Pesq. Vet. Bras. 38(8):1511-1517, agosto 2018 DOI: 10.1590/1678-5150-PVB-5233 Original Article Animais de Produção/Livestock Diseases ISSN 0100-736X (Print) ISSN 1678-5150 (Online)

Vet 2651 pvb-5233 LD MALDI-TOF MS as a tool for the identification of alginolyticus from Perna perna mussels (Linnaeus, 1758)1 Greiciane F. Bronzato2 2 2, Bruno R. Pribul3, Dália P. Rodrigues3 2 2 and Miliane M.S. Souza2* , Marcelo S. Oliva , Marisol G. Alvin ABSTRACT.- , Shana M.O. Coelho , Irene S. Coelho MALDI-TOF MS as a tool for the identification of Vibrio alginolyticus Bronzato from Perna G.F., perna Oliva musselsM.S., Alvin (Linnaeus, M.G., Pribul 1758). B.R., PesquisaRodrigues Veterinária D.P., Coelho Brasileira S.M.O., Coelho38(8):1511-1517. I.S. & Souza M.M.S. 2018. Federal Rural do Rio de Janeiro, BR-465 Km 7, Seropédica, RJ 23897-970, Brazil. E-mail: Departamento de Microbiologia e Imunologia Veterinária, Universidade Vibrio [email protected]. Vibrio alginolyticus species are ubiquitous in aquatic environments, including coastal and marine is an opportunistic pathogen for fish, crustaceans and mussels and their identification by biochemicalVibrio tests spp. may isolates be impaired associated due with their mussels nutritional (Perna requirements. perna The study used Matrix-Assisted Laser Desorption/Ionizationrpo Time-of-Flight Mass Spectrometry (MALDI-TOFVibrio MS) to identify 49 ) from differentV. alginolyticus locations along the Rio de Janeiro coast. The A gene was used as a genus-specific marker of Shewanella spp. and putrefaciens was positive in all 209 isolates. MALDI-TOF MSV. parahaemolyticusconfirmed 87.8% of when compared to the results of the biochemical tests. FourpyrH isolates sequencing were identified as (8.16%) and one was identified as (2.0%). Just one isolate was not identified by this technique (2.0%). The 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, INTEX TERMS: MALDI-TOF MS, mitiliculture, mussels. our study confirms its high specificity andVibrio sensitivity alginolyticus, in these Perna marine perna, identification.

RESUMO.- [MALDI-TOF MS como ferramenta na identificação Ionização/Desorção por Laser Assistida por Matriz (MALDI-TOF de Vibrio alginolyticus isolados de mexilhões Perna perna Vibrio (Linnaeus, 1758).] Espécies de Vibrio são ubiquitárias de mexilhões (Perna perna em ambientes aquáticos, incluindo habitats costeiros e longoMS) para da costa identificar do Rio diferentes de Janeiro. espécies O gene rpode provenientes marinhos. A espécie Vibrio alginolyticus é oportunista para ) coletadosVibrio em diferentes locais ao A foi utilizado como de testes bioquímicos pode ter a qualidade prejudicada deum V. marcador alginolyticus gênero-específico quando comparados de com spp. os resultadossendo positivo dos peixes, crustáceos e moluscos e a sua identificação através em todos os 209 isolados. MALDI-TOF MS confirmou 87,75% utilizou Espectrometria de Massa por Tempo de Vôo de Shewanella putrefaciens V. parahaemolyticus devido às suas exigências nutricionais. O presente estudo testes bioquímicos. Quatro isolados foram identificados como proteômica. E o sequenciamento (8,16%), um do como pyr 1 dos(2,0%) resultados e apenas da um técnica (2,0%) proteomica. não foi identificado MALDI-TOF pela MS técnica tem sido considerada uma excelente opção paraH confirmoua caracterização 75% 2 Received on July 20, 2017. FederalAccepted Rural for do publication Rio de Janeiro on September (UFRRJ), 7,BR- 2017. 465 Km 7, Seropédica, RJ Departamento de Microbiologia e Imunologia Veterinária, Universidade author: bacteriana, por ser eficiente, rápida, de fácil aplicação e este 23897-970,3 Brazil. E-mail: [email protected]; *Corresponding estudo confirmou a sua elevada especificidade e sensibilidade [email protected] naTERMOS identificação DE INDEXAÇÃO: de bactérias MALDI-TOF marinhas. MS, militicultura, Vibrio Rio Laboratório de Janeiro, RJde 21045-900,Enterobactérias, Brazil. Departamento de Bacteriologia, Pavilhão alginolyticus, Perna perna, mussels. Rocha Lima, Instituto Oswaldo Cruz, Fiocruz, Av. Brasil 4365, Manguinhos,

1511 1512 Greiciane F. Bronzato et al.

