Production of Polyhydroxyalkanoates (PHAs) by Vibrio alginolyticus Strains Isolated from Salt Fields.

Vibrio alginolyticus is a halophilic organism usually found in marine environments. It has attracted attention as an opportunistic pathogen of aquatic animals and humans, but there are very few reports on polyhydroxyalkanoate (PHA) production using V. alginolyticus as the host. In this study, two V. alginolyticus strains, LHF01 and LHF02, isolated from water samples collected from salt fields were found to produce poly(3-hydroxybutyrate) (PHB) from a variety of sugars and organic acids. Glycerol was the best carbon source and yielded the highest PHB titer in both strains. Further optimization of the NaCl concentration and culture temperature improved the PHB titer from 1.87 to 5.08 g/L in V. alginolyticus LHF01. In addition, the use of propionate as a secondary carbon source resulted in the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). V. alginolyticus LHF01 may be a promising host for PHA production using cheap waste glycerol from biodiesel refining.

Tetrodotoxins (TTXs) and Vibrio alginolyticus in Mussels from Central Adriatic Sea (Italy): Are They Closely Related?

Tetrodotoxins (TTXs), potent neurotoxins, have become an increasing concern in Europe in recent decades, especially because of their presence in mollusks. The European Food Safety Authority published a Scientific Opinion setting a recommended threshold for TTX in mollusks of 44 µg equivalent kg-1 and calling all member states to contribute to an effort to gather data in order to produce a more exhaustive risk assessment. The objective of this work was to assess TTX levels in wild and farmed mussels (Mytilus galloprovincialis) harvested in 2018-2019 along the coastal area of the Marche region in the Central Adriatic Sea (Italy). The presence of Vibrio spp. carrying the non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) genes, which are suspected to be involved in TTX biosynthesis, was also investigated. Out of 158 mussel samples analyzed by hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry (HILIC-MS/MS), 11 (7%) contained the toxins at detectable levels (8-26 µg kg-1) and 3 (2%) contained levels above the EFSA safety threshold (61-76 µg kg-1). Contaminated mussels were all harvested from natural beds in spring or summer. Of the 2019 samples, 70% of them contained V. alginolyticus strains with the NRPS and/or PKS genes. None of the strains containing NRPS and/or PKS genes showed detectable levels of TTXs. TTXs in mussels are not yet a threat in the Marche region nor in Europe, but further investigations are surely needed.

Treatment Trial of Nile Tilapia (Oreochromis niloticus) Experimentally Infected with Vibrio alginolyticus Isolated from Sea bass (Dicentrarchus labrax).

BACKGROUND AND OBJECTIVE: In Egypt, Nile tilapia represents the main cultured type due to its economical price, palatability and easy culturing. This study was aimed to elucidate the pathogenicity of V. alginolyticus isolated from diseased sea bass and experimentally infected healthy Nile tilapia fish. MATERIALS AND METHODS: Healthy Nile tilapia fish were injected I/P with V. alginolyticus isolated from diseased sea bass. Symptoms and mortality rates of infected Nile tilapia fish were recorded during the experimental period. Re-isolation of V. alginolyticus was done from infected tilapia fish by bacteriological methods. For confirmation the pathogenicity of Vibrio isolated either from marine fish or tilapia fish, PCR test was done using tdh and bla gens. Liver and kidney function tests with histopathological examinations of some organs were performed. Treatment trial was done according to the antibiotic sensitivity test. RESULTS: The isolated Vibrio is highly pathogenic to Nile tilapia fish causing deterioration in all parameters which finished by severe mortalities. Treatment with florfenicol, enrofloxacin, or oxytetracycline reduced the mortality rate and improved liver and kidney function parameters of infected Nile tilapia fish. CONCLUSION: V. alginolyticus can infect both marine and fresh water fish inducing a high mortality rate. Treatment of infected fish with florfenicol, enrofloxacin, or oxytetracycline reduces the mortality rate.

Comprehensive depiction of novel heavy metal tolerant and EPS producing bioluminescent Vibrio alginolyticus PBR1 and V. rotiferianus PBL1 confined from marine organisms.

