Genotypic assessment of a dichotomous key to identify Vibrio coralliilyticus, a coral pathogen.

Vibrio coralliilyticus is a known pathogen to corals and larvae of bivalves. Its identification is made based on phenotypic and genotypic characters of isolated strains. To evaluate the efficiency of the phenotypic identification, 21 strains identified as V. coralliilyticus using a widely used dichotomous key were analyzed by qualitative PCR and sequencing of the 16S rDNA region. The results obtained by the behavioral test, amino acids usage, allow us to distinguish 3 A/L/O profiles: (1) A+/L-/O+; (2) A+/L+/O+; and (3) A-/L+/O+. In the genotypic tests, all strains tested positive with primers specific for the Vibrio genus. However, when primers were used for species identification, the results did not match those obtained with the dichotomous key chosen. The phenotypic characteristics taken into account to set apart V. coralliilyticus and other species were not proven to be efficient. More information about the morphological diversity of colonies and enzymatic activities should be considered in the formulation of phenotypic keys for V. coralliilyticus and related species.

Pathogenic Vibrio species isolated from estuarine environments (Ceará, Brazil) - antimicrobial resistance and virulence potential profiles

ABSTRACT Detection of virulent strains associated with aquatic environment is a current concern for the management and control of human and animal health. Thus, Vibrio diversity was investigated in four estuaries from state of Ceará (Pacoti, Choró, Pirangi and Jaguaribe) followed by antimicrobial susceptibility to different antimicrobials used in aquaculture and detection of main virulence factors to human health. Isolation and identification were performed on TCBS agar (selective medium) and dichotomous key based on biochemical characteristics, respectively. Nineteen strains of genus Vibrio were catalogued. Vibrio parahaemolyticus (Choró River) and V. alginolyticus (Pacoti River) were the most abundant species in the four estuaries. All strains were submitted to disk diffusion technique (15 antimicrobials were tested). Resistance was found to: penicillin (82%), ampicillin (54%), cephalotin (7%), aztreonan (1%), gentamicin, cefotaxime and ceftriaxone (0.5%). Five pathogenic strains were chosen to verification of virulence factors. Four estuaries showed a high abundance of species. High number of tested positive strains for virulence is concerning, since some of those strains are associated to human diseases, while others are known pathogens of aquatic organisms.

Pathogenic Vibrio species isolated from estuarine environments (Ceará, Brazil) - antimicrobial resistance and virulence potential profiles.

Detection of virulent strains associated with aquatic environment is a current concern for the management and control of human and animal health. Thus, Vibrio diversity was investigated in four estuaries from state of Ceará (Pacoti, Choró, Pirangi and Jaguaribe) followed by antimicrobial susceptibility to different antimicrobials used in aquaculture and detection of main virulence factors to human health. Isolation and identification were performed on TCBS agar (selective medium) and dichotomous key based on biochemical characteristics, respectively. Nineteen strains of genus Vibrio were catalogued. Vibrio parahaemolyticus (Choró River) and V. alginolyticus (Pacoti River) were the most abundant species in the four estuaries. All strains were submitted to disk diffusion technique (15 antimicrobials were tested). Resistance was found to: penicillin (82%), ampicillin (54%), cephalotin (7%), aztreonan (1%), gentamicin, cefotaxime and ceftriaxone (0.5%). Five pathogenic strains were chosen to verification of virulence factors. Four estuaries showed a high abundance of species. High number of tested positive strains for virulence is concerning, since some of those strains are associated to human diseases, while others are known pathogens of aquatic organisms.

Antioxidant, hemolytic, antimicrobial, and cytotoxic activities of the tropical Atlantic marine zoanthid Palythoa caribaeorum.

