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)

Cloning and expression of alginate lyase genes from Vibrio alginolyticus and characterization of the alginate lyase / 生物工程学报

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 Fe²⁺, Fe³⁺ ions promoted enzyme activity. SDS and Cu²⁺ 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.

Aislamiento y caracterización del bacteriófago Va1 específico a Vibrio alginolyticus

Vibrio alginolyticus está asociado a enfermedades en acuicultura. El uso indiscriminado de antibióticos ha conllevado a la búsqueda de alternativas en el tratamiento de enfermedades bacterianas, entre ellas la aplicación de bacteriófagos, los cuales infectan y destruyen selectivamente bacterias. En ese sentido, en este trabajo se aisló un bacteriófago altamente lítico a V. alginolyticus el cual fue denominado Va1, con el objetivo de evaluar los parámetros físicos químicos en los cuales es viable. Para esto, se evaluó al bacteriófago Va1 en diferentes condiciones de pH, temperatura, cloroformo. El fago Va1 presenta mayores títulos a 20 y 30 °C y pH de 5 a 8 disminuyendo su viabilidad a partir de 40 °C y en unidades de pH menores de 5. La exposición al cloroformo redujo la viabilidad del fago Va1 en un 25%. A partir de la curva de un paso se determinó que el periodo de latencia y el tamaño de la explosión fueron de 20 minutos y 192 UFP/centro infectivo respectivamente. Al microscopio electrónico de transmisión el fago Va1 evidencio una cabeza icosaedrica y una cola no contráctil, características propias de la familia Podoviridae. En conclusión, el fago Va1 presenta características potenciales para su uso en fagoterapia

Vibrio alginolyticus is associated with diseases in aquaculture. The misuse of antibiotics has led to the search for alternatives in the treatment of bacterial diseases, among them the application of bacteriophages that infect and destroy bacteria selectively. In this way, a highly lytic V. alginolyticus bacteriophage, termed Va1, was isolated, with the aim to evaluate its physical chemical parameters. For this purpose, different temperature, pH, chloroform exposure and host range conditions were evaluated. The temperature stability of phage Va1 showed higher titers at 20 and 30 °C decreasing from 40 °C. With respect to pH, the highest titers for the bacteriophage were between 5 and 8, and chloroform exposure reduced viability of the Va1 phage by 25%. The one-step curve determined that the latency period and the burst size were 20 minutes and 192 PFU / infective center respectively. Under the transmission electron microscope, the Va1 phage showed an icosahedral head and a non-contractile tail, belonging to the Podoviridae family. In conclusion, Va1 phage presents potential characteristics for use in phage therapy

The changes of inflammatory factors in mice infected with Vibrio alginolyticus / 国际检验医学杂志

Objective To investigate the changes of inflammatory factors in mice infected with Vibrio alginolyticus .Methods Established a V .alginolyticus ATCC17749 T‐infected mouse model through intraperitoneal injection ,then performed the median le‐thal dose experiment ,the detection of hematological and liver function indicators ,histopathologic evaluation and inflammatory cyto‐kine detection .Results The median lethal dose of V .alginolyticus ATCC17749T was 1 × 109 CFU by intraperitoneal injection .The dose of 1 × 109 CFU per mouse was adopted in the following study .WBC and platelet numbers significantly decreased after infection (P<0 .05) ,RBC number and hemoglobin content significantly increased(P<0 .05) and ALT ,AST significantly increased after V . alginolyticus infection(P<0 .05);light microscopy observation revealed that ,hepatic cell and pulmonary tissue suffered serious inju‐ry after infection.Through the detection of antibody array ,the levels of 20 inflammatory factors were found changed significantly . 8 of them including KC ,IL‐6 ,RNATES ,IL‐12 ,Eotaxin ,G‐CSF ,MIP‐1a ,Mig ,the expression of which increased more than 10 times .Conclusion The inflammatory factors analysed in the paper provide a theoretical basis for the study of the inflammatory mechanism of Vibrio alginolyticus infection .