INTRODUCTION Vibrio gene sequencing, yielding unique biochemical fingerprints species are Gram-negative and halophilic bacteria for species sub-typing (Emami et al. 2012). comprising a highly diverse metabolic group of autochthons in Mass spectrometry (MS) is a technique based in the analysis the marine environment (Makino et al. 2003, Haley et al. 2010). of ionized molecules in a gaseous phase. Measurements of Thethe World Health and VibrioOrganization Illness (WHO)Surveillance considers System pathogenic (COVIS) the mass are obtained by measuring the acceleration of Vibrioof the Centers for Disease Control and Prevention (CDC), and ions in an electric field. Since proteins and peptides are not volatile,Penque the2009). generation Lasch et of al.a sufficiently (2009) described high concentration a proteomic of species to be a public healthVibrio threat species and includingannually intacttechnique ionized using molecules whole cells is a to big detect challenge reproducible (Stryer et standards al. 2007, V.reports alginolyticus the number, V. parahaemolyticus of human infections, V. vulnificus during, V. mimicus Vibrio outbreaks.and other Vibrio (vibriosis by pathogenic V. cholerae) of microbial protein in order to support phenogenotypical Vibrio alginolytius species; Cholera due to toxigenic assays. Several reports have shown that MALDI-TOF MS is (Scallan et al. 2011, CDC 2012, WHO 2013,2014). an option for the identification of at distribution. It is considered belongs a ubiquitous to the saprophytic organism microbiotain seawater the Thespecies minimal (Carbonnelle sample preparation, et al. 2007, sampleBarbuddhe acquisition et al. 2008, and of several marine inhabitants and has wide geographical Dieckmann et al. 2008, Ilina et al. 2009, Nagy et al. 2009). and an opportunistic pathogen for fish, shellfish, crustaceans, the speed of the data acquisition combined with its potential coral reefs, mussels and it has also been reported causing for high-throughput sample automation may turn MALDI-TOF otitis and wounds infections in humans (Cavallo & Stabili MS into a valuable effectiveVibrio method alginolyticus, (Bruyne consideringet al. 2011). its 2002, Sganga et al. 2009, Wang et al. 2012, Zhenyu et al. 2013, importanceIn this study, in human we analyzed and animal MALDI-TOF health. MS as a tool for Kong et al. 2015). As reported, the temperature, salinity, reliable identification of pH,Yi et and al. 2008, starvation Yeh et are al. 2010,the primary López-Hernández environmental et al. factors2015). MATERIALS AND METHODS of epidemic vibriosis in Vibrioaquaculture alginolytius (Li & in Chen aquiculture 2008, Sampling. Perna species were collected at three