The current study depicts the isolation of luminescent bacteria from fish and squid samples that were collected from Veraval fish harbour. From Indian mackerel, total 14 and from squid, total 23 bioluminescent bacteria were isolated using luminescence agar medium. Two bioluminescent bacteria with highest relative luminescence intensity PBR1 and PBL1 were selected. These two isolates were subjected to detailed biochemical characterization and were tested positive for 5 out of 13 biochemical tests. Furthermore, both PBR1 and PBL1 were able to ferment cellobiose, dextrose, fructose, galactose, maltose, mannose, sucrose and trehalose with acid production. Based on 16S rRNA partial gene sequence analysis, PBR1 was identified as Vibrio alginolyticus and PBL1 as V. rotiferianus. Antibiotic susceptibility test using paper-disc method showed that PBR1 and PBL1 were sensitive to chloramphenicol, ciprofloxacin, co-trimoxazole, gatifloxacin, levofloxacin, linezolid ad roxithromycin out of 18 antibiotics tested. Moreover, both strains were evaluated for their exopolysachharide (EPS) producing ability where PBR1 and PBL1 were able to yield 1.34 g% (w/v) and 2.45 g% (w/v) EPS respectively from 5 g% (v/v) sucrose concentration. Heavy metal toxicity assessment was carried out using agar well diffusion method with eight heavy metals and both the strains were sensitive to As(III), Cd(II), Ce(II), Cr(III), Cu(II), Hg(II) and while they showed resistance to Pb(II) and Sr(II). Based on these results, a study was conducted to demonstrate bio-removal of Pb and Sr by EPS of PBR1 and PBL1. Fourier transform infrared (FTIR) spectra revealed the functional groups of EPS involved in interaction with the heavy metals. Owing to the sensitivity for the remaining heavy metals, these bioluminescent bacteria can be used further for the development of luminescence-based biosensor.

Identification of Vibrio alginolyticus virulent strain-specific DNA regions by suppression subtractive hybridization and PCR.

AIMS: Vibrio alginolyticus was frequently isolated from diseased farmed fish in the coaster waters of Hainan Island over the past two decades. In this study, we attempted to identify candidates of virulent strain-specific DNA regions for this pathogen. METHODS AND RESULTS: Suppression subtractive hybridization (SSH) and PCR were successively performed between the typical virulent strain and avirulent strain of V. alginolyticus, in which they shared 99·54% homology of 16S rDNAs. Out of 2873 subtracted clones, nine clones were finally indicated to harbour virulent strain-specific DNA fragments. The receivable functions of the major fragments in the nine clones were believed to encode methyl-accepting chemotaxis protein (n = 1), type VI secretion system-associated FHA domain protein TagH (n = 1), diguanylate cyclase (n = 1), AraC family transcriptional regulator (n = 1), ABC-type uncharacterized transport system permease component (n = 1) and hypothetical proteins (n = 4). Two hypothetical proteins contain several disordered regions. CONCLUSIONS: Some specific DNA regions existed in the virulent strain of V. alginolyticus, and the SSH assay could be a highly sensitive method for identifying virulent regions in pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: This report is the first to describe the identification of virulent strain-specific DNA regions in the V. alginolyticus genome, which is helpful in developing virulent strain-specific rapid detection methods and is a pivotal precondition for clarifying the molecular virulence mechanism of V. alginolyticus.

Genomic variation among closely related Vibrio alginolyticus strains is located on mobile genetic elements.

