In vitro resistance of Enterobacter cloacae isolated from fresh seafood to colistin

Abstract INTRODUCTION: Enterobacter cloacae is a clinically important bacterium from the Enterobacteriaceae family. This study evaluated resistance of E. cloacae strains from fish (n=14) and shrimp (n=9) to colistin. METHODS: Biochemical identification and antimicrobial susceptibility tests were carried out in an automated Vitek®2 instrument. RESULTS: Colistin resistance was observed in 21.4% and 66.7% of the strains from fish and shrimp, respectively. We observed minimum inhibitory concentrations of ≥16 mg/L and ≤5 mg/L in 8 and 15 of all strains, respectively. CONCLUSIONS: Fish and shrimp can carry drug-resistant enterobacteria, which can be of clinical interest.

Antibacterial and cytotoxicity activity in macroalgae extracts: perspectives for the use against pathogenic bacteria from shrimp farms (Litopenaeus vannamei) / Atividade antibacteriana e citotóxica em extratos de macroalgas: perspectivas de uso contra bactérias patogênicas de ambiente de cultivo de camarões (Litopenaeus vannamei)

Some infections caused by pathogenic microorganisms might shows high prevalence in farmed shrimp environments, compromising production and causing economic losses. Therefore, the search for compounds with antibiotic activity has become intensive, following the record of new antimicrobial-resistant bacteria. The study of those bioactive compounds in marine macroalgae has produced satisfactory results, such as the discovery of antibacterial activity against multiresistant strains. Accordingly, this study aims to research antibiotic activity in macroalgae extracts of Chlorophyta, Phaeophyta and Rhodophyta found in the coast of Ceará and also to evaluate the cytotoxicity activity against bacterial strains (Vibrio sp.) from shrimp farms (Litopenaeus vannamei). The extracts cytotoxicity was also evaluated. The results prove that there was antibacterial activity in ethanolic, acetonic, hexanic and methanolic extracts against bacterial strains of Vibrio with multiple resistance profile as well as displaying low cytotoxicity.

Algumas infecções causadas por micro-organismos patogênicos podem apresentar alta prevalência em ambientes de cultivo de camarões marinhos, comprometendo a produção e causando prejuízos econômicos aos aquicultores. Assim, tem-se tornado intensa a busca por compostos com atividade antibiótica pelo registro cada vez mais frequente de bactérias com perfil de resistência a antimicrobianos. A presença desses compostos com bioatividade em macroalgas marinhas tem revelado resultados satisfatórios, como a descoberta de ação antibacteriana contra cepas multirresistentes. Desta forma, decidiu-se pesquisar as propriedades antibióticas dos extratos de macroalgas das classes Chlorophyta, Phaeophyta e Rhodophyta, coletadas no litoral cearense, bem como avaliar a citotoxicidade destes extratos, frente a cepas bacterianas (Vibrio sp.) isoladas e provenientes de ambientes de cultivo de camarões marinhos (Litopenaeus vannamei). Os resultados comprovaram que houve atividade antibacteriana dos extratos etanólicos, acetônicos, hexânicos e metanólicos contra cepas bacterianas de Vibrio, além de apontar que os extratos de todas as espécies apresentaram baixa citotoxicidade.

Isolamento de Vibrio spp de camarões comercializados in natura na cidade de São Gonçalo - RJ / Isolation of Vibrio spp from shrimp commercialized in natura in the city of Sao Gonçalo - RJ