Marine invertebrates are capable of synthesizing bioactive compounds, which may be beneficial to human health. The aim of this study was to evaluate the antioxidant, hemolytic, antimicrobial and cytotoxic activities of crude extract (70% EtOH), and dichloromethane (DCM), ethyl acetate (EtOAc), and aqueous (Aq) fractions of the marine zoanthid Palythoa caribaeorum. The phenolic compound contents of the crude extract, DCM, EtOAc and Aq fractions were 12.33, 18.17, 10.53, and 3.18 mg GAE per gram, respectively. DPPH radical scavenging activity showed slight variation. IC50 of crude extract, DCM, EtOAc and Aq fractions were 11.13, 11.25, 11.74, and 11.28 µg mL(-1), respectively. Among the sample, ferrous ion chelating was the highest in crude extract (IC50 302.90 µg mL(-1)), followed by EtOAc, Aq, and DCM fractions with 457.77, 547.91, and 641.82 µg mL(-1), respectively. Ferric-reducing antioxidant power showed optical density at about 0.5. The samples tested exhibited low hemolytic activity under 10% up to a concentration of 50 µg mL(-1). No antimicrobial activity was observed against any of the tested bacterial strains. For the cytotoxic activity, LC50 of DCM, crude extract, EtOAc, and Aq were 52.10, 83.06, 86.34, and 117.45 µg mL(-1), showing high toxicity.

Antibiotic Resistance of Salmonella spp. Isolated from Shrimp Farming Freshwater Environment in Northeast Region of Brazil.

This study investigated the presence and antibiotic resistance of Salmonella spp. in a shrimp farming environment in Northeast Region of Brazil. Samples of water and sediments from two farms rearing freshwater-acclimated Litopenaeus vannamei were examined for the presence of Salmonella. Afterwards, Salmonella isolates were serotyped, the antimicrobial resistance was determined by a disk diffusion method, and the plasmid curing was performed for resistant isolates. A total of 30 (16.12%) of the 186 isolates were confirmed to be Salmonella spp., belonging to five serovars: S. serovar Saintpaul, S. serovar Infantis, S. serovar Panama, S. serovar Madelia, and S. serovar Braenderup, along with 2 subspecies: S. enterica serovar houtenae and S. enterica serovar enterica. About twenty-three percent of the isolates were resistant to at least one antibiotic, and twenty percent were resistant to at least two antibiotics. Three strains isolated from water samples (pond and inlet canal) exhibited multiresistance to ampicillin, tetracycline, oxytetracycline, and nitrofurantoin. One of them had a plasmid with genes conferring resistance to nitrofurantoin and ampicillin. The incidence of bacteria pathogenic to humans in a shrimp farming environment, as well as their drug-resistance pattern revealed in this study, emphasizes the need for a more rigorous attention to this area.

Kanagawa-negative, tdh- and trh-positive Vibrio parahaemolyticus isolated from fresh oysters marketed in Fortaleza, Brazil.

Between October 2008 and June 2009, 15 samples of 10 live oysters each (Crassostrea rhizophorae) measuring 8.31-10.71 cm were purchased from a restaurant on the seashore of Fortaleza, Brazil. The Vibrio count ranged from 75 (estimated) to 43,500 CFU/g. Fourteen species were identified among the 56 isolated Vibrio strains, with V. parahaemolyticus as the most prevalent. Two of the 17 V. parahaemolyticus strains were urease-positive and tdh- and trh-positive on multiplex PCR, but neither produced ß-hemolysis halos in Wagatsuma agar. Thus, fresh oysters served in natura in Fortaleza, Brazil, were found to contain Vibrio strains known to cause gastroenteritis in humans.

Antibacterial activity of GUAVA, Psidium guajava Linnaeus, leaf extracts on diarrhea-causing enteric bacteria isolated from Seabob shrimp, 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.

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.

Practical use of immobilized lysozyme for the remediation process of Escherichia coli in aqueous solution

The lysozyme enzyme was immobilized on vitreous surface (fragments with diameters of 0.3 and 1.0 mm) for remediation of the microorganism Escherichia coli JM 109 into fresh water and saline solutions with 0.9 percent NaCl (w/v). Characterization of enzymatic film was carried out by infrared spectroscopy and atomic force microscopy techniques. Bactericide activity of the enzyme was evaluated by spectrophotometric analysis. It was verified that the enzymatic film was strongly coupled with the vitreous surface. The topographic analysis demonstrated that the deposited film was uniform and homogeneous. It was observed bactericide activity of film deposited on vitreous surface with 0.3 mm in fresh and saline solutions. This fact was not verified to vitreous fragments with 1.0 mm of diameter.