BACKGROUND: Species of the genus Vibrio, one of the most diverse bacteria genera, have undergone niche adaptation followed by clonal expansion. Niche adaptation and ultimately the formation of ecotypes and speciation in this genus has been suggested to be mainly driven by horizontal gene transfer (HGT) through mobile genetic elements (MGEs). Our knowledge about the diversity and distribution of Vibrio MGEs is heavily biased towards human pathogens and our understanding of the distribution of core genomic signatures and accessory genes encoded on MGEs within specific Vibrio clades is still incomplete. We used nine different strains of the marine bacterium Vibrio alginolyticus isolated from pipefish in the Kiel-Fjord to perform a multiscale-comparative genomic approach that allowed us to investigate [1] those genomic signatures that characterize a habitat-specific ecotype and [2] the source of genomic variation within this ecotype. RESULTS: We found that the nine isolates from the Kiel-Fjord have a closed-pangenome and did not differ based on core-genomic signatures. Unique genomic regions and a unique repertoire of MGEs within the Kiel-Fjord isolates suggest that the acquisition of gene-blocks by HGT played an important role in the evolution of this ecotype. Additionally, we found that ~ 90% of the genomic variation among the nine isolates is encoded on MGEs, which supports ongoing theory that accessory genes are predominately located on MGEs and shared by HGT. Lastly, we could show that these nine isolates share a unique virulence and resistance profile which clearly separates them from all other investigated V. alginolyticus strains and suggests that these are habitat-specific genes, required for a successful colonization of the pipefish, the niche of this ecotype. CONCLUSION: We conclude that all nine V. alginolyticus strains from the Kiel-Fjord belong to a unique ecotype, which we named the Kiel-alginolyticus ecotype. The low sequence variation of the core-genome in combination with the presence of MGE encoded relevant traits, as well as the presence of a suitable niche (here the pipefish), suggest, that this ecotype might have evolved from a clonal expansion following HGT driven niche-adaptation.

Metabolic mechanism of colistin resistance and its reverting in Vibrio alginolyticus.

Colistin is a last-line antibiotic against Gram-negative multidrug-resistant bacteria, but the increased resistance poses a huge challenge to this drug. However, the mechanisms underlying such resistance are largely unexplored. The present study first identified the mutations of two genes encoding AceF subunit of pyruvate dehydrogenase (PDH) and TetR family transcriptional regulator in colistin-resistant Vibrio alginolyticus (VA-RCT ) through genome sequencing. Then, gas chromatography-mass spectroscopy-based metabolomics was adopted to investigate metabolic responses since PDH plays a role in central carbon metabolism. Colistin resistance was associated with the reduction of the central carbon metabolism and energy metabolism, featuring the alteration of the pyruvate cycle, a recently characterized energy-producing cycle. Metabolites in the pyruvate cycle reprogramed colistin-resistant metabolome to colistin-sensitive metabolome, resulting in increased gene expression, enzyme activity or protein abundance of the cycle and sodium-translocating nicotinamide adenine dinucleotide-ubiquinone oxidoreductase. This reprogramming promoted the production of the proton motive force that enhances the binding between colistin and lipid A in lipopolysaccharide. Moreover, this metabolic approach was effective against VA-RCT in vitro and in vivo as well as other clinical isolates. These findings reveal a previously unknown mechanism of colistin resistance and develop a metabolome-reprogramming approach to promote colistin efficiency to combat with colistin-resistant bacteria.

Fast, simple and highly specific molecular detection of Vibrio alginolyticus pathogenic strains using a visualized isothermal amplification method.

BACKGROUND: Vibrio alginolyticus is an important pathogen that has to be closely monitored and controlled in the mariculture industry because of its strong pathogenicity, quick onset after infection and high mortality rate in aquatic animals. Fast, simple and specific methods are needed for on-site detection to effectively control outbreaks and prevent economic losses. The detection specificity towards the pathogenic strains has to be emphasized to facilitate pointed treatment and prevention. Polymerase chain reaction (PCR)-based molecular approaches have been developed, but their application is limited due to the requirement of complicated thermal cycling machines and trained personnel. RESULTS: A fast, simple and highly specific detection method for V. alginolyticus pathogenic strains was established based on isothermal recombinase polymerase amplification (RPA) and lateral flow dipsticks (LFD). The method targeted the virulence gene toxR, which is reported to have good coverage for V. alginolyticus pathogenic strains. To ensure the specificity of the method, the primer-probe set of the RPA system was carefully designed to recognize regions in the toxR gene that diverge in different Vibrio species but are conserved in V. alginolyticus pathogenic strains. The primer-probe set was determined after a systematic screening of amplification performance, primer-dimer formation and false positive signals. The RPA-LFD method was confirmed to have high specificity for V. alginolyticus pathogenic strains without any cross reaction with other Vibrio species or other pathogenic bacteria and was able to detect as little as 1 colony forming unit (CFU) per reaction without DNA purification, or 170 fg of genomic DNA, or 6.25 × 103 CFU/25 g in spiked shrimp without any enrichment. The method finishes detection within 30 min at temperatures between 35 °C and 45 °C, and the visual signal on the dipstick can be directly read by the naked eye. In an application simulation, randomly spiked shrimp homogenate samples were 100% accurately detected. CONCLUSIONS: The RPA-LFD method developed in this study is fast, simple, highly specific and does not require complicated equipment. This method is applicable for on-site detection of V. alginolyticus pathogenic strains for the mariculture industry.