Molecular variations in Vibrio alginolyticus and V. harveyi in shrimp-farming systems upon stress

A study was performed to investigate the genomic variations in the shrimp farm isolates of Vibrio alginolyticus and V. harveyi when the isolates were subjected to environmental stress. Samples of shrimps, water and sediment were collected from Southern Indian coastal shrimp farms. Vibrio isolates were biochemically identified and confirmed using 16S rDNA and gyrB gene specific PCR. The bacterial strains were genotyped by PCR fingerprinting using GTG(5) and IS (Insertion Sequence) primers. Seven strains each of V. alginolyticus and V. harveyi were subjected to 10 passages through trypticase soya broth (TSB), which contained different NaCl concentrations (3, 6 and 8%) and trypticase soya agar (TSA). V. alginolyticus was also passaged through TSB with a 12% NaCl concentration. PCR fingerprinting, which was performed on the strains that were passaged through different salt concentrations, confirmed that V. alginolyticus and V. harveyi could affect the genomic variations, depending on the environmental conditions of the culture. The study highlights the complex genotypic variations that occur in Vibrio strains of tropical aquatic environment because of varied environmental conditions, which result in genetic divergence and/or probable convergence. Such genetic divergence and/or convergence can lead to the organismal adaptive variation, which results in their ability to cause a productive infection in aquatic organisms or generation of new strains.

Distribution of Vibrio alginolyticus-like species in Shenzhen coastal waters, China

We investigated the distribution of vibrios in Shenzhen coastal waters in order to obtain valuable information for the aquaculture industry and a health warning system. Quantities of vibrios from surface waters ranged from 0 to 4.40×10(4) CFUs mL-1 in April (spring), while from 0 to 2.57×10³ CFUs mL-1 in September (autumn); the abundance of V. alginolyticus-like species from surface water ranged from 0 to 6.72×10³ CFUs mL-1 in April (spring) and from 0 to 1.28×10³ CFUs mL-1 in September (autumn); higher counts were observed in spring. The V. alginolyticus-like species was dominant in Shenzhen coastal waters, with the highest abundance in the clean region (stations YMK001 and GDN064) in April, suggesting that Vibrio spp. were naturally occurring bacteria in marine environments. The correlation between the abundance of vibrios (including V. alginolyticus-like species) and environmental factors varied in different regions and different seasons. There were no vibrios detected when the salinity was less than 11.15ë in the Zhujiang River estuary, which indicated that salinity played a key role in the distribution of vibrios and V. alginolyticus-like species.

Enzymatic characterization of Vibrio alginolyticus strains isolated from bivalves harvested at Venice Lagoon (Italy) and Guanabara Bay (Brazil) / Caracterização enzimática de cepas de Vibrio alginolyticus isoladas de bivalves coletados na Lagoa de Veneza (Itália) e Baía de Guanabara (Brasil)

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 percent of sodium chloride (NaCl) and APW plus 3 percent NaCl incubated at 37 ºC for 18-24h. 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 percent), V. harveyi (19 percent) and V. parahaemolyticus (7.6 percent), 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.

O ecossistema aquático é o habitat natural de microrganismos incluindo aqueles dos gêneros Vibrio e Aeromonas os quais são patogênicos para o homem e animais. Na presente investigação foi avaliada a freqüência destas bactérias e a característica enzimática de 34 cepas de Vibrio alginolyticus isoladas de bivalves coletados na Lagoa de Venice (Itália) e Baía de Guanabara (Brasil) durante o período de Novembro-2003 a Fevereiro-2004. As amostras de mexilhões foram submetidas a enriquecimento em Água Peptonada Alcalina (APA) adicionada de 1 por cento de Cloreto de Sódio (NaCl) e APA com 3 por cento de NaCl (37 ºC/18-24h). Em seguida as amostras foram semeadas em Agar TCBS (Agar Tiossulfato Citrato Bile Sacarose) e as colônias suspeitas foram submetidas à caracterização bioquímica. As cepas de Vibrio alginolyticus foram avaliadas quanto à produção das enzimas colagenase, elastase e condroitinase. Os resultados demonstraram o isolamento de 127 microrganismos assim distribuídos: 105 cepas de Vibrio das quais V. alginolyticus (32,4 por cento), V. harveyi (19 por cento) e V. parahaemolyticus (7,6 por cento), 20 cepas de Aeromonas e 2 Plesiomonas shigelloides foram os principais patógenos isolados. Observou-se a produção das três enzimas a partir de V. alginolyticus, consideradas principais fatores de virulência da bactéria, em especial em casos de infecção dermatológica humana.