Aeromonas spp. isolated from oysters (Crassostrea rhizophorea) from a natural oyster bed, Ceará, Brazil.

Between April and October 2002, thirty fortnightly collections of oysters (Crassostrea rhizophorea) from a natural oyster bed at the Cocó River estuary in the Sabiaguaba region (Fortaleza, Ceará, Brazil) were carried out, aiming to isolate Aeromonas spp. strains. Oyster samples were submitted to the direct plating (DP) and the presence/absence (P/A) methods. Aeromonas were identified in 15 (50%) samples analyzed by the DP method and in 13 (43%) analyzed by the P/A method. A. caviae, A. eucrenophila, A. media, A. sobria, A. trota, A. veronii bv. sobria, A. veronii bv. veronii and Aeromonas sp. were isolated. The predominant species was A. veronii (both biovars), which was identified in 13 (43%) samples, followed by A. media in 11 (37%) and A. caviae in seven (23%). From the 59 strains identified, 28 (48%) presented resistance to at least one of the eight antibiotics tested.

Aeromonas spp. isolated from oysters (Crassostrea rhizophorea) from a natural oyster bed, Ceará, Brazil

Foram realizadas 30 coletas quinzenais, entre abril e outubro de 2002, de ostras (Crassostrea rhizophorea) de um criadouro natural, no estuário do rio Cocó (Fortaleza/Ceará/Brasil), objetivando-se isolar cepas de Aeromonas spp. As amostras de ostras foram submetidas aos métodos de plaqueamento direto (PD) e presença/ausência (P/A). Foram identificadas Aeromonas em 15 (50%) amostras analisadas pelo método PD e em 13 (43%) pelo método P/A. Foram isoladas: A. caviae, A. eucrenophila, A. media, A. sobria, A. trota, A. veronii bv. sobria, A. veronii bv. veronii e Aeromonas sp. A espécie predominate foi A. veronii (ambos biovars), identificada em 13 (43%) amostras, seguida de A. media em 11 (37%) e A. caviae em 7 (23%). Das 59 cepas identificadas, 28 (48%) apresentaram resistência a pelo menos um, dos oitos antibióticos testados.

Fecal pollution in water from storm sewers and adjacent seashores in Natal, Rio Grande do Norte, Brazil.

A study on the distribution patterns of enteropathogenic bacteria polluting the shoreline in Natal, Rio Grande do Norte, Brazil, was carried out based on 72 samples obtained from three storm sewers and adjoining beach locations, Praia do Meio (PM), Areia Preta (AP) and Ponta Negra (PN). From each location, 12 water samples were taken and analyzed for fecal coliforms (FC) and Escherichia coli. In AP, two (16.7%) of the seawater samples and five (41.7%) of the storm sewer samples yielded values above 1.1 x 107 FC/100 ml, whereas only one (8.3%) of the samples from PM reached this level. There was no correlation (p > 0.05) between rainfall indices and FC values. A total of 64 E. coli isolates were obtained: 37 from the storm sewer samples and 27 from the seawater samples. Of these isolates, four (O143, two O112ac, and O124) were enteroinvasive and two (O111 and O125) were enteropathogenic. Resistance to antibiotics and to heavy metals was also analyzed. Almost 36% of the E. coli strains isolated were resistant to more than one antibiotic. All strains were resistant to zinc and copper at the highest concentration tested (250 microg/ml), and several (23.4%) were resistant to mercury at 50 microg/ml. Our results agreed with previous reports that antibiotic resistance is commonly associated with heavy-metal resistance in pathogens.

Vibrio spp. and Salmonella spp., presence and susceptibility in crabs Ucides cordatus.

