases with a dimeric structure. Mutational assays confirmed both the putative di-zinc allocations and the architecture of substrate recognition. In addition, the catalytic domain-alone truncated PepD(CAT) exhibited substrate specificity to l-homocarnosine compared with that of the wild-type PepD, indicating a potential value in applications of PepD(CAT) for GABAergic therapies or neuroprotection.

Identification of a luxO-regulated extracellular protein Pep and its roles in motility in Vibrio alginolyticus.

The fish pathogen Vibrio alginolyticus virulence factors and regulation mechanism are barely known except the quorum sensing regulated extracellular products. In the present study, the cleavable isotope-coded affinity tags (cICATs) were used to analyze the differentially expressed extracellular proteins of V. alginolyticus wild-type and a ΔluxO mutant, and a putative peptidase Pep was identified. Quantitative real-time reverse transcription PCR confirmed that the transcription of pep was down-regulated by LuxO. Furthermore, western blotting demonstrated that the expression of Pep was cell-density dependent and quorum sensing regulated in V. alginolyticus. The pep in-frame mutant abolished the swimming and swarming motility, suggesting that it was involved in motility regulation in V. alginolyticus. In liquid medium, the polar flagellum was observed intact in ∆pep mutant while the chemotaxis related gene cheY, which is a response regulator causing change in direction of flagellar rotation, was down-regulated in ∆pep mutant. When ∆pep mutant was grown on hard agar plate, the lateral flagella disappeared and the transcriptions of flagellar biogenesis genes flaK, fliS, lafK, and lafA were down-regulated in ∆pep mutant. These results suggested that Pep was a luxO-regulated extracellular protein and involved in the motility of V. alginolyticus.

Functional characterization of Vibrio alginolyticus twin-arginine translocation system: its roles in biofilm formation, extracellular protease activity, and virulence towards fish.

The bacterial twin-arginine translocation (Tat) system contributes to translocate folded proteins and plays pleiotropic roles in growth, motility, and the secretion of some virulent factors. In this study, the authors identified the Tat gene cluster in fish pathogen Vibrio alginolyticus and explored its roles in pathogenesis toward fish. Vibrio alginolyticus Tat mutants showed growth deficiency in TMAO medium, while the complement strain restored the ability to grow in the medium, demonstrating the conservative function of the Tat system in translocation of redox enzymes or cofactors in this bacterium. In V. alginolyticus, deletion of the tatABC genes led to a drastic decrease in biofilm biogenesis. Interestingly, the secretion of extracellular protease Asp, an established exotoxin of the bacterium, was significantly decreased in the TatC mutant, suggesting that TatC might play a part in the production of virulence factors in the bacterium. Furthermore, the Tat mutants displayed attenuated virulence toward the fish model and EPC cells. These findings suggest that the Tat secretion related to the extracellular protease activity as well as virulence in V. alginolyticus provided new insights into the pathogenesis of vibriosis in fish.

pH-Controlled Release of Antigens Using Mesoporous Silica Nanoparticles Delivery System for Developing a Fish Oral Vaccine.

The development of effective vaccines and delivery systems in aquaculture is a long-term challenge for controlling emerging and reemerging infections. Cost-efficient and advanced nanoparticle vaccines are of tremendous applicability in prevention of infectious diseases of fish. In this study, dihydrolipoamide dehydrogenase (DLDH) antigens of Vibrio alginolyticus were loaded into mesoporous silica nanoparticles (MSN) to compose the vaccine delivery system. Hydroxypropyl methylcellulose phthalate (HP55) was coated to provide protection of immunogen. The morphology, loading capacity, acid-base triggered release were characterized and the toxicity of nanoparticle vaccine was determined in vitro. Further, the vaccine immune effects were evaluated in large yellow croaker via oral administration. In vitro studies confirmed that the antigen could be stable in enzymes-rich artificial gastric fluid and released under artificial intestinal fluid environment. In vitro cytotoxicity assessment demonstrated the vaccines within 120 µg/ml have good biocompatibility for large yellow croaker kidney cells. Our data confirmed that the nanoparticle vaccine in vivo could elicit innate and adaptive immune response, and provide good protection against Vibrio alginolyticus challenge. The MSN delivery system prepared may be a potential candidate carrier for fish vaccine via oral administration feeding. Further, we provide theoretical basis for developing convenient, high-performance, and cost-efficient vaccine against infectious diseases in aquaculture.