Bactérias do gênero Vibrio fazem parte da microbiota de camarões, pois têm capacidade de associar-se à quitina presente no exoesqueleto destes animais e ao zooplancton, que por sua vez são consumidos por estes animais. O gênero contém pelo menos 12 espécies patogênicas, incluindo V. cholerae, responsável por várias pandemias de cólera. A contaminação humana acontece através do consumo de alimentos, principalmente de origem marinha, crus ou mal cozidos. Por se tratar de um tipo de pescado amplamente consumido pela população, este trabalho teve como objetivo investigar a presença de espécies de Vibrio em camarões comercializados in natura na cidade de São Gonçalo-RJ. Os camarões foram adquiridos em duas peixarias da cidade e caracterizados por metodologia convencional e molecular; cento e vinte e nove amostras testaram positivamente para as provas bioquímicas realizadas e, destas, cinquenta e duas testaram positivamente para os testes moleculares. Visando investigar a identidade das espécies de Vibrio, as amostras foram submetidas ao PCR multiplex para 4 espécies (V. cholerae, V. mimicus, V. parahaemolyticus, V. vulnificus). Doze isolados foram identificados como V. parahaemolyticus e 9 como V.cholerae não O1. Dentre os demais isolados, 31 demonstram se tratar de outras espécies de Vibrio spp. O sítio com o maior número de isolados foi a casca, seguida pelo hepatopâncreas/ hemolinfa. A ribotipagem por PCR das 21 cepas demonstrou claramente a separação das cepas de V.parahaemolyticus e V.cholerae. As cepas de V. cholerae e V. parahaemolyticus demonstraram alto índice de resistência a ampicilina (83,33%) e 100% de sensibilidade à nitroflurantoína e tetraciclina. Sete cepas (38,8%) apresentaram perfil de multirresistência a dois antimicrobianos. Nossos resultados demonstram a presença de espécies patogênicas de Vibrio em amostras de pescados amplamente consumidos pela população.

Utilização comparativa de enrofloxacino, probiótico (Lactobacilus plantarum) e propionato de sódio na larvicultura de camarão branco do pacífico / Comparative use of enrofloxacin, probiotic (Lactobacillus plantarum) and sodium propionate in pacific white shrimp larvae / Utilización comparativa de enrofloxacino, probiótico (Lactobacillus plantarum) y propionato de sodio en larvicultura de camarón blanco del pacífico

O objetivo do presente trabalho foi avaliar métodos de controle e tratamento de bacterioses na larvicultura de Litopenaeus vannamei, avaliando o uso contínuo de probiótico e o uso pontual de enrofloxacino 15mg.L-1 e propionato de sódio 0,5 mM.L-1 nos momentos de metamorfose sobre os, parâmetros zootécnicos e microbiológicos (larvas e da água). Foram utilizadas 16 unidades de 60L, povoadas na densidade de 325 náuplios.5L-1, divididos em três tratamentos: enrofloxacino, probiótico, propionato de sódio e controle. O enrofloxacino e o propionato foram ministrados em protozoea 3, misis 3 e pós-larva 4 e o probiótico foi ministrado na ração ao longo de todo o experimento. O probiótico aumentou as contagens de bactérias ácido-láticas em relação aos demais tratamentos na água de cultivo (p=0,00001) e em relação ao enrofloxacino e o proprionato nas larvas (p=0,0048). A água do tratamento com probiótico apresentou menor contagem de Vibrio spp. que o enrofloxacino (p=0,0011) e as larvas tratadas com probiótico apresentaram menor contagem de Vibrio spp. que o propionato (p=0,0158). Contudo, não foi observada diferença nos índices zootécnicos avaliados. Assim, os aditivos na dose utilizada não alteram parâmetros zootécnicos da larvicultura do camarão L. vannamei.

The purpose of this study was to assess control and treatment methods for bacterial diseases in Litopenaeus vannamei, evaluating the continuous use of probiotics and occasional use of 15mg-L-1 enrofloxacin and 0.5-mM.L-1 sodium propionate at the morphological change moments on the performance and microbiological parameters of larvae and water. A total of 16 60-L units were used, stocked with 325 nauplii/5L-1, divided into three treatments and one control. Enrofloxacin and propionate were administered into protozoea 3, misis 3 and 4, and post-larvae 4, while the probiotic was administered in the feed throughout the experiment. The probiotic increased the counts of lactic acid bacteria in relation to the other treatments in the culture water (p = 0.00001) and in relation to enrofloxacin and propionate in larvae (p = 0.0048). The treatment water with probiotic had lower counts of Vibrio ssp. than enrofloxacin (p = 0.0011) and larvae treated with probiotic showed lower counts of Vibrio ssp. that propionate (p = 0.0158). However, no difference was observed in the performance indexes assessed. Thus, it can be concluded that additives in the assessed doses did not influence the performance parameters of L. vannamei.