Molecular Identification of Vibrio alginolyticus Causing Vibriosis in Shrimp and Its Herbal Remedy.

Penaeus monodon is highly susceptible to vibriosis disease. Aims of the study were to identify the pathogen causing vibriosis in P. monodon through molecular techniques and develop a biocontrol method of the disease by application of herbal extracts. Shrimp samples were collected aseptically from the infected farm and the bacteria were isolated from the infected region of those samples. Based on phenotypic identification, several isolates were identified as Vibrio sp. 16S rRNA gene sequences of the selected isolates exhibited 100% homology with V. alginolyticus strain ATCC 17749. An in vivo infection challenge test was performed by immersion method with V. alginolyticus where these isolates caused high mortality in juvenile shrimp with prominent symptoms of hepatopancreatic necrosis. Antibiogram profile of the isolates was determined against eleven commercial antibiotic discs whereas the isolates were found resistant to multiple antibiotics. A total of twenty-one herbal extracts were screened where Emblica officinalis, Allium sativum, and Syzygium aromaticum strongly inhibited the growth of V. alginolyticus in in vitro conditions. In in vivo conditions, the ethyl acetate extracts of E. officinalis and A. sativum successfully controlled the vibriosis disease in shrimp at a dose of 10 mg/g feed. This is the first report on molecular identification and biocontrol of V. alginolyticus in shrimp in Bangladesh.Penaeus monodon is highly susceptible to vibriosis disease. Aims of the study were to identify the pathogen causing vibriosis in P. monodon through molecular techniques and develop a biocontrol method of the disease by application of herbal extracts. Shrimp samples were collected aseptically from the infected farm and the bacteria were isolated from the infected region of those samples. Based on phenotypic identification, several isolates were identified as Vibrio sp. 16S rRNA gene sequences of the selected isolates exhibited 100% homology with V. alginolyticus strain ATCC 17749. An in vivo infection challenge test was performed by immersion method with V. alginolyticus where these isolates caused high mortality in juvenile shrimp with prominent symptoms of hepatopancreatic necrosis. Antibiogram profile of the isolates was determined against eleven commercial antibiotic discs whereas the isolates were found resistant to multiple antibiotics. A total of twenty-one herbal extracts were screened where Emblica officinalis, Allium sativum, and Syzygium aromaticum strongly inhibited the growth of V. alginolyticus in in vitro conditions. In in vivo conditions, the ethyl acetate extracts of E. officinalis and A. sativum successfully controlled the vibriosis disease in shrimp at a dose of 10 mg/g feed. This is the first report on molecular identification and biocontrol of V. alginolyticus in shrimp in Bangladesh.

Detection of Vibrio anguillarum and Vibrio alginolyticus by Singleplex and Duplex Loop-Mediated Isothermal Amplification (LAMP) Assays Targeted to groEL and fklB Genes

Singleplex and duplex loop-mediated isothermal amplification (LAMP) assays were developed for detecting Vibrio anguillarum, a major bacterial pathogen of fish, and Vibrio alginolyticus, a pathogen of fish and humans, separately and simultaneously from contaminated seawater by targeting the groEL gene of V. anguillarum, which encodes a molecular chaperone protein, and the fklB gene of V. alginolyticus, which encodes a 22 kilodalton (kDa) peptidyl prolyl isomerase. The optimal reaction conditions to produce consistent results were 65°C for 30 min, 63°C for 30 min, and 63°C for 40 min for the groEL (singleplex for V. anguillarum), fklB (singleplex for V. alginolyticus), and groEL + flkB (duplex) LAMP assays, respectively, analyzed via visual detection methods (use of calcein, and SYBR Green I) and agarose gel electrophoresis. The assays were found to be species-specific, as closely related Vibrio spp. were not detected. The limits of detection (LoDs) of the LAMP assays for DNA template from pure culture and artificially contaminated seawater were 10 and 14 fg (groEL assay; for V. anguillarum), 12.5 and 17 fg (fklB assay; for V. alginolyticus), and 50 and 70 fg (duplex assay) per reaction, respectively, which were much better than the LoDs of conventional polymerase chain reaction (PCR). Singleplex and duplex LAMP assays were found to be rapid, species-specific, and sensitive for the detection of V. anguillarum and V. alginolyticus and are applicable to laboratory and field diagnostics