The presence of Vibrio spp. and Salmonella spp. in crabs marketed at the Bezerra de Menezes Ave., Fortaleza, State of Ceará, Brazil, was assessed between February and May, 2003. The number of individuals sampled in each one of the fifteen weekly samplings ranged between four and eight. Seven strains of Salmonella, from four different samplings, were identified, being five of them identified as serotype S. Senftenberg and two as S. Poona. All strains of Salmonella were sensitive to the tested anti-microbial drugs, with the exception of tetracycline and nalidixic acid, for which an intermediary sensibility was found. The MPN's for Vibrio ranged between 110/g and 110,000/g. Of the forty five Vibrio strains isolated from the crab samples, only 10 were identified up to the species level: two V. alginolyticus and eight V. parahaemolyticus. Bacteria belonging to the Enterobacteriaceae and Pseudomonaceae families were also identified, namely Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, Pantoea agglomerans and Pseudomonas aeruginosa. The proper cooking of the animals is recommended in order to avoid problems for the consumers of this crustacean.

Vibrio spp. and Salmonella spp., presence and susceptibility in crabs Ucides cordatus

Foram pesquisadas a presença de Vibrio spp. e de Salmonella spp. em caranguejos comercializados na Av. Bezerra de Menezes, Fortaleza, Ceará, Brasil, no período entre fevereiro e maio de 2003. O número de indivíduos em cada, das quinze coletas realizadas, semanalmente, variava entre quatro e oito dependendo do tamanho dos animais, totalizando um número de 90 (noventa) animais examinados. Foram identificadas sete cepas de Salmonella spp. provenientes de quatro coletas: cinco foram identificadas como sorovar S. Senftenberg e duas como S. Poona. Todas as cepas de Salmonella, isoladas das amostras de caranguejos, apresentaram sensibilidade aos antimicrobianos testados, com exceção de tetraciclina e ácido nalidíxico para os quais elas apresentaram uma sensibilidade intermediária. Os NMPs para Vibrio spp. variaram entre 110 e 110.000/g. Das quarenta e cinco cepas de Vibrio spp. isoladas das amostras de caranguejo, foram identificadas, até espécie, somente 10: duas de V. alginolyticus e oito de V. parahaemolyticus. Foram também identificadas bactérias pertencentes às famílias Enterobacteriaceae e Pseudomonadaceae: Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, Pantoea agglomerans e Pseudomonas aeruginosa. Recomenda-se que a cocção dos animais seja bem feita a fim de se evitar problemas para os consumidores dos crustáceos.

Bacteria of fecal origin in mangrove oysters (Crassostrea rhizophorae) in the Cocó River estuary, Ceará State, Brazil

O objetivo deste trabalho foi avaliar a qualidade microbiológica da ostra de mangue (Crassostrea rhizophorae) originária de um criadouro natural no estuário do Rio Cocó, Fortaleza, Ceará, Brasil. Para isso, foram realizadas as estimativas do Número Mais Provável (NMP) de Coliformes Totais (CT) e de Fecais (CF) e de Enterococcus spp. Os valores encontrados para CT e CF no músculo (com líquido intervalvar) variaram de <1,8 a >1.600 e <1,8 a 920 por grama, respectivamente. O valor do NMP de Enterococcus spp. variou de <3,0 a >1.100/g. Não houve correlação entre os parâmetros físico-químicos (temperatura, salinidade e pH) da água na área do criadouro e os níveis de contaminação encontrados nas ostras. Somente houve correlação entre os valores de CT e CF. Cepas de Enterococcus spp. foram isoladas e submetidas a testes bioquímicos para identificação das espécies e, posteriormente, foram testadas para verificar a produção de substância inibitória semelhante à bacteriocina utilizando a cepa-teste Escherichia coli ATCC 25922. De um total de 121 cepas de Enterococcus spp. testadas apenas uma, E. faecalis, apresentou atividade inibitória.