El objetivo del presente trabajo fue evaluar métodos de control y tratamiento de enfermedades bacterianas en larvicultura de Litopenaeus vannamei, evaluando el uso continuo de probiótico y el uso puntual de 15 mg.L-1 de enrofloxacino y 0,5 mM.L-1 de propionato de sodio en momentos de metamorfosis sobre los parámetros zootécnicos y microbiológicos (larvas y del agua). Se han utilizado 16 unidades de 60L, pobladas en la densidad de 325 nauplios.5L-1, divididos en tres tratamientos: enrofloxacino, probiótico, propionato de sodio y control. El enrofloxacino y el propionato fueron suministrados en protozoea 3, misis 3 y postlarva 4 y el probiótico suministrado en alimento durante el transcurso del experimento. El probiótico aumentó el contaje de bacterias ácido-lácticas en correlación a los demás tratamientos en agua de cultivo (p=0,0001) y las larvas en relación al enrofloxacino y el propionato en las larvas (p=0,0048). El agua del tratamiento con probiótico presentó menor contaje de Vibrio spp. que el enrofloxacino (p=0,0011) y las larvas tratadas con probiótico presentaron menor contaje de Vibrio spp. que el propionato (p=0,0158). Sin embargo, no se ha observado diferencia en los índices zootécnicos de larvicultura de camarón L. vannamei.

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.

Detección de protozoos parásitos entéricos, bacteriófagos de E. coli y organismos indicadores de contaminación en camarones comercializados en el estado Zulia / Detection of enteric protozoa parasites, E. coli bacteriophages and fecal pollution Indicator organisms in shrimp sold in the state of Zulia

Se determinó la presencia de Giardia intestinalis y Cryptospodidium parvum, bacteriófagos de Escherichia coli y organismos indicadores de contaminación (OIC), en muestras de camarones para el consumo humano comercializados en el estado Zulia. Los parásitos se concentraron a partir de sistemas digestivos de pools de camarones por la técnica de formol-éter y se cuantificaron por inmunofluorescencia directa. La concentración de los bacteriófagos de E. coli F+ y los OIC se evaluó por técnicas estándar. En este trabajo se detectó la presencia de G. intestinalis, C. parvum, bacteriófagos y E. coli en camarones comercializados en el estado Zulia que cumplían los criterios de la normativa venezolana de calidad sanitaria e inocuidad. Del total de muestras analizadas el 91,5% fueron positivas para G. intestinalis (promedio: 36,6 quistes/100g), 95,3% para C. parvum (promedio: 32,8 ooquistes/100g), 100% para los bacteriófagos de E coli F+ (promedio de 2,8 x 103 UFP/100 g) y 71,5% para E. coli (promedio de 4,3 x 104 NMP/g). Los resultados obtenidos indican que los camarones pueden convertirse en un vehículo para la transmisión de patógenos al hombre y dejan en evidencia la necesidad de la inclusión de un parámetro parasitológico y viral en el control de la calidad microbiológica de estos productos alimenticios.