Enzymatic, outer membrane proteins and plasmid alterations of starved Vibrio parahaemolyticus and Vibrio alginolyticus cells in seawater.

The marine bacteria Vibrio parahaemolyticus and V. alginolyticus were incubated in seawater for 8 months to evaluate their adaptative responses to starvation. The starved cells showed an altered biochemical and enzymatic profiles, respectively, on Api 20E and Api ZYM systems and an evolution to the filterable minicells state capable to pass membrane pore size 0.45 microm. Outer membrane proteins patterns of stressed bacteria were also altered. Indeed, these modifications were manifested by the appearance and/or disappearance of bands as well as in the level of expression of certain proteins. Plasmids profiles analysis showed that V. alginolyticus ATCC 33787 lost three plasmids, whereas other tested strains conserved their initial profiles.

Purification, crystallization and preliminary X-ray analysis of an aminoacylhistidine dipeptidase (PepD) from Vibrio alginolyticus.

The aminoacylhistidine dipeptidase (PepD) protein encoded by Vibrio alginolyticus pepD was successfully overexpressed and characterized and the putative active-site residues responsible for metal binding and catalysis were identified. The purified enzyme contained two zinc ions per monomer. The recombinant dipeptidase enzyme, which was identified as a homodimer in solution, exhibited broad substrate specificity for Xaa-His dipeptides, with highest activity towards the His-His dipeptide. The purified protein was crystallized using the hanging-drop vapour-diffusion method. Preliminary crystallographic analysis showed that the crystal belonged to space group P6(1) or P6(5), with unit-cell parameters a = b = 80.42, c = 303.11 A. The crystal contained two molecules per asymmetric unit and the predicted solvent content was 53.4%.

Investigation of several virulence properties among Vibrio alginolyticus strains isolated from diseased cultured fish in Tunisia.

We analysed 34 Vibrio alginolyticus strains isolated from gilthead sea bream Sparus aurata L. and sea bass Dicentrarchus labrax L. cultured in fish farms on the Tunisian Mediterranean coast for the presence of several virulence properties such as extracellular products (ECP) production, growth in iron-limiting conditions and survival in fish serum. The results obtained with different substrates showed that ECP of V. alginolyticus were hydrolytic. The virulence was correlated with the ability of strains to grow in the presence of non-immune fish serum or under conditions of iron limitation. We further examined the presence of virulence genes homologous to those in V. cholerae (toxR, toxS, VPI and ace); toxR was found in 16 V. alginolyticus strains and toxS in 17 strains out of 34 analysed. A positive amplification for the virulence pathogenicity island (VPI) was produced by 12 V alginolyticus strains. Finally, the ace expected amplification fragment was found in 7 V. alginolyticus isolates. Thus, the pathogenicity of V. alginolyticus may be the result of a combination of all these factors.

Structure of Vibrio FliL, a New Stomatin-like Protein That Assists the Bacterial Flagellar Motor Function.

Many motile bacteria swim or swarm using a filamentous rotating organelle, the flagellum. FliL, a component protein of the flagellar motor, is known to enhance the motor performance under high-load conditions in some bacteria. Here we determined the structure of the periplasmic region of FliL (FliLPeri) of the polar flagellum of Vibrio alginolyticus FliLPeri shows a remarkable structural similarity to the stomatin/prohibitin/flotillin/HflK/C (SPFH) domain of stomatin family proteins, some of which are involved in modulation of ion channel activities in various organisms. FliLPeri forms a ring assembly in the crystal with an inner diameter of around 8 nm, which is comparable to the size of the stator unit. Mutational analyses suggest that the FliL ring forms a complex with the stator unit and that the length of the periplasmic linkers of FliL and the stator B-subunit is essential for the complex formation. We propose a model of the FliL-stator complex to discuss how Vibrio FliL modulates stator function in the bacterial flagellar motor under conditions of high viscosity.IMPORTANCE Some flagellated bacteria regulate motor torque in response to the external load change. This behavior is critical for survival, but the mechanism has remained unknown. Here, we focused on a key protein, FliL of Vibrio alginolyticus, and solved the crystal structure of its periplasmic region (FliLPeri). FliLPeri reveals striking structural similarity to a conserved domain of stomatin, which is involved in ion channel regulation in some organisms, including mammals. FliLPeri forms a ring with an inner diameter that is comparable in size to the stator unit. The mutational analyses suggested that the presence of the ring-like assembly of FliL around the stator unit enhances the surface swarming of Vibrio cells. Our study data also imply that the structural element for the ion channel regulation is conserved from bacteria to mammals.