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Development of novel aptamer-based enzyme-linked apta-sorbent assay (ELASA) for rapid detection of mariculture pathogen Vibrio alginolyticus.

As the major opportunistic pathogen to both marine animals and humans, Vibrio alginolyticus (V. alginolyticus) has caused heavy economic losses to mariculture. ssDNA aptamer VA2 targeting live V. alginolyticus was generated by systematic evolution of ligands by exponential enrichment (SELEX) technology in our previous study. In this study, we first developed aptamer (VA2)-based enzyme-linked apta-sorbent assay (VA2-ELASA) for rapid detection of mariculture pathogen V. alginolyticus. The VA2-ELASA could achieve the rapid detection for V. alginolyticus infection with high specificity and sensitivity. The VA2-ELASA could specifically identify V. alginolyticus, but not other non-target bacterial strains. VA2-ELASA could detect V. alginolyticus at the concentration of 5 × 104 /ml, the incubation time short to 1 min and the incubation temperature as high as 45°C, which proved sensitivity and stability of the novel VA2-ELASA in this study. It took less than one hour to accomplish the detection process by VA2-ELASA. The characteristics of specificity, sensitivity and easy operation make VA2-ELASA a novel useful technology for the rapid diagnosis of pathogen V. alginolyticus in mariculture.

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.

Isolation and characterisation of pVa-21, a giant bacteriophage with anti-biofilm potential against Vibrio alginolyticus.

There is an increasing emergence of antibiotic-resistant Vibrio alginolyticus, a zoonotic pathogen that causes mass mortality in aquatic animals and infects humans; therefore, there is a demand for alternatives to antibiotics for the treatment and prevention of infections caused by this pathogen. One possibility is through the exploitation of bacteriophages. In the present study, the novel bacteriophage pVa-21 was classified as Myoviridae and characterised as a candidate biocontrol agent against V. alginolyticus. Its morphology, host range and infectivity, growth characteristics, planktonic or biofilm lytic activity, stability under various conditions, and genome were investigated. Its latent period and burst size were estimated to be approximately 70 min and 58 plaque-forming units/cell, respectively. In addition, phage pVa-21 can inhibit bacterial growth in both the planktonic and biofilm states. Furthermore, phylogenetic and genome analysis revealed that the phage is closely related to the giant phiKZ-like phages and can be classified as a new member of the phiKZ-like bacteriophages that infect bacteria belonging to the family Vibrionaceae.

Isolation and characterization of a virulence related Vibrio alginolyticus strain Wz11 pathogenic to cuttlefish, Sepia pharaonis.

Vibrio alginolyticus is a ubiquitous marine opportunistic pathogen that can infect various hosts in marine environment. In the present study, V. alginolyticus strain Wz11 was isolated from diseased cuttlefish, Sepia pharaonis, with 20% of promoted death and high survival capacity in skin mucus and tissue liquid. Its growth, siderophore production, and expressions of haemolysin and swarming related genes were characterized under iron limited conditions. The minimal inhibitory concentration (MIC) of 2,2'-dipyridyl (DP) to V. alginolyticus strain Wz11 was 640 µM. While growth of V. alginolyticus strain Wz11 was inhibited by DP, production of iron-seizing substances, haemolytic activity and swarming motility were increased. Moreover, expressions of haemolysin related genes tlh, tdh and vah and flagellar related genes flgH, fliC, fliD and fliS were also characterized using real-time reverse transcriptase PCR. Expression of tdh was up-regulated to 7.7-fold, while expressions of tlh and vah were down-regulated to 0.016-fold and 0.03-fold, respectively. The expression of fliC, flgH, fliD and fliS was up-regulated to 4.9-, 3.8-, 8.6- and 4.5-fold, respectively. Concluded from our results suggested that V. alginolyticus strain Wz11 was considered as a potential pathogen of S. pharaonis, and iron level played an important role in the production of iron-seizing substances, and activities of haemolysin and bacterial swarming as well as their related gene expressions.