The presence of G. intestinalis and C. parvum, E. coli F+ bacteriophages and fecal pollution indicator organisms was determined in shrimp for human consumption marketed in the State of Zulia. Parasites were concentrated from the digestive systems of shrimp pools, detected by formalin-ether and quantified by direct immunofluorescence. E. coli F + bacteriophage and pollution indicator organism concentrations were determined by standard techniques. In this work, G. intestinalis, C. parvum, E. coli F + bacteriophages and E. coli were detected in shrimp for human consumption marketed in the State of Zulia that met the quality criteria of Venezuelan health and safety regulations. 91.5% of the samples analyzed were positive for G. intestinalis (average: 36.6 cyst/100g), 95.3% for C parvum (average: 32.8 oocyst/100g), 100% for E coli F + bacteriophages (average: 2.8 x 103 FPFU/100g) and 71.5% for E. coli (average: 4.3 x 104 MPN/g). Results of this research indicate that shrimp can become a vehicle for transmitting pathogens to humans and demonstrate the need for including a parasitic and viral parameter in microbiological quality control for seafood.

Isolation and characterization of infectious Vibrio sinaloensis strainsfrom the Pacific shrimp Litopenaeus vannamei (Decapoda: Penaeidae)

Infectious diseases especially those caused by bacterial and viral pathogens are serious loss factors in shrimp farming. In this study, bacteria were isolated from the gut and hepatopancreas of stressed shrimps obtained from a commercial farm. The isolates were screened on Thiosulfate citrate bile salt sucrose (TCBS) agar plates for the selection of Vibrio species. Presumptive vibrios were characterized through tests for hemolytic and enzymatic activity, hydrophobicity, growth and molecular identification. Three experimental infections were conducted in order to confirm the pathogenicity of selected bacterial strains VHPC18, VHPC23, VHPC24 and VIC30. In the third experimental challenge the LD50 was obtained, it lasted 10 days with 10 shrimp, weighing 6.9±1.1g, per tank. The treatments in triplicate were: (1) saline solution (control group); (2) 2×10(5)CFU/shrimp; (3) 4×10(5)CFU/shrimp; (4) 2×10(6)CFU/shrimp; (5) 4×10(6)CFU/shrimp, and (6) 8×10(6)CFU/shrimp. In all challenges, water parameters measured during the experimental period remained within optimum ranges. Pathogenicity tests confirmed that the mixture of four vibrio isolates, identified as Vibrio sinaloensis, was virulent for L. vannamei. The LD50 value was 1.178×10(5)CFU/g body weight. V. sinaloensis may act as opportunistic pathogens for cultured L. vannamei. Rev. Biol. Trop. 60 (2): 567-576. Epub 2012 June 01.

Las enfermedades de etiología infecciosa, especialmente las causadas por patógenos bacterianos y virales ocasionan graves pérdidas en el cultivo de camarón blanco Litopenaeus vannamei. En este estudio se caracterizo: la actividad enzimática y hemolítica; hidrofobicidad; crecimiento e identificación molecular de vibrios aislados del intestino y hepatopancreas de camarones estresados, obtenidos de una granja comercial, en medio Agar Tiosulfato Citrato Bilis Sacarosa. Además, se realizaron tres infecciones experimentales para confirmar la patogenicidad de las cepas bacterianas seleccionadas VHPC18, VHPC23, VHPC24 y VIC30. En la tercera infección experimental se obtuvo la LD50, el reto duro 10 días, con 10 camarones por tanque con un peso de 6.9±1.1g. Los tratamientos se realizaron por triplicado: (1) solución salina (grupo control); (2) 2×10(5)UFC/camarón; (3) 4×10(5)UFC/camarón; (4) 2×10(6)UFC/camarón; (5) 4×10(6)UFC/camarón y (6) 8×10(6)UFC/camarón. En todos los retos, los parámetros del agua permanecieron dentro de los intervalos óptimos. Las pruebas de patogenicidad confirmaron que la mezcla de cuatro aislados de Vibrio, identificados como Vibrio sinaloensis, fue virulenta para L. vannamei. El valor de la LD50 fue de 1.178×10(5)UFC/g de peso corporal. Los resultados permiten establecer que las cepas de V. sinaloensis pueden actuar como patógenos oportunistas en L. vannamei cultivado.