Outbreaks of infections by Mussels of the have been reported in the United States, Spain, Mexico, Japan different times and places in the state of Rio de Janeiro, totalizing and China (Cavallo & Stabili 2002). seven different sampling being three from the rocks of the Santana V. alginolyticusGlobal warming outbreaks is implicated (Sganga et inal. the2009). increase The pathogenesis of average Archipelago, Macae, two from maricultureFig.1 longline). In each in thecolletion Bay of were Ilha ocean temperature, whichV. alginolyticus favors the incidence of unseasonal Grande, Angra dos Reis and finally, two collections were made on the beach in Arraial do Cabo, Rio de Janeiro ( and epidemiology of infections are under obtained 2 lots containing 25 adult mussels with closed valves and investigation.Yoder et al. 2008). Nonetheless, it is clearly correlated with the rise the same size as used commercially (greater than 6cm). The mussels of seafood consumption (WangVibrio alginolyticus& Chen 2005, to Wang others et al.species, 2008, were washed individually with brush in running drinkingPerna water perna to). such as V. parahaemolyticus, requires additional techniques remove the dirt. During this process were rejected the animals with The close relation of open valves, totalizing two hundred samples of mussels ( The samples were placed in polyethylene boxes and transported at for its differentiation. Also, this species undergoes significant 6-10°C to the Veterinary Bacteriology Laboratory of Federal Rural genetic, physiological and morphological changes due to University of Rio de Janeiro. The samples were properly kept in laboratorial conditions or in nutrient-restrictedVibrio environmentsspecies makes polyethylene boxes. They were identified, placed in an ice chest (Srinivasan & Kjelleberg 1998, Bauer & Rørvik 2007). The great and transported in temperature conditions between 6-10 °C to the variability(Thompson of biochemicalet al. 2004, Tarrcharacteristics et al. 2007). of For these reasons, Veterinary Bacteriology Laboratory of Federal Rural University of it difficult to standardize an accurate phenotypic identification Rio de Janeiro. The preparation of the mussels such as the removal Vibrio of dirt, shell openings, crushing of the body mass, removal of the it is necessary to develop more specific, rapid, and sensitive intravalvarMicrobiological liquid and analysis. inoculation were performed according to the methods for species identification. protocol described by FDA (2004). Different modern molecular technologies that analyses The body mass andVibrio intravalvular alginolyticus liquid biological materials have increased potentials for the detection, were shaked in beaker to held crushing of the solid parts and promotes identification and enumeration of bacterial strains in water, homogenization of the material. Aiming detect food, clinical and environmental samples (Gilbride et al. 2006). a 25-gram sample -2was dilution added in to APW, 225mL used of as alkaline enrichment peptone broth, water and Molecular techniques such as restriction fragment length the(APW) samples containing were incubated 1% NaCl. atSerial 37o dilution of each sample was polymorphism (RFLP), 16S rDNA gene sequencing and performed until 10 pulsed-field gel electrophoresis (PFGE) are discriminating C for 18 hours. After that, they and(Sur informativeet al. 2007). for measuring strain relatedness, diversity 37wereo inoculated in plates containing VibrioThiosulfate alginolyticus Citrate Bileisolates Sucrose was and can profitably be used to trace epidemiological sources agar (TCBS) with the addition of 1, 2 and 3% NaCl and incubated at C for 18 to 24 hours (FDA, 2004). Recently, proteomic technologies have been widely used considered as those that presented yellow colonies characteristic of for research into microbial gene expression and physiology. sucrose fermentation. These isolates were submitted to biochemical Even considering the recent advances in sequencing complete tests for species confirmation. Five to ten colonies from each plate bacterial genomes, proteomic analysis provides information atwere 37 oinoculated into tubes containing nutrient agar, LIA (lysineVibrio spp.iron not available solely from genomic analysis (Van Schaik & agar) and KIA (Kligler iron agar), along with 1% NaCl, and incubated Willems 2010, Cash 2011). This method can complement C for 24 hours for differential diagnosis between Pesq. Vet. Bras. 38(8):1511-1517, agosto 2018 Vibrio alginolyticus Perna perna mussels (Linnaeus, 1758) 1513

MALDI-TOF MS as a tool for the identification of from

Fig.1. Map of the State of Rio de Janeiro, indicating the three regions studied.

®) using molecular Vibrio spp. and members. Additionally, the isolatesVibrio were documentedMALDI-TOF by the MS programtechnique. QuantiOne The samples (BioRad were inoculated in BHI submitted to the cytochrome-oxidase test to distinguish weight markers of 100 bp (Fermentas). from Enterobacteriaceae members (Koneman et al. 2008). species identification was performed through biochemical tests based agar at 37°C for 24h and each culture was transferred to a microplate on exposure to two different concentrations (10µg and 150µg) of (96 MSP, Bruker - Billerica, USA). The bacterial sediment was covered vibriostatic agent O/129 resistance (2,4 diamine-6,7 diisopropilpteridine); by a lysis solution (70% formic acid, Sigma-Aldrich). Futhermore, glucose,production sucrose, of ortho-nitrophenyl-β-galactoside arabinose, lactose, cellobiose, (ONPG); mannitol, production maltose a 1-μL aliquot of matrix solution (alpha-ciano-4-hidroxi‑cinamic of acetoine by using the Voges-Proskauer test; fermentation of sampleacid diluted were ingenerated 50% acetonitrile in a mass andspectrometer 2.5% trifluoracetic (MALDI-TOF acid, LT Sigma‑Aldrich) was added to each sediment. The spectra of each and mannose; presence of lysine and ornithine decarboxylase; presence of arginine dehydrogenase; gelatin hydrolysis; presence wereMicroflex, collected Bruker) in a equippedmass range with between a 337-nm 2,000-20,000m/s, nitrogen laser in and a linear then reductionof halophilia (Sewell in different 2002). VibrioNaCl APW alginolyticus concentrations (0%, 3%, 6%, path, controlled by the FlexControl 3.3 (Bruker) program. The spectra 8% and 10%); motility observation, indole production; and nitrate Genetic analysis. ATCC 17749rpoA was geneused were analyzed by the MALDI Biotyper 3.1 (Bruker) program, using as reference strain. Vibrio the standard configuration for bacteria identification, by which Polymerase chain reaction for the spectrum of the sample is compared to the references in the was carried out to confirm spp. identification. Bacterial database. The results vary on a 0-3 scale, where the highest value DNA extraction was performed by thermal lysis according to means a more precise match and reliable identification. In this lthe-1 method proposed by Dalmasso et al. (2009). The PCR was study,pyr weH sequencing.accepted values for matching greater than or equal to 2 as performed-1 in a final -1volume of 20μl containing 10X buffer (10mmol proposed by the manufacturer. l 2, 0,3mmol l pyr TrisHCl, pH 9.0, 50mM KCl, and 0.1% Triton X-100), 2.5mmol Polymerase Chain Reaction The bacterial DNA was extracted after thermal Korea MgCl South), 0.2mmol of l-1 each primer 5′-AATCAGGCTCGGGCCCT-3′ lysis and the 541 bp H fragment amplification wasV. alginolyticus obtained by and 5′ GCAATTTT(A/G)TC(A/G/T)AC(C/T)GG-3′ (Bioneer, Seoul, technique (PCR) in representative isolates dNTP (Fermentas), 1 U of Taq polymerase to elucidate questions about the characterization of (Fermentas) and 5μl the DNA extract. Amplicons of 242 bp were by validating the results found in both biochemical and proetomic detected by 1.5% agarose gel, stained with SYBR green (Invitrogen) techniques (Thompson et al. 2005). The concentrations used for and examined under a UV transilluminator (UvTrans) and then the reactions were: buffer 10X (10mM Tris-HCl, pH 9.0; 50mM KCl Pesq. Vet. Bras. 38(8):1511-1517, agosto 2018 1514 Greiciane F. Bronzato et al.