Septic Shock due to Vibrio alginolyticus in a Cirrhotic Patient: The First Case in Korea

We describe a case of septic shock due to Vibrio alginolyticus presenting with fever and bilateral leg pain. Despite intensive management with antibiotics and inotropic agents, the patient died from septic shock 1 day after hospitalization. V. alginolyticus was isolated from both leg wounds and a blood culture. To the best of our knowledge, this is the first reported case of V. alginolyticus bacteremia in Korea.

Characterization of AcrA gene from Vibrio alginolyticus Strain HY9901 / 微生物学通报

A 460 bp internal fragment of the AcrA gene from Vibrio alginolyticus strain HY9901 was amplified by PCR with designed primers and the unknown flanking sequence of 5 '- and 3 '- ends of the AcrA gene was finally characterized by inverse PCR and nested PCR. Sequence analysis showed the AcrA gene contained 1101 bp ORF encoding 366 amino acids and the deduced amino acid sequence of the precursor from Vibrio alginolyticus strain HY9901 showed significant homology with the putative protein of other Vibrio species. The AcrA shows 76%, 73%, 71% and 70% homology with V.vulnificus strain YJ016, V. parahaemolyticus strain RIMD 2210633, V. splendidus strain 12B01 and V. cholerae O1 biovar eltor str. N16961 respectively.

Misidentification as Vibrio alginolyticus with Vitek GNI+ Card in Three Cases / 대한임상미생물학회지

The introduction of a new, fully automated system into the clinical microbiology laboratory contributes to a rapid identification of microorganisms with accurate and reliable results, but such a system requires a high cost and additional tests for identification of some species. For instance, additional tests on oxidase, indole, motility, hemolysis, and pigmentation are needed in the correct identification by using Vitek GNI+ system (bioMerieux Vitek Inc., MO, USA). In particular, Vibrio and Aeromonas species are occasionally identified incorrectly when an automated system is used, and thus conventional biochemical tests may be more reliable in the identification of such species. We experienced three cases of incorrect identification of Vibrio parahaemolyticus, Vibrio cholerae, and Aeromonas veronii biovar sobria as Vibrio alginolyticus by using Vitek GNI+ card.

Construction and expression of single chain variable Fv of anti-idiotype monoclonal antibody against Vibrio alginolyticus / 中国免疫学杂志

Objective:To clone the single chain variable Fv of anti-idiotype monoclonal antibody against vibrio alginolyticus.Methods:Total RNA was extracted from hybridoma cell 2F4 secreting MAb against vibrio alginolyticus and cDNA was amplified by retropolymerase chain reaction(RT-PCR),the expression vector pTAT-AL1 was constructed for the recombinant V_H-V_L expression.The transformed E.coli BL21 cells were propagated and induced by IPTG.Results:The V_H gene contained 369 base pairs and encoded 123 amine acid residues;The V_L gene contained 339 base pairs and encoded 113 amine acid residues;There were four FRs three CDRs and two characteristic cysteine residues in the V_H and V_L gene,respectively.ELISA results showed the ScFv retained almost the same antigen affinity and specificity as its parent monoclonal anitbody.Conclusion:The single chain variable Fv of anti-idiotype monoclonal antibody against vibrio alginolyticus was constructed successfully and expression products was found in the periplasmic space and inclusion bodies.This ScFv might be a new generation of gene engineering vaccine of the anti-idiotype monoclonal antibody against vibrio alginolyticus in fishery.