The stormwater drain system as a pollution vector of the seashore in Fortaleza (Ceará state, Brasil)

The aim of this study was to make a diagnosis of coastal pollution through bacteriological analysis of water taken from stormwater drain systems and the nearby seashore. The results were submitted to statistical analysis as to differences in the Most Probable Number (MPN) of fecal coliform (FC) found between the stormwater drain systems and adjacent seashore locations both at low and high tides. The main conclusions were: (a) most water samples collected from the stormwater systems presented MPN values for FC above 1,000 per 100ml; (b) only 20 percent of the samples from the adjacent seashore presented MPN values for FC above 1,000 per 100ml, a fact which may be explained by the distance between the drain system outlets and the actual seashore; (c) FC/100 ml values were invariably higher in the stormwater drain systems than in seawater; (d) out of the 180 strains isolated, 118 were Escherichia coli; (e) the highest in-sewer pollution rate was found at the sampling location in front of Imperial Othon Palace Hotel (IO-SEW), characterized by the presence of an everlasting puddle, while the most polluted seawater sample was taken in front of Statue of Iracema (SI-SEA); (g) the high tide regime enhances the chance of pollution of the coastal zone by fecal coliforms; (h) the high pollution indices by fecal coliforms can only be accounted for by the existence of illegal sewage pipelines connected to the stormwater drain systems.

Detecçäo de vibrio parahaemolyticus em cauda de lagosta, (panulirus laevicauda L.) / Vibrio parahaemolyticus in lobster panulirus (laevicauda latreille L.)

This study verifies the presence of Vibrio parahaemolyticus on 16 tails samples of the lobster Panulirus laevicauda(Latreille)species collected at the same location in 1990 and on 16 samples collected at a local Fish Industry.The MPN of V.parahaemolyticus obtained on samples from all sources varied from <3.0 to 21.0/g. of 39 strains identified as V.parahaemolyticus on the Fish Market samples collected in 1988, 11(28.2 por cento)showed to be "non agglutinable" and among isolates of 1990, 5(35.7 por cento)of them agglutinated with anti-K sera. Only one isolate among Industry samples was typable.The serogroups found among V.parahaemolyticus strain were: KI, KII, KIII, KIV, KV, KVI, KVII, KVIII and KIX.From a total of 56 V.parahaemolyticus strains isolated on Fish Market samples collected in 1988 and 1990, 29(51.8 por cento) were Kanagawa-positive, whereas those isolated on samples the from Industry(3) were Kanagawa-negative

Investigaçäo sobre a microbiota de caudas de lagostas Panulirus argus (Latreille) ou Panulirus laevicauda (Latreille) processadas com tripolifosfato de sódio / Research about microbiota of the tail of the lobster Panulirus argus (Latreille) or Panulirus laevicauda (Latreille) processed with sodium tripoliphosphate

Foram avaliadas as condiçöes higiênico-sanitárias de 100 amostras de caudas de lagostas, durante a etapa de adiçäo de Tripolifosfato de Sódio (TPF), em uma indústria de pesca, do município de Fortaleza-CE. Paralelamente foram analisadas 75 amostras da soluçäo de TPF a 5%, correspondentes às amostras de lagostas retiradas, através da quantificaçäo das bactérias psicrotróficas e de S. aureus; da determinaçäo do Número Mais Provável (NMP) de coliformes fecais e da pesquisa de Salmonela. Os resultados, comparados aos padröes microbiológicos brasileiros vigentes, indicam ausência de Salmonella em todas as amostras. S. aureus, em lagostas, foi detectado numa variaçäo de zero a 1,7 x 10**3 UFC/g na etapa I e zero nas demais etapas. As soluçöes de TPF apresentaram zero na etapa I e numa variaçäo de zero a 8,05 x 10 UFC/ml e zero a 2,8 x 10**3 UFC/ml nas etapas II e III, respectativamente. Em lagostas e soluçöes de RPF, os NMPs de coliformes fecais obtidos variaram de <3a> 1,1 x 10**/g e as contagens de psicrotróficas nestas amostras oscilaram de 10**9 UFC/g e 10**3 a 10**8 UFC/ml respectivamente