New Invasive Nemertean Species (Cephalothrix Simula) in England with High Levels of Tetrodotoxin and a Microbiome Linked to Toxin Metabolism.

The marine nemertean Cephalothrix simula originates from the Pacific Ocean but in recent years has been discovered in northern Europe. The species has been associated with high levels of the marine neurotoxin Tetrodotoxin, traditionally associated with Pufferfish Poisoning. This study reports the first discovery of two organisms of C. simula in the UK, showing the geographical extent of this species is wider than originally described. Species identification was initially conducted morphologically, with confirmation by Cox 1 DNA sequencing. 16S gene sequencing enabled the taxonomic assignment of the microbiome, showing the prevalence of a large number of bacterial genera previously associated with TTX production including Alteromonas, Vibrio and Pseudomonas. LC-MS/MS analysis of the nemertean tissue revealed the presence of multiple analogues of TTX, dominated by the parent TTX, with a total toxin concentration quantified at 54 µg TTX per g of tissue. Pseudomonas luteola isolated from C. simula, together with Vibrio alginolyticus from the native nemertean Tubulanus annulatus, were cultured at low temperature and both found to contain TTX. Overall, this paper confirms the high toxicity of a newly discovered invasive nemertean species with links to toxin-producing marine bacteria and the potential risk to human safety. Further work is required to assess the geographical extent and toxicity range of C. simula along the UK coast in order to properly gauge the potential impacts on the environment and human safety.

MALDI-TOF MS as a tool for the identification of Vibrio alginolyticus from Perna perna mussels (Linnaeus, 1758) / MALDI-TOF MS como ferramenta na identificação de Vibrio alginolyticus isolados de mexilhões Perna perna (Linnaeus, 1758)

Vibrio species are ubiquitous in aquatic environments, including coastal and marine habitats. Vibrio alginolyticus is an opportunistic pathogen for fish, crustaceans and mussels and their identification by biochemical tests may be impaired due their nutritional requirements. The study used Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) to identify 49 Vibrio spp. isolates associated with mussels (Perna perna) from different locations along the Rio de Janeiro coast. The rpoA gene was used as a genus-specific marker of Vibrio spp. and was positive in all 209 isolates. MALDI-TOF MS confirmed 87.8% of V. alginolyticus when compared to the results of the biochemical tests. Four isolates were identified as Shewanella putrefaciens (8.16%) and one was identified as V. parahaemolyticus (2.0%). Just one isolate was not identified by this technique (2.0%). The pyrH sequencing confirmed 75% of the proteomic technique results. MALDI-TOF MS is an excellent option for characterization of bacterial species, as it is efficient, fast and easy to apply. In addition, our study confirms its high specificity and sensitivity in these marine bacteria identification.(AU)

Espécies de Vibrio são ubiquitárias em ambientes aquáticos, incluindo habitats costeiros e marinhos. A espécie Vibrio alginolyticus é oportunista para peixes, crustáceos e moluscos e a sua identificação através de testes bioquímicos pode ter a qualidade prejudicada devido às suas exigências nutricionais. O presente estudo utilizou Espectrometria de Massa por Tempo de Vôo de Ionização/Desorção por Laser Assistida por Matriz (MALDI-TOF MS) para identificar diferentes espécies de Vibrio provenientes de mexilhões (Perna perna) coletados em diferentes locais ao longo da costa do Rio de Janeiro. O gene rpoA foi utilizado como um marcador gênero-específico de Vibrio spp. sendo positivo em todos os 209 isolados. MALDI-TOF MS confirmou 87,75% de V. alginolyticus quando comparados com os resultados dos testes bioquímicos. Quatro isolados foram identificados como Shewanella putrefaciens (8,16%), um como V. parahaemolyticus (2,0%) e apenas um (2,0%) não foi identificado pela técnica proteômica. E o sequenciamento do pyrH confirmou 75% dos resultados da técnica proteomica. MALDI-TOF MS tem sido considerada uma excelente opção para a caracterização bacteriana, por ser eficiente, rápida, de fácil aplicação e este estudo confirmou a sua elevada especificidade e sensibilidade na identificação de bactérias marinhas.(AU)

Vibrio alginolyticus 16S-23S intergenic spacer region analysis, and PCR assay for identification of coral pathogenic strain XSBZ03.

