Oscarina Viana de Sousa

Antimicrobial resistance profile of Vibrio species isolated from marine shrimp farming environments (Litopenaeus vannamei) at Ceara ´ , Brazil

Brazilian shrimp culture industry has a great economic importance mainly to the northeast region. However, the accelerated development of this activity has resulted in the emergency of outbreaks of diseases from farming shrimp, and as a consequence the use of antimicrobial drugs to minimize the potential adverse effect under the shrimp production. The inappropriate use of antibiotics in aquaculture is one of the causes for the high incidence of antimicrobial resistant bacteria isolated from aquatic environments that represent a danger for aquatic organisms and human health. There is little information available on the level of antimicrobial resistance in pathogenic bacteria from shrimp farming environment. Therefore, this study aimed to evaluate the phenotypic resistance profile among Vibrio isolates from hatcheries water samples and from cultivated marine shrimp hepatopancreas (L. vannamei). Antimicrobial susceptibility testing was carried out by a standard disc diffusion method and the minimum inhibitory concentration (MIC) of oxytetracycline (OTC) for resistant Vibrio isolates was determinate by broth dilution method. The results showed a high incidence of resistance to ampicillin (45.2%) and to the tetracycline class (38.7%). Florfenicol and nitrofurantoin were 100% effective against Vibrio isolates. In this study, the OTC-resistant Vibrio spp. showed MIC values of more than 400mg/L and the presence of seawater did not influence the oxytetracycline bioactivity. The occurrence of antimicrobial multiresistance patterns was observed in 29% of Vibrio isolates. Fifty-five percent of multiresistant isolates of Vibrio lost one or more antibiotic resistance phenotype after procedure to curing of resistance plasmids. The oxytetracycline resistance was the phenotype most often lost among plasmid-cured isolates.

Antibacterial activity of GUAVA, Psidium guajava Linnaeus, leaf extracts on diarrhea-causing enteric bacteria isolated from Seabob shrimp, Xiphopenaeus kroyeri (Heller)

O cha de folhas de goiaba Psidium guajava Linnaeus e comumente usado como remedio nas gastrenterites e diarreias infantis por aqueles que nao tem acesso a antibioticos. Esta pesquisa estudou o efeito antibacteriano sobre bacterias causadoras de diarreias, do oleo 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 bacterias testadas foram isoladas de camarao sete-barbas Xiphopenaeus kroyeri (Heller) usando-se como controle cepas padrao, de cada especie. Das bacterias testadas, o melhor efeito inibitorio foi observado sobre Staphylococcus aureus. O extrato de metanol apresentou maior inibicao bacteriana. O oleo essencial mostrou acao inibitoria contra S. aureus e Salmonella spp. Foi demonstrada alguma resistencia das cepas isoladas de camarao aos antibioticos comerciais testados. Estes dados confirmam que remedios a base de folhas de goiabeira podem ser utilizados em casos de diarreia provocada por essas bacterias, quando o uso de antibiotico for restrito. Conclui-se que extratos de folhas de goiaba e o seu oleo essencial sao muito ativos contra S. aureus, tornando-os importantes fontes em potencial de novos compostos antimicrobianos.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.

Microbicidal effect of medicinal plant extracts (Psidium guajava Linn. and Carica papaya Linn.) upon bacteria isolated from fish muscle and known to induce diarrhea in children

Out of the twenty-four samples of shrimp and fish muscle used for this study, twelve were collected near a large marine sewer for waste disposal, 3 km off the coast of Fortaleza (Brazil) and used for the isolation of E. coli. Other twelve were collected at the Mucuripe fresh fish market (Fortaleza, Brazil) and used for the isolation of Staphylococcus aureus. Ethanol, water and acetone-diluted extracts of guava and papaya leaf sprouts were tested on the bacteria in order to verify their microbicidal potential. The E. coli strains used in the trials were rated LT positive. The papaya leaf extracts (Carica papaya Linn) showed no microbicidal activity while the guava sprout extracts (Psidium guajava Linn) displayed halos exceeding 13 mm for both species, an effect considered to be inhibitory by the method employed. Guava sprout extracts by 50% diluted ethanol most effectively inhibited E. coli (EPEC), while those in 50% acetone were less effective. It may be concluded that guava sprout extracts constitute a feasible treatment option for diarrhea caused by E. coli or by S. aureus-produced toxins, due to their quick curative action, easy availability in tropical countries and low cost to the consumer.

Multiple drug resistant Staphylococcus aureus strains isolated from a fish market and from fish handlers

The aim of this study was to investigate the presence of antibiotic resistant Staphylococcus aureus strains in fish stalls and in hands and nasal and oral cavities of fish handlers of the Mucuripe Fish Market, Fortaleza, Ceara, Brazil. All S. aureus isolates were resistant to Ampicillin and 44 % were multi-drug resistant.

Antimicrobial susceptibility of Escherichia coli isolated from shrimp (Litopenaeus vannamei) and pond environment in northeastern Brazil.