Antibiotic resistance of Vibrio parahaemolyticus isolated from pond-reared Litopenaeus vannamei marketed in Natal, Brazil

Ten out of fifty fresh and refrigerated samples of shrimp (Litopenaeus vannamei) collected from retailers in Natal (Rio Grande do Norte, Northeastern Brazil) tested positive for Vibrio parahaemolyticus. The Kanagawa test and multiplex PCR assays were used to detect TDH and TRH hemolysins and the tdh, trh and tlh genes, respectively. All strains were Kanagawa-negative and tlh-positive. Antibiotic susceptibility testing was done for seven antibiotics by the agar diffusion technique. Five strains (50 percent) presented multiple antibiotic resistance to ampicillin (90 percent) and amikacin (60 percent), while two strains (20 percent) displayed intermediate-level resistance to amikacin. All strains were sensitive to chloramphenicol. Intermediate-level susceptibility and/or resistance to other antibiotics ranged from 10 to 90 percent, with emphasis on the observed growing intermediate-level resistance to ciprofloxacin. Half our isolates yielded a multiple antibiotic resistance index above 0.2 (range: 0.14-0.29), indicating a considerable risk of propagation of antibiotic resistance throughout the food chain.

Antibacterial effect (in vitro) of Moringa oleifera and Annona muricata against Gram positive and Gram negative bacteria / Efeito antibacteriano (in vitro) de Moringa oleifera (moringa) e Annona muricata (graviola) frente a bactérias Gram-negativas e Gram-positiva

Antibacterial effects of aqueous and ethanolic extracts of seeds of moringa (Moringa oleifera) and pods of soursop (Annona muricata) in the concentration of 1:5 and 1:10 in volumes 50, 100, 150 and 200 µL were examined against Staphylococcus aureus, Vibrio cholerae, Escherichia coli (isolated from the organism and the aquatic environment) and Salmonella Enteritidis. Antibacterial activity (inhibition halo > 13 mm) against S. aureus, V. cholerae and E. coli isolated from the whiteleg shrimp, Litopenaeus vannmaei, was detected in aqueous and ethanolic extracts of moringa. E. coli isolated from tilapiafish, Oreochromis niloticus, was sensitive to the ethanolic extract of moringa. The aqueous extracts of soursop showed an antibacterial effect against S. aureus and V. cholerae, but the antibacterial activity by the ethanol extracts of this plant was not demonstrated.

Para avaliação do efeito bactericida frente à Staphylococcus aureus, Vibrio cholerae, Escherichia coli (isolada de pescados e ambiente aquático) e Salmonella Enteretidis, foram testados extratos aquosos e etanólicos de sementes de moringa (Moringa oleifera) e casca de graviola (Annona muricata) na concentração de 1:5 e 1:10, nos volumes de 50, 100, 150 e 200 µL. Os resultados mostraram efeito antibacteriano (halo de inibição > 13mm) dos extratos aquosos e etanólicos de moringa frente a S. aureus, V. cholerae e E. coli isoladas de camarão cinza Litopenaeus vannmaei. A cepa de E. coli isolada do pescado Oreochromis niloticus apresentou sensibilidade frente ao extrato etanólico de moringa. Os extratos aquosos de graviola apresentaram efeito bactericida frente a S. aureus e V. cholerae, entretanto, os extratos etanólicos dessa planta não mostraram atividade antibacteriana.