Roles of LuxR in regulating extracellular alkaline serine protease A, extracellular polysaccharide and mobility of Vibrio alginolyticus.

In marine Vibrio species, the Vibrio harveyi-type LuxR protein, a key player in a quorum-sensing system, controls the expression of various genes. In this study, the luxR homologue in Vibrio alginolyticus was identified and named luxR(val), whose expression was greatly induced by the increase of cell number. The luxR(val) in-frame deletion mutant showed a significant downregulation of total extracellular protease activity, and especially caused a 70% decrease in the transcript levels of extracellular alkaline serine protease A (proA), which was an important virulent factor of V. alginolyticus. Complementation in trans with luxR(val) could restore the expression of proA to the level of the wild-type strain. Deletion of the luxR(val) gene also resulted in changes of colony morphology, extracellular polysaccharide production and mobility. Therefore, another member of the V. harveyi-type LuxR regulator family has been characterized in V. alginolyticus.

Enzymatic characterization of Vibrio alginolyticus strains isolated from bivalves harvested at Venice Lagoon (Italy) and Guanabara Bay (Brazil).

The aquatic ecosystem is the natural habitat of microorganisms including Vibrio and Aeromonas genus which are pathogenic to human and animals. In the present investigation the frequency of these bacteria and the enzymatic characteristics of 34 Vibrio alginolyticus strains isolated from bivalves harvested in Venice Lagoon (Italy) and Guanabara Bay (Brazil) were carried out from November 2003 to February 2004. The mussels' samples were submitted to enrichment in Alkaline Peptone Water (APW) added with 1% of sodium chloride (NaCl) and APW plus 3% NaCl incubated at 37 degrees C for 18-24 h. Following the samples were streaked onto TCBS Agar (Thiossulfate Citrate Bile Sucrose Agar) and the suspected colonies were submitted to biochemical characterization. Also, the Vibrio alginolyticus strains were evaluated to collagenase, elastase and chondroitinase production. The results showed the isolation of 127 microorganisms distributed as follows: 105 Vibrio strains such as V. alginolyticus (32.4%), V. harveyi (19%) and V. parahaemolyticus (7.6%), 20 Aeromonas strains and two Plesiomonas shigelloides were the main pathogens isolated. We observed the production of the three enzymes from V. alginolyticus strains considered as the main virulence factors of the bacteria, especially in cases of human dermatological infection.

[Cloning and expression of alginate lyase genes from Vibrio alginolyticus and characterization of the alginate lyase].

With the discovery of the significant medicinal value of alginate oligosaccharides and bioethanol produced by microalgae, alginate lyase has been the focus of research in all fields. Five alginate lyase genes in cluster from Vibrio alginolyticus were cloned and expressed in Escherichia coli. SDS-PAGE and enzyme activity showed that four of the five genes have the activity to degrade alginate. Optimization of the induction conditions, protein purification and enzyme properties of rAlgV3 with the highest enzyme activity were studied. The results showed that the enzyme activity of recombinant enzyme rAlgV3 increased from 2.34×104 U/L to 1.68×105 U/L, which was 7.3 times higher than before. The optimal reaction temperature was 40 °C, and the enzyme was relatively stable between 4 °C and 20 °C. The enzyme had a higher activity between pH 6.5 and 9.0, with the optimum pH 8.0. It showed a wide range of pH that the alginate lyase can exist stably between pH 4.5 and 9.5. Appropriate concentrations of NaCl and Fe²âº, Fe³âº ions promoted enzyme activity. SDS and Cu²âº ions inhibited the enzyme activity. The enzyme degraded Poly-M fragments and Poly-G fragments, with a wide range of substrate properties. The degraded product of sodium alginate of rAlgV3 analyzed by ESI-MS mainly was oligosaccharides with a polymerization degree of 2 to 3, which means that rAlgV3 was an endo-type alginate lyase. This enzyme has the potential in the development of third-generation bioethanol and the production of alginate oligosaccharides.