2 DISCUSSION and 0.1% Triton X-100), 2.0mM of MgCl , 0.5µM of each primer, The rpo 0.2mM of dNTP (ThermoScience) and 2U of Taq polymerase (Kapa) in a total reaction volume of 20µL with 20ng of the extracted DNA. A gene has been used as phylogenetic markers for The fragments were evaluated by electrophoresis in agarose gel (1.5%) bacterial classification because it is ubiquitous in bacteria, and reveled with the SYBR green dye (Invitrogen), with the image presented as a single copy and due to its resistanceVibrio to lateralgenus, capture system L-PIX EX (Loccus Biotecnologia). The PCR products gene transfer. Moreover, it belongs to the bacterial core genome were purified using an Exo-Sap (USB Corporation, Cleveland, Ohio), as Grimontiaand although, Listonella it is highly, Photobacteria conserved amongand Aeromonas the . (Zeigler recommended by the manufacturer and submitted to the sequencer it shows a good variability against relatedrpo Agenera gene detection such as ABI 3130xl of Applied Biosystems (Biotechnology Laboratory of the Vibrio Genomic Sciences Post-Graduation Program of the Catholic University 2003, Thompson et al. 2005). Therefore the of Brasilia). The sequences were edited using the Bioedit program is considered a specific, sensitive and fast method for (Hall 1999) and Mega version 4.0 (Kumar et al. 2004) and compared identification (Thompson et al. 2005, Dalmasso et al. 2009, with other sequences in the NCBI database (GenBank: http://www. Jeyasekaran et al. 2011). ncbi.nlm.nih.gov/) using the BLAST algorithm (Altschul et al. 1997). BasedVibrio on isolate the findings as Vibrio in alginolyticus the present. Instudy, particular, we could this RESULTS demonstrate that MALDI-TOF was able to identify 87.75% Vibrio ofmicroorganisms. the Hazen et al. (2009) used the MALDI-TOF Vibrio alginolyticus. technique can be used as an Vibrioidentification tool for aquatic From the amount of 209rpo spp. isolates, 49 (23%) V. alginolyticus, V. parahaemolyticus, were phenotypically characterized as V.MS harveyi to compare and spectraV. campbelli of 10 isolates to evaluate the PCR amplification of the A gene tested V.positive alginolyticus in all discriminatory potential for 209 isolates, corroborating the genus phenotypic identification. Böhme et al. (2010) used MALDI-TOF identification, MS to characterizesince these The MALDI‑TOF MS was performed inShewanella all 49 putrefaciens species cannot be distinguished by 16S rRNA gene analysis.Proteus, phenotypic identified and confirmedV. thisparahaemolyticus identification in 87.75% Pseudomonas, Vibrio, Serratia and Shewanella. The authors (n=43). Four isolates were identified as bacterialreported speciesthat this associated technique with was ableseafoods, to distinguish including between (8.16%), one was identified as (2.0%). andisolates one isolatewere submitted was not identified to pyr by this technique (2.0%). marine microbiota. After the proteomic identification, eight representative2 and 39A) closely related species, contributing to the identification of H gene that confirmed the Vibrio species, but not to distinguish results in 75%. In 62,5% (85A, 11C, 6AM, 3MAC V. alginolyticusIn our study, the S.phenotypic putrefaciens protocol employed was able of isolates, the coherence betweenV. alginolyticus the bioquimical sequences tests to differentiate between and MALDI TOF MS techniquepyr Hpresented sequence. the One percentage isolate (6B’) of from , which only happened after maximum identity (100%) with V. alginolyticus, but its MALDI-TOF MS analysis.V. alginolyticus In accordance withV. parahaemolyticus the metabolic pyrdeposited in the NCBI by its V. parahaemolyticus, characteristics of these microorganisms, biochemical tests was identified by phenotypic tests as able of differentiating from isolatesH sequence did not showed presented 100% correlatedidentity to data between the are the sucrose and arabinose fermentation and ornithine corroboringtechniques used with being the MALDIisolate 30,TOF characterized MS technique. as V. Only mimicus two decarboxylation (Koneman et al. 2008, Noguerola & Blanch pyr 2008). Elliot et al. (1995) presented differential characteristics of the species frequently associated with the human byas ShewanellaH sequence putrefaciens and not identified by MALDI TOF MS. Finally, disease related to the consumption of seafood.V. alginolyticus In a table V.one alginolyticus isolate (72B)pyr identifiedH sequence by the(Table proteomic 1). technique demonstratedand V. mimicus .by Shewanella the authors putrefaciens it was possible has been to observe described the showed 100% of identity with differentiation through biochemical tests of by Table 1. Identification of isolates of Vibrio alginolyticus according to phenotypic, proteomic, genotypic and biochemical tests able of differentiating the microorganisms identified by the techniques Biochemical BLASTn Strains MALDI-TOF a