Propriedades físico-químicas, sensoriais e bacteriológicas de camarões (Litopenaeus brasiliensis) irradiados e armazenados sob refrigeração / Physical, chemical, sensorial and bacterial properties of irradiated shrimp (Litopenaeus brasiliensis) stored under refrigeration

A irradiação de alimentos é uma opção tecnológica para minimizar problemas de perdas de alimentos, pois reduz asalterações provenientes da atividade bacteriana, estendendo a validade do produto. Neste estudo foram irradiados camarões(Litopenaeus brasiliensis) com doses de 1,5 e 2,5 kGy, após 30 dias de armazenamento sob refrigeração (2°C ± 2°C), como objetivo de avaliar os efeitos da irradiação sobre a validade comercial de camarões através de parâmetros físico-químicos,microbiológicos e sensoriais. Foram avaliados pH, bases voláteis totais (BVT), contagem de bactérias heterotróficas aeróbiasmesófilas (BHAM) e heterotróficas aeróbias psicrófilas (BHAP) e teste de aceitação sensorial. Os valores de pH não variaramentre as amostras controle e as amostras irradiadas apresentando média de 7,08 ± 0,21 durante os 30 dias de armazenamento.A produção de BVT nas amostras controle extrapolou o limite aceitável para o consumo no 15o dia de armazenamentoenquanto nas amostras irradiadas com 2,5 kGy, este limite não foi alcançado em 28 dias de armazenamento, demonstrandoo efeito positivo da utilização da irradiação. Na análise sensorial, não ocorreu diferença significativa (p> 0,01) na aceitação,quanto aos atributos de sabor, aparência, aroma e impressão global entre as amostras.

Food irradiation is currently regarded as a technological option to mitigate food losses due to its potential in reducing microbiological load and its associated spoilage effects, consequently extending the shelf life of many products. Samples of shrimp (Litopenaeus brasiliensis) have been exposed to gamma doses of 0, 1.5 and 2.5 kGy and monitored changes in sensorial characteristics (30 judges), pH and total volatile bases (TVB) levels. All samples were kept at 2°C ± 2°C through the experiments. No significant change in pH has been observed during the experiments regardless of the gamma dose applied. There was statistical difference between the non-irradiated samples and those irradiated with 2.5 kGy (p 0.01), though no significant difference has been found between the non-irradiated samples and those irradiated with 1.5 kGy nor between the samples irradiated with 1.5 kGy and those irradiated with 2.5 kGy. Based on the upper commercial limit for NB, non-irradiated samples achieved the limit in 15 days while the samples irradiated with 2.5 kGy didnt achieve the limit in 28 days. In sensorial analyses, there was no significant difference (p> 0.01) in the acceptance, according to the attributes flavor, appearance, aroma and global impression among the samples.

PCR-based detection of enterotoxigenic isolates of Bacillus cereus from tropical seafood.

Background & objectives: Bacillus cereus is an important enterotoxigenic food borne pathogen. The present study was undertaken to assess the occurrence of B. cereus in tropical fish and evaluation of virulent gene specific PCR for differentiation of diarrhoeal enterotoxin producing isolates of B. cereus from non enterotoxigenic isolates. Methods: Selective plating on polymixin-pyruvate-egg yolk-mannitol-bromocresol purple agar (PEMPA) was used for isolation of B. cereus from finfish, prawn and clams. Enterotoxin producing ability of all 42 isolates obtained from the samples was judged by reverse passive latex agglutination (RPLA) test and the presence of different virulent genes i.e. hbla, bceT and entFM was screened by PCR. Results: B. cereus and enterotoxigenic B. cereus were found to be in 36.7 and 29.41 per cent of fish samples, respectively. All the diarrhoeal enterotoxin producing isolates showed the presence of hbla gene, but hbla gene was not present in any of the non-enterotoxigenic isolates tested in this study. Interpretation & conclusions: Our findings indicated that hbla gene specific PCR can be employed for differentiation of enterotoxigenic B. cereus isolates from non-enterotoxigenic isolates.

