Antigenic and genetic characterization of the first rabies virus isolated from the bat Eumops perotis in Brazil

Although the main transmitters of rabies in Brazil are dogs and vampire bats, the role of other species such as insectivorous and frugivorous bats deserves special attention, as the rabies virus has been isolated from 36 bat species. This study describes the first isolation of the rabies virus from the insectivorous bat Eumops perotis. The infected animal was found in the city of Ribeirão Preto, São Paulo. The virus was identified by immunofluorescence antibody test (FAT) in central nervous system (CNS) samples, and the isolation was carried out in N2A cell culture and adult mice. The sample was submitted to antigenic typing using a panel of monoclonal antibodies (CDC/Atlanta/USA). The DNA sequence of the nucleoprotein gene located between nucleotides 102 and 1385 was aligned with homologous sequences from GenBank using the CLUSTAL/W method, and the alignment was used to build a neighbor-joining distance-based phylogenetic tree with the K-2-P model. CNS was negative by FAT, and only one mouse died after inoculation with a suspension from the bat's CNS. Antigenic typing gave a result that was not compatible with the patterns defined by the panel. Phylogenetic analysis showed that the virus isolated segregated into the same cluster related to other viruses isolated from insectivorous bats belonging to genus Nyctinomops ssp. (98.8% nucleotide identity with each other).

Genotypic identification of Cryptosporidium spp. isolated from hiv-infected patients and immunocompetent children of São Paulo, Brazil

Cryptosporidium isolates identified in fourteen stool samples, collected from five HIV-infected patients and nine immunocompetent children, living in the Sate of São Paulo, Brazil, were submitted to a molecular analysis using a nested PCR followed of restriction fragment length polymorphism (RFLP), for genetic characterization. The analysis was based on digestion with RsaI restriction enzyme of a DNA fragment amplified from the Cryptosporidium oocyst wall protein (COWP) gene. Based on this analysis, four samples were identified as Cryptosporidium parvum, eight as Cryptosporidium hominis and two presented a profile that correspondedto Cryptosporidium meleagridis when compared to the standards used in the analysis. The use of molecular methods can be helpful to identify source of infections and risk factors related to Cryptosporidium infection in our communities.

Enzymatic characterization of Vibrio alginolyticus strains isolated from bivalves harvested at Venice Lagoon (Italy) and Guanabara Bay (Brazil)

The aquatic ecosystem is the natural habitat of microorganisms including Vibrio and Aeromonas genus which are pathogenic to human and animals. In the present investigation the frequency of these bacteria and the enzymatic characteristics of 34 Vibrio alginolyticus strains isolated from bivalves harvested in Venice Lagoon (Italy) and Guanabara Bay (Brazil) were carried out from November 2003 to February 2004. The mussels' samples were submitted to enrichment in Alkaline Peptone Water (APW) added with 1% of sodium chloride (NaCl) and APW plus 3% NaCl incubated at 37 ºC for 18-24h. Following the samples were streaked onto TCBS Agar (Thiossulfate Citrate Bile Sucrose Agar) and the suspected colonies were submitted to biochemical characterization. Also, the Vibrio alginolyticus strains were evaluated to collagenase, elastase and chondroitinase production. The results showed the isolation of 127 microorganisms distributed as follows: 105 Vibrio strains such as V. alginolyticus (32.4%), V. harveyi (19%) and V. parahaemolyticus (7.6%), 20 Aeromonas strains and two Plesiomonas shigelloides were the main pathogens isolated. We observed the production of the three enzymes from V. alginolyticus strains considered as the main virulence factors of the bacteria, especially in cases of human dermatological infection.

Molecular characterization of environmental Cryptococcus neoformans isolated in Vitoria, ES, Brazil

Cryptococcus neoformans is the major cause of fungal meningitis, a potentially lethal mycosis. Bird excreta can be considered a significant environmental reservoir of this species in urban areas, thirty-three samples of pigeon excreta were collected within the city of Vitoria, Brazil. Cryptococcus neoformans was isolated and identified using standard biochemical assays in ten samples. PCR amplification with primer M13 and orotidine monophosphate pyrophosphorylase (URA5) gene-restriction fragment length polymorphism (RFLP) analysis discerned serotypes and genotypes within this species. All isolates were serotype A (C. neoformans var. grubii) and genotype VNI. The two alternative alleles a and α at the mating type locus were determined by PCR amplification and mating assays performed on V8 medium. All isolates were MAT α mating type but only 50% were able to mate in vitro with the opposite mating type MAT a tester strains (JEC20, KN99a and Bt63). This study adds information on the ecology and molecular characterization of C. neoformans in the Southeast region of Brazil.

In vitro susceptibility testing of dermatophytes isolated in Goiania, Brazil, against five antifungal agents by broth microdilution method

The antifungal activities of fluconazole, itraconazole, ketoconazole, terbinafine and griseofulvin were tested by broth microdilution technique, against 60 dermatophytes isolated from nail or skin specimens from Goiania city patients, Brazil. In this study, the microtiter plates were incubated at 28 ºC allowing a reading of the minimal inhibitory concentration (MIC) after four days of incubation for Trichophyton mentagrophytes and five days for T. rubrum and Microsporum canis. Most of the dermatophytes had uniform patterns of susceptibility to the antifungal agents tested. Low MIC values as 0.03 µg/mL were found for 33.3%, 31.6% and 15% of isolates for itraconazole, ketoconazole and terbinafine, respectively.

Extended-spectrum beta-lactamases among Enterobacteriaceae isolated in a public hospital in Brazil

Extended-spectrum β-lactamases (ESBL) in enterobacteria are recognized worldwide as a great hospital problem. In this study, 127 ESBL-producing Enterobacteriaceae isolated in one year from inpatients and outpatients at a public teaching hospital at São Paulo, Brazil, were submitted to analysis by PCR with specific primers for blaSHV, blaTEM and blaCTX-M genes. From the 127 isolates, 96 (75.6%) Klebsiella pneumoniae, 12 (9.3%) Escherichia coli, 8 (6.2%) Morganella morganii, 3 (2.3%) Proteus mirabilis, 2 (1.6%) Klebsiella oxytoca, 2 (1.6%) Providencia rettgeri, 2 (1.6%) Providencia stuartti, 1 (0.8%) Enterobacter aerogenes and 1 (0.8%) Enterobacter cloacae were identified as ESBL producers. BlaSHV, blaTEM and blaCTX-M were detected in 63%, 17.3% and 33.9% strains, respectively. Pulsed field gel eletrophoresis genotyping of K. pneumoniae revealed four main molecular patterns and 29 unrelated profiles. PCR results showed a high variety of ESBL groups among strains, in nine different species. The results suggest the spread of resistance genes among genetically different strains of ESBL-producing K. pneumoniae in some hospital wards, and also that some strongly related strains were identified in different hospital wards, suggesting clonal spread in the institutional environment.

Análise da estrutura e da função da proteína morfogenética RodZ de Bacillus subtilis

Resumo: RodZ