6B’ V. alginolyticus V. parahaemolyticus V. parahaemolyticus NCBI0.0 Biochemical tests responsible for misidentification identification b Sucrose; arabinose fermentation, ornithine 30 V. alginolyticus V. mimicus 0.0 b, descarboxylationONPGc and VP Not identified 0%NaCl, 6%NaCl, 8%NaCl and 10%NaCl growth, VP 72B V. alginolyticus S. putrefaciens V. alginolyticus 0.0 , 10µg O/129 and sucrose Maltose; mannose fermentation and lysine 85A V. alginolyticus V. alginolyticus V. alginolyticus 6e-178 - descarboxylation V. alginolyticus V. alginolyticus V. alginolyticus 0.0 - 6AM V. alginolyticus V. alginolyticus V. alginolyticus 6e-178 - 11C 2 V. alginolyticus V. alginolyticus V. alginolyticus 1e-170 - 39A V. alginolyticus V. alginolyticus V. alginolyticus 0.0 - 3MAC a b VP = Voges-Proskauer, c

All isolates showed maximum identity (100%) for a species previously identified, ONPG = o-nitrophenyl-beta-D- galactopyranoside.

Pesq. Vet. Bras. 38(8):1511-1517, agosto 2018 Vibrio alginolyticus Perna perna mussels (Linnaeus, 1758) 1515

MALDI-TOF MS as a tool for the identification of from CONCLUSIONS as an important bacterium in the degradation of organic Vibrio alginolyticus Kalawat 2010). and need sodium compounds, especiallyV. alginolyticus concerningS. putrefaciens aquatic animals found PernaThe Perna study mussel. showed the effectiveness of the MALDI-TOF in foods, sewageS. putrefaciensand estuarine/marine water (Sharma & technique for evaluation of isolated from for growth, but can grow in NaCl-free media As laboratories adopt MALDI-TOF mass spectrometry (Teixeira 2009). Maltose and mannoseTable 1). fermentation along for routine bacterial identification, it is important to define with lysine decarboxylationS. putrefaciens are the biochemical assays able whether or not the technology is useful for identification to differentiate these species ( of specific species. The identification of environmental As mentioned above, was only identified by MALDI-TOF MS, corroborating the need to employ more precise bacteria is difficult due to several factors, such as salinity, pH, techniques in the analysis of aquatic microbiota. MALDI-TOF temperature and metabolic diversity. In the present study, MShealth. proved Since to 46be isolatesa reliable obtained and useful a score tool tohigher understand than 2, thewe the results of the MALDI-TOFV. MSalginolyticus technique corresponded to marineconsidered environment this a reliable and technique.its impact on animal and public related87.75% species with the V. resultsparahaemolyticus of the phenotypic. test, being very efficientThe pyr to distinguishing from the closely