Characterization and experimental infection of Flexibacter maritimus (Wakabayashi et al. 1986) in hatcheries of post-larvae of Litopenaeus vannamei Boone, 1931

A preliminary study to characterize filamentous bacteria, whose presence is related to high mortality of Litopenaeus vannamei larvae cultured in Santa Catarina State, Brazil, is reported. The extract of infected larvae was diluted in different concentrations, cultured in marine agar (DifcoTM, Marine Agar 2216) and incubated at 30 °C for 48 hours. The biochemical characterization included hydrolytic reactions of starch, gelatin and tyrosine, growth in TCBS agar, growth in 0 and 37‰ salinity, pigment production in tyrosine agar, production of H2S, nitrate reduction, congo red reaction, oxidase and catalase. The isolated bacteria belong to the species Flexibacter maritimus, Gram-negative bacilli of 0.4-0.5 µm width and 15 µm length. Experiments were carried out on pathogenicity of F. maritimus in post-larvae of L. vannamei. Survival and symptoms in L. vannamei post-larvae 24 hours after inoculation with F. maritimus and its growth in marine agar were evaluated. Mortality was detected around 92,5 percent as well as symptoms like melanized lesions in several parts of body, discolouration of gills, bad formation of appendages and of the last abdominal segment, low motility and feeding reduction. The experimental infection results suggested that isolated bacteria of the genus Flexibacter are pathogenic to the shrimp Litopenaeus vannamei post-larvae.

Neste trabalho, relata-se um estudo preliminar para caracterizar bactérias filamentosas associadas a elevadas mortalidades de larvas e pós-larvas cultivadas no Estado de Santa Catarina, Brasil. Um extrato de larvas infectadas foi diluído em diferentes concentrações, cultivado em Agar Marine (DifcoTM, Marine Agar 2216) e incubado a 30° C por 48 horas. A caracterização bioquímica inclui reações de amido, gelatina e tirosesine, crescimento em Agar TCBS,crescimento a 0 e 37‰ de salinidade, produção de pigmentos em Agar Tirosine, produção de H2S, redução de nitrato, reação vermelho congo, oxidase e catalase. A bactéria isolada apresentou características de Flexibacter maritimus, um bacilo Gram negativo de 0,4-0,5 µm de largura e 15 µm de comprimento. Os experimentos foram realizados sobre a patogenicidade de F. Maritimus, em pós-larvas de L. vannamei. Foram avaliados a sobrevivência e os sintomas das pós-larvas 24 horas após a inoculação com F. Maritimus, além do seu crescimento em Agar Marine. Foram encontradas mortalidades de 92,5 por cento e sintomas próprios da enfermidade, tais como lesões melanizadas em várias partes do corpo, descoloração das brânquias e diminuição da motilidade e da alimentação. Com base nos resultados da infecção experimental, pode se concluir que a bactéria isolada do gênero Flexibacter é patogênica para pós-larvas de L. vannamei.

Antibacterial activity of GUAVA, Psidium guajava Linnaeus, leaf extracts on diarrhea-causing enteric bacteria isolated from Seabob shrimp, Xiphopenaeus kroyeri (Heller) / Avaliação da atividade antibacteriana de extrato de folhas de goiabeira, Psidium guajava Linnaeus, sobre bactérias entéricas diarreiogênicas, isoladas de camarão sete-barbas, Xiphopenaeus kroyeri (Heller)

Guava leaf tea of Psidium guajava Linnaeus is commonly used as a medicine against gastroenteritis and child diarrhea by those who cannot afford or do not have access to antibiotics. This study screened the antimicrobial effect of essential oils and methanol, hexane, ethyl acetate extracts from guava leaves. The extracts were tested against diarrhea-causing bacteria: Staphylococcus aureus, Salmonella spp. and Escherichia coli. Strains that were screened included isolates from seabob shrimp, Xiphopenaeus kroyeri (Heller) and laboratory-type strains. Of the bacteria tested, Staphylococcus aureus strains were most inhibited by the extracts. The methanol extract showed greatest bacterial inhibition. No statistically significant differences were observed between the tested extract concentrations and their effect. The essential oil extract showed inhibitory activity against S. aureus and Salmonella spp. The strains isolated from the shrimp showed some resistance to commercially available antibiotics. These data support the use of guava leaf-made medicines in diarrhea cases where access to commercial antibiotics is restricted. In conclusion, guava leaf extracts and essential oil are very active against S. aureus, thus making up important potential sources of new antimicrobial compounds.