Porites andrewsi white syndrome (PAWS), caused by Vibrio alginolyticus strains XSBZ03 and XSBZ14, poses a serious threat to corals in the South China Sea. To obtain a specific target against which to develop a rapid PCR detection method for the coral pathogenic strain XSBZ03, the 16S-23S rRNA gene intergenic spacer (IGS) region of 4 strains of V. alginolyticus, including the XSBZ03 and XSBZ14 strains, was amplified, sequenced and analyzed. Six types of IGS were found: IGS0, IGSG, IGSIA, IGSAG, IGSGLV, and IGSGLAV. IGS0, IGSG, IGSIA, IGSAG and IGSGLV appeared to be the most prevalent forms in the 4 strains and the percentage identity range within each type was 91.4-100%, 89.3-98.5%, 83.0-99.8%, 91.5-95.6%, and 88.7-99.3%, respectively. IGSGLAV was found only in the HN08155 strain, a causative agent of fish disease. IGSGLAV, IGSGLV and IGSAG are reported here for the first time in V. alginolyticus. An IGS sequence specific to the XSBZ03 strain was identified following alignment of the homologous IGSs, and used to design strain-specific primers for its rapid identification by PCR. The results from PCR analysis suggest that the method is a rapid, practical, and reliable tool for the identification of the XSBZ03 strain in samples of isolated bacteria, as well as seawater and coral samples spiked with the bacterial strain. This is the first report of a rapid diagnostic assay for a causative agent of PAWS, based on PCR detection of a coral pathogen at the strain level. After applying this assay in coral transplantation, the survival rates of transplanted corals were significantly increased. This diagnostic assay should aid with both the elucidation of the cause of the disease, and transplantation of PAWS-free P. andrewsi in the South China Sea.

Efficacy of cell free supernatant from Bacillus licheniformis in protecting Artemia salina against Vibrio alginolyticus and Pseudomonas gessardii.

Bacterial diseases are widespread in aquaculture farms and causative agents often adapt to biofilm mode of growth. These biofilms are detrimental to aquaculture species as they resist antibiotics and other agents that are used to control them. Two bacterial pathogens isolated from infected prawn samples were identified as Vibrio alginolyticus and Pseudomonas gessardii on the basis of morphological features, biochemical characteristics, 16S r RNA gene sequencing and phylogenetic analysis. Their pathogenic nature was confirmed by performing in vivo challenge experiments using Artemia salina as a model system. Seven days post infection, the mortality observed with V. alginolyticus and P. gessardii was 97 ±â€¯4.08% and 77.5 ±â€¯5.24%, respectively. The isolates formed extensive biofilms on polystyrene and glass surfaces. These infections could be controlled in an effective manner by using the cell free supernatant (CFS) of a tropical marine epizoic strain of Bacillus licheniformis D1 that is earlier reported to contain an antimicrobial protein (BLDZ1). The CFS inhibited biofilms in an efficient manner (82.35 ±â€¯1.69 and 82.52 ±â€¯1.11% for V. alginolyticus and P. gessardii, respectively) on co-incubation. In addition, pre-formed biofilms of V. alginolyticus and P. gessardii were also removed (84.53 ±â€¯1.26 and 67.08 ±â€¯1.43%, respectively). Fluorescence and scanning electron microscopic studies confirmed the antibiofilm potential of this protein on glass surfaces. The antibiofilm nature was due to the anti-adhesion and antimicrobial properties exhibited by the CFS. Treatment of A. salina with CFS (6 h prior to infections) was effective in protecting larvae against infections by field isolates. This study highlights the significance of marine natural products in providing alternative biofilm controlling agents to tackle infections and decreasing the usage of antibiotics in aquaculture settings.