Bruker Daltonics Inc. provides a large Biotyper TM reference H gene sequencing confirmed the proteome database for species classification. Each database entry contains results in 75%, validating this technique as a high potential a main spectrum generated from at least 20 summarized for routine automated analysis, allowing the identification of and processed single spectra of individual bacterial strains. isolates from clinical and environmental sources on a large This database was mainly generated for medical laboratory scale. It is also a quick and easy method with high specificity Conflict of interest statement. diagnostics because the main spectra largely correspond and sensitivity. tostrains, infective which bacterial has led species to limited isolated ecological from studiesclinical basedsamples. on Acknowledgements. - The authors have no competing interests. Therefore,wholecell MALDI-TOF this database MS-data lacks (Erlercoverage et al. of 2015). environmental pyr - The authors are grateful to Larissa A.B. Botelho, Beatriz M. Moreira and the Coordination of Improvement of Higher Education In this study H sequencing confirmed the proteomic Personnel (CAPES) for financial and technical support of this study. results in 75%. MALDI-TOF MS and DNA sequence-based REFERENCES species classifications are comparable (Alatoom et al. 2011, AeromonasRezzonico et al. 2010, Verroken et gyral. 2010), for instance Benagli et al. (2012) validated 93% of MALDI-TOF MS-based Alatoom A.A., Cunningham S.A., Ihde S.M., Mandrekar J. & Patel R. 2011. Comparison of direct colony method versus extraction method for species classifications by B sequence analysis; identification of gram-positive cocci by use of Bruker Biotyper matrix- considering that the remaining 7% were not identifiedgyrB sequencing because assisted laser desorption ionization-time of flight mass spectrometry. J. ofand missing the MALDI-TOF reference MS spectra. technique These produce results similar corroborate results also and Clin. Microbiol. 49(8):2868-2873. Altschul S.F., Madden T.L., Schäffer A.A., Zhang J., Zhang Z., Miller W. & Lipman D.J. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein for evaluating the sensitivity and specificity of thepyr phenotypic database search programs. Nucleic Acids Res. 25(17):3389-3402.

Vibrio Carbonnelle E., Beretti J.L., Cottyn S., Quesne G., Berche P., Nassif X. & Ferroni A. Hazenparahaemolyticus T.H., Martinez R.J., Chen Y., Lafon P.C., Garrett N.M., Parsons M.B., 2007. Rapid identification of staphylococci isolated in clinical microbiology Bopp C.A., Sullards M.C. & Sobecky P.A. 2009. Rapid identification of laboratories by Matrix-Assisted Laser Desorption Ionization-Time of Flight by whole-cell Matrix-Assisted Laser Desorption ionization- Mass Spectrometry. J. Clin. Microbiol. 45(7):2156-2161. 6756. Cash P. 2011. Investigating pathogen biology at the level of proteome. Proteomics Ilina E.N., Borovskaya A.D., Malakhova M.M., Vereshchagin V.A., Kubanova 11(15):3190-3202. A.A., Kruglov A.N., Svistunova T.S., Gazarian A.O., Maier T., Kostrzewa M. Vibrio in seawater and Mytilus & Govorun V.M.Neisseria. 2009. Direct bacterial profiling by Matrix-Assisted Laser galloprovincialis Desorption-Ionization Time-of-Flight Mass Spectrometry for identification Cavallo R.A. & Stabili L. 2002. Presence of of pathogenic J. Molecular Diagnostics 11(1):75-86. Water Research 36(15):3719-3726. Jeyasekaran G., Thirumalai Raj K., Jeya ShakilaVibrio R., cholerae Jemila Thangarani A. & Sukumar D. 2011. Multiplex polymerase chain reaction-based assay for CDC 2012. National Cholera and Vibriosis Surveillance: Cholera and other the specific detection of toxin-producing in fish and fishery Vibrio llness Surveillance (COVIS) Annual Summary. Centers for Disease products. Appl. Microbiol. Biotechnol. 90(3):1111-1118. nationalsurveillance/choleravibrio-surveillance.html> Access on July Koneman6th ed. E.,Lippincott Winn W.J., Williams Allen S., Janda and Wilkins, W., Procop Philadelphia, G., Schreckenberger p.387-422. P. & Woods G. 2008. Koneman’s Color Atlas and Text Book of Diagnostic Microbiology. Chimetto L.A., BrocchiMussismulia M., Thompson hispida. C.C., Martins R.C.R., Ramos H.B. & Thompson F.L. 2008. Vibrios dominate as culturable nitrogen-fixing bacteria of Brazilian coral System. Appl. Microbiol. 31(4):312- Kong W., Huang L.X., Su Y.Q.,Vibrio Qin alginolyticus Y.X., Ma Y., Xu X.J., Lin M., Zheng J. & Yan 319. Q. 2015. Investigation of possible molecular mechanisms underlying the regulation of adhesion in with comparative transcriptome Dalmasso A., Civera T. & Bottero M.T. 2009. Multiplex primer-extension analysis. Antonie van Leeuwenhoek 107(5):1197-1206. 129(1):21-25. Kumar S., Tamura K. & Nei M. 2004. MEGA3: integrated software for molecular Vibrio evolutionary genetics analysis and sequence alignment. Briefings in Dieckmann R., Strauch E. & Alter T. 2010. Rapid identification and characterization Bioinformatics 5(2):150-163. of species using whole-cell MALDI-TOF mass spectrometry. J. Appl. Microbiol. 109(1):199-211. Bacillus anthracis Lasch P., Beyer W., Nattermann H., Stammler M., Siegbrecht E., Grunow R. Dieckmann R., Helmuth R., Erhard M. & Malorny B. 2008. Rapid classification & Naumann D. 2009. Identification of by using Matrix- and identification of Salmonellae at the species and subspecies levels by Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry whole-cell matrix-assisted laser desorption ionization-time of flight mass and Artificial Neural Networks. Appl. Environ. Microbiol. 75(22):7229- spectrometry. Appl. Environ. Microbiol. 74(24):7767-7778. Litopenaeus org/10.1128/AEM.01402-08> , vannamei Vibrio alginolyticus under low and V. parahaemolyticus, V. vulnificus and other Vibrio spp., p.9.01-9.27. In: Li C.C. & Chen J.C. 2008. The immune response of white shrimp Elliot E.L., Kaysner C.A., Jackson L. & Tamplin M.L. 1995. and its susceptibility to high pH stress. Fish Shellfish Immunol. 25(6):701-709. K.M., Pardío-Sedas V.T., Lizárraga-Partida L., Williams FDA Bacteriological Analytical Manual. 8th ed. AOAC International,