O chá de folhas de goiaba Psidium guajava Linnaeus é comumente usado como remédio nas gastrenterites e diarréias infantis por aqueles que não têm acesso a antibióticos. Esta pesquisa estudou o efeito antibacteriano sobre bactérias causadoras de diarréias, do óleo essencial e do extrato de folhas de goiabeira usando como diluente: metanol, hexano e acetato de etila. Os extratos foram testados sobre Staphylococcus aureus, Salmonella spp. e Escherichia coli. As bactérias testadas foram isoladas de camarão sete-barbas Xiphopenaeus kroyeri (Heller) usando-se como controle cepas padrão, de cada espécie. Das bactérias testadas, o melhor efeito inibitório foi observado sobre Staphylococcus aureus. O extrato de metanol apresentou maior inibição bacteriana. O óleo essencial mostrou ação inibitória contra S. aureus e Salmonella spp. Foi demonstrada alguma resistência das cepas isoladas de camarão aos antibióticos comerciais testados. Estes dados confirmam que remédios à base de folhas de goiabeira podem ser utilizados em casos de diarréia provocada por essas bactérias, quando o uso de antibiótico for restrito. Conclui-se que extratos de folhas de goiaba e o seu óleo essencial são muito ativos contra S. aureus, tornando-os importantes fontes em potencial de novos compostos antimicrobianos.

White spot syndrome virus isolates of tiger shrimp Penaeus monodon (Fabricious) in India are similar to exotic isolates as revealed by polymerase chain reaction and electron microscopy.

Microbiological analysis of samples collected from cases of white spot disease outbreaks in cultured shrimp in different farms located in three regions along East Coast of India viz. Chidambram (Tamil Nadu), Nellore (Andhra Pradesh) and Balasore (Orissa), revealed presence of Vibrio alginolyticus, Vibrio parahaemolyticus, and Aeromonas spp. but experimental infection trials in Penaeus monodon with these isolates did not induce any acute mortality or formation of white spots on carapace. Infection trials using filtered tissue extracts by oral and injection method induced mortality in healthy P. monodon with all samples and 100% mortality was noted by the end of 7 day post-inoculation. Histopathological analysis demonstrated degenerated cells characterized by hypertrophied nuclei in gills, hepatopancreas and lymphoid organ with presence of intranuclear basophilic or eosino-basophilic bodies in tubular cells and intercellular spaces. Analysis of samples using 3 different primer sets as used by other for detection of white spot syndrome virus (WSSV) generated 643, 1447 and 520bp amplified DNA products in all samples except in one instance. Variable size virions with mean size in the range of 110 x 320 +/- 20 nm were observed under electron microscope. It could be concluded that the viral isolates in India involved with white spot syndrome in cultured shrimp are similar to RV-PJ and SEMBV in Japan, WSBV in Taiwan and WSSV in Malaysia, Indonesia, Thailand, China and Japan.

Recombinant expression of marine shrimp lysozyme in Escherichia coli

Shrimp Lysozyme (Lyz) is a key component of the antibacterial response as part of the innate defense in Crustacea; however, it has not been possible to purify this protein because of the very low amount present in the shrimp blood cells (hemocytes). In an effort to produce enough protein to study its function and biochemical properties we have overexpressed Lysozyme from marine shrimp (Penaeus vannamei) in E. coli. A bacterial protein expression system based on the T7 polymerase promoter was used. Although Lyz was produced as insoluble protein in inclusion bodies, its refolding led to an active protein with a yield of ~10 percent. Details of the protein recombinant expression techniques applied to this shrimp protein are presented.