A Case of Vibrio alginolyticus Isolated from Otorrhea of Chronic Otitis Media / 감염

Vibrio alginolyticus is a microorganism of marine environment that occasionally occurs as a human pathogen. We isolated V. alginolyticus from a patient with otitis media. A 37-year-old man had been exposed to seawater one month before admission. The isolate showed typical biochemical and characteristics of this organism such as positive Voges-Proskauer reaction, fermentation of sucrose, growth on 10% sodium chloride media. In vitro susceptibility test shows the isolate was resistant to ampicillin and carbenicillin, but was susceptible to other antimicrobial agents. The patient improved with ciprofloxacin and ofloxacin therapy.

Enumeração e identificação de víbrios sacarose-positivos em amostras de lulas frescas comercializadas no município de Niterói-RJ / Enumeration and identification of sucrose-positive vibrios in samples of fresh squids commercialized at Niterói county-RJ

Foi efetuado um estudo sobre a enumeração e a identificação de víbrios sacarose-positivos em lulas frescas obtidas no comércio varejista do município de Niterói-RJ. No experimento, em 50 amostras de lulas, identificadas como pertencentes à espécie Oorytheutis brasiliensis Blainville, 1823, foram isoladas Unidades Formadoras de Colônias (UFC) sacarose-positivas em 28 (56%) amostras. Destas, foram identificados o Vibrio a/ginolyticus e víbrios do grupo NAG (não aglutináveis). A média dos Números Mais Prováveis (NMP) dos víbrios totais (com exceção da amostra com NMP > 2400 bacts./g) foi 101,70 bacts./g. Do total de 29 isolamentos nas 28 amostras, o V alginolyticus do grupo I de Heiberg obteve o maior percentual (62,07%), o V alginolyticus do grupo 111 apenas 10,30% e os víbrios NAG com 27,63%.

From 50 samples of squids of lhe species Dorytheutis brasiliensis Blainville, 1823, Colonies Formed Units (CFUs) sucrose-positives Víbrio alginolyticus and NAG vibrios (non-agglutinable) were isolated. The average of the Most Probabble Number of total vibrios (except the sample with MPN > 2400 bacts./g) was 101,70 bacts./g. From 28 samples, 29 isolations were carried out and lhe V. alginolyticus group I of Heiberg's Classification showed the greatest percentage (62,07%). The V. alginolyticus group 111 showed only 10,30% and NAG vibrios 27,63%.

Growth,Siderophore Production and Outer Membrane Protein Expression of Vibrio alginolyticus by Iron Regulation / 微生物学通报

Iron uptake mechanism of Vibrio alginolyticus was primarily investigated. V.alginolyticus could survive in the medium with high-concentration iron chelator. The strain of V. alginolyticus isolated from diseased fish produced more siderophore than that from marine environment. The extract of siderophore from V. alginolyticus could stimulate the growth of Escherichia coli mutant AN93. Under iron limitation,the growth rate was decreased and several outer membrane proteins were induced. Adding iron into the iron-limited medium the normal growth could be recovered.

THE STUDY OF PATHOGENIC BACTERIA AND WHOLE CELL VACCINE ON SPARUS LATUS VIBRIO DISEASE / 微生物学通报

Physiological and biochemical characteristics,drug sensitivity of pathogenic bacteria isolated from the diseased Sparus latus in Zhanjiang,Guangdong,and inactivated whole cell vaccine were studied.The results show that the isolated strain is Vibrio alginolyticus and is highly sensitive to tetracycline,gentamycin,chloramphenicol,and so on.But it is not sensitive to erythromycin and penicillin K.It also shows that the pathogen is inactivated more easily at pH6.0 than at pH8.4 when formalin is used.The whole cell vaccine can strengthen Sparus latus's immuno function and lessen their death rate when fish are infected by the Vibrio alginolyticus.