totalJ.D.J., Martínez-Herreraand pathogenic Vibrio D., Flores-Primo parahaemolyticus A., Uscanga-Serrano densities in R. Crassostrea & Rendón- Emami K., Askari V., Ullrich M., Mohinudeen K., Anil A.C., Khandeparker L., virginicaCastro K. 2015. Environmental parameters influence on the dynamics of Burgess J.G. & Mesbahi E. 2012. characterization of bacteria in ballast water using MALDI-TOF Mass Spectrometry. PLoS One 7(6):e38515. harvested from Mexico’s Gulf coast. Marine Pollution Bulletin 91(1):317-329. ErlerVibrio R., Wichels A., Heinemeyer E.A., Hauk G., Hippelein M., Reyes N.T. & Y., Najima M., Nakano M., Yamashita A., Kubota Y., Kimura S., Yasunaga Gerdts G. 2015. VibrioBase: a MALDI-TOF MS database for fast identification Makino K., Oshima K., Kurokawa K., Yokoyama K., Uda T., Tagomori K., Iijima of spp. that are potentially pathogenic in humans. System. Appl. Vibrio parahaemolyticus Microbiol. 38(1):16-25. T., V.Honda cholerae. T., Shinagawa H., Hattori M. & Iida T. 2003. Genome sequence of : a pathogenic mechanism distinct from that of Lancet 361(9359):743-749. Maryland. Available at Access on July 20, 2017 Nagy E., Maier T., Urban E., Terhes G. & Kostrzewa M. 2009. Species identification wastewater: understanding microbial communities, detecting pathogens of clinical isolates of Bacteroides by matrix-assisted laser-desorption⁄ionization Gilbride K.A., Lee D.Y. & Beaudette L.A. 2006. Molecular techniques in time-of-flight mass spectrometry. Clin. Microbiol. Infection 15(8):796-802. Vibrio and real-time process control. J. Microbiol. Methods 66(1):1-20. Noguerola I. & Blanch A.R. 2008. Identification of spp. with a set of dichotomous keys. J. Appl. Microbiol. 105(1):175-185. D.C., ChallacombeV. J.F., cholerae. Detter J.C., Han C.S., Huq A. & Colwell R.R. 2010. Comparative genomic analysis reveals evidence of two novel Penque D. 2009. Two-dimensional gel electrophoresis and mass spectrometry closely related to BMC Microbiol. 10(1):154. . Hall T.A. 1999. BioEdit: a user-friendly biological sequence alignment editor and Rezzonico F., Vogel G., Duffy B. & Tonolla M. 2010. Application of whole-cell analysis program for Windows 95/98/NT Nucl. Acids, Symp. Ser. 41:95-98. matrix-assisted laser desorption ionization-time of flight mass spectrometry Pesq. Vet. Bras. 38(8):1511-1517, agosto 2018 Vibrio alginolyticus Perna perna mussels (Linnaeus, 1758) 1517