This study aimed to test the susceptibility of Escherichia coli strains isolated from the water, bottom sediments and individuals cultivated in shrimp farm ponds, to antibiotics belonging to different families, namely B-Lactams: Imipenem (IPM; 10 μ g), Ampicillin (AMP; 10 μ g), Cephalothin (CEP; 30 μ g), Cefoxitin (FOX; 30 μ g), Ceftriaxone (CRO; 30 μ g); Tetracycline: Tetracycline (TCY; 30 μ g); Aminoglycosides: Gentamicin (GEN; 10 μ g), Amikacin (AMK; 30 μ g); Chloramphenicol: Chloramphenicol (CHO; 30 μ g); Fluoroquinolones: Ciprofloxacin (CIP; 5 μ g); Nitrofurans: Nitrofurantoin (NIT; 300 μ g); Sulfonamides: Trimethoprim-Sulfamethoxazole (SXT; 30 μ g); Quilononas: Nalidixic Acid (NAL; 30 μ g). In the laboratory, the method of dissemination (Test Kirby-Bauer) was performed in order to fulfill the antibiogram tests. The results showed high indices of resistance to Imipenem, Cephalothin and Ampicillin. Chloramphenicol, Nitrofurantoin, Cefoxitin, Ceftiaxone and Ciprofloxacin have displayed the highest index of sensitive strains. The antibiotic resistance index (ARI) and the multiple resistance index (MAR) varied within the ranges of 0.068–0.077 and 0.15–0.39, respectively. More than 90.5% of strains of Escherichia coli showed a variety of resistance profiles to the tested antibiotics. The high indices of resistance may be a consequence of indiscriminate use of antibiotics, but also the transfer of resistance through mobile genetic elements found in shrimp farms.

HGA-2, a novel galactoside-binding lectin from the sea cucumber Holothuria grisea binds to bacterial cells.

A novel lectin, HGA-2, was isolated from the sea cucumber Holothuria grisea. The protein was isolated by a single chromatographic step using a column of Guar Gum as affinity. HGA-2 showed an apparent molecular mass of 17 kDa and 34 kDa under reducing and nonreducing conditions, respectively. The hemagglutinating activity was specific for rabbit erythrocytes, showing no activity for human blood A, B and O. Its hemagglutinating activity was inhibited by carbohydrates containing galactose, with higher affinity for GalNAc and glycoprotein porcine stomach mucin (PSM). HGA-2 was stable at pH 6-10, significantly declining at pH 5 and a temperature of 40°C, with its activity being abolished at 100 °C. The HGA-2 protein was found to be Ca(2+)-dependent; it was highly toxic against Artemia nauplii and able to recognize and agglutinate cells of Escherichia coli. Amino acid sequences of tryptic peptides of HGA-2 strongly suggest that HGA-2 is a member of the C-type lectin family.

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.

Population dynamics and antimicrobial susceptibility of Aeromonas spp. along a salinity gradient in an urban estuary in Northeastern Brazil

The main objective of this study was to quantify population and identify culturable species of Aeromonas in sediment and surface water collected along a salinity gradient in an urban estuary in Northeastern Brazil. Thirty sediment samples and 30 water samples were collected from 3 sampling locations (A, B and C) between October 2007 and April 2008. The Aeromonas count was 10-7050CFU/mL (A), 25-38,500CFU/mL (B) and<10CFU/mL (C) for water samples, and ∼100-37,500CFU/g (A), 1200-43,500CFU/g (B) and<10CFU/g (C) for sediment samples. Five species (Aeromonas caviae, A. sobria, A. trota, A. salmonicida and A. allosaccharophila) were identified among 41 isolates. All strains were sensitive to chloramphenicol and ceftriaxone, whereas 33 (80, 4%) strains were resistant to at least 2 of the 9 antibiotics tested. Resistance to erythromycin was mostly plasmidial. In conclusion, due to pollution, the Cocó River is contaminated by pathogenic strains of Aeromonas spp. with a high incidence of antibacterial resistance, posing a serious risk to human health.

DETECTION OF VIRULENCE GENES IN ENVIRONMENTAL STRAINS OF Vibrio cholerae FROM ESTUARIES IN NORTHEASTERN BRAZIL

The objectives of this study were to detect the presence of Vibrio cholerae in tropical estuaries (Northeastern Brazil) and to search for virulence factors in the environmental isolates. Water and sediment samples were inoculated onto a vibrio-selective medium (TCBS), and colonies with morphological resemblance to V. cholerae were isolated. The cultures were identified phenotypically using a dichotomous key based on biochemical characteristics. The total DNA extracted was amplified by PCR to detect ompW and by multiplex PCR to detect the virulence genes ctx, tcp, zot and rfbO1. The results of the phenotypic and genotypic identification were compared. Nine strains of V. cholerae were identified phenotypically, five of which were confirmed by detection of the species-specific gene ompW. The dichotomous key was efficient at differentiating environmental strains of V. cholerae. Strains of V. cholerae were found in all four estuaries, but none possessed virulence genes.

l-rhamnose-binding lectin from eggs of the Echinometra lucunter: Amino acid sequence and molecular modeling

An L-rhamnose-binding lectin named ELEL was isolated from eggs of the rock boring sea urchin Echinometra lucunter by affinity chromatography on lactosyl-agarose. ELEL is a homodimer linked by a disulfide bond with subunits of 11 kDa each. The new lectin was inhibited by saccharides possessing the same configuration of hydroxyl groups at C-2 and C-4, such as L-rhamnose, melibiose, galactose and lactose. The amino acid sequence of ELEL was determined by tandem mass spectrometry. The ELEL subunit has 103 amino acids, including nine cysteine residues involved in four conserved intrachain disulfide bonds and one interchain disulfide bond. The full sequence of ELEL presents conserved motifs commonly found in rhamnose-binding lectins, including YGR, DPC and KYL. A three-dimensional model of ELEL was created, and molecular docking revealed favorable binding energies for interactions between ELEL and rhamnose, melibiose and Gb3 (Galα1-4Galβ1-4Glcβ1-Cer). Furthermore, ELEL was able to agglutinate Gram-positive bacterial cells, suggesting its ability to recognize pathogens.