MALDI-TOF MS as a tool for the identification of from

for rapid identification and clustering analysis of Pantoea species. Appl. Microbiol. 71(9):5107-5115. RodriguesAEM.03112-09> N.M., Bronzato G.F., Santiago G.S., Botelho L.A., Moreira B.M., Van Schaik W. & Willems R.J.L. 2010. Genome-based insights into the evolution of enterococci. Clin. Microbiol. Infect. 16(6):527-532. Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) identification biochemical tests: a study with enterobacteria from Verroken A., Janssens M., Berhin C., Bogaerts P., Huang T.D., WautersNocardia G. a dairy cattle environment. Braz. J. Microbiol. 48(1):132-138. ionization-time of flight mass spectrometry for identification of species. J. Clin. Microbiol. 48(11):4015-4021. Litopenaeus Jones J.L. & Griffin P.M. 2011. Foodborne illness acquired in the united vannamei Vibrio alginolyticus states major pathogens. Emerg. Infect. Dis. 17(1):7-15. and its susceptibility to at different salinity levels. Fish Shellfish Immunol. 18(4):269-278. (Eds), Clinical Laboratory Medicine. 2nd ed. Lippincott Willians and Vibrio alginolyticus: their roles in Wang Q., Liu Q., Cao X., Yang M. & Zhang Y. 2008. Characterization of two Vibrio alginolyticus TonB systems in marine fish pathogen Sganga G., Cozza V., Spanu T., Spada P.L. & Fadda G. 2009. Global climate change iron utilization and virulence. Archs Microbiol. 190(5):595-603. Vibrio alginolyticus rpoS in a healthy man. Ostomy Wound Manage. 55(4):60-62.Shewanella Wang S.X., Wei J.T. & Li T.B. 2012. Susceptibility of a Sharma K.K. & Kalawat U. 2010. Emerging infections: -a series of mutant to environmental stresses and its expression of OMPs. J. Basic five cases. J. Lab. Physicians 2(2):61-65. 2727.72150> Vibrio. J. Biosciences 23(4):501-511. Srinivasan S. & Kjelleberg S. 1998. Cycles of famine and feast: the starvation WHO 2013/2014. Cholera. Wkly Epidemiol. Rec. 89:345-356. Available at and outgrowth strategies of a marine Access on July 20, 2017. Litopenaeus vannamei that had been immersed in the Yeh S.T., Li C.C., Tsui W.C.,Gracilaria Lin Y.C. & tenuistipitata Chen J.C. 2010. and The subjected protective to combinedimmunity Stryer L., Tymoczko J.L. & Berg J.M. 2007. Biochemistry. 6th ed. W.H. Freeman of white shrimpVibrio alginolyticus and Company, USA. 1026p. hot-water extract of Sur D., Dutta S., Sarkar B.L., Manna B., Bhattacharya M.K., Datta K.K., Saha A., stresses of injection and temperature change. Fis Shellfish Dutta B., Pazhani G.P., Choudhuri A.R. & Bhattacharya S.K. 2007. Occurrence, Immunol. 29(2):271-278. significance & molecular epidemiology of cholera outbreaks in West Bengal. in natural seawater. Acta Indian J. Med. Res. 125(6):772-776. Vibrio alginolyticus Vibrio rpoB Yi OceanologicaJ.B., Chen Q., Zou Sinica W.Z., 27:120-129. Yan Q.P., Zhuang Z.X. & Wang X.R. 2008. Starvation Tarr C.L., Patel J.S., Puhr N.D., Sowers E.G., Bopp C.A. & Strockbine N.A.A. effects on pathogenic 2007. Identification of isolates by a multiplex PCR assay and sequence determination. J. Clin. Microbiol. 45(1):134-140. disease and outbreaks associated with recreational water use and other N.T., Calderon R.L., Roy S.L. & Beach M.J. 2008. Surveillance for waterborne Teixeira C.E. 2009. EvaluationO. niloticus of the combined) cold atmosphere. effect of theDoctoral processes Thesis, of irradiation and modified in bacteriological, physico-chemical and sensory aquatic facility-associated health events-United States, 2005/2006. MMWR quality of the file tilapia ( Surveillance Summary 57(9):1-29. Federal Fluminense University, Niterói. Vibrios. Microbiol. Molec. Zeigler D.R. 2003. Gene sequences useful for predicting relatedness of whole Thompson F.L., Iida T. & Swings J. 2004. Biodiversity of genomes in bacteria. Int. J. Syst. Evol. Microbiol. 53(Pt 6):1893-1900. Biol. Rev. 68(3):403-431. 431.2004> Vibrio alginolyticus Porites Zhenyuandrewsi X., Shaowen K., Chaoqun H., Zhixiong Z., Shifeng W. & Yongcan Z. 2013. Thompson F.L., Gevers D., Thompson C.C., Dawyndt P., Naser S., Hoste B., First characterization of bacterial pathogen, , for Munn C.B. & Swings J. 2005. Phylogeny and molecular identification white syndrome in the South China Sea. PLoS One 8(9):e75425. of vibrios on the basis of multilocus sequence analysis. Appl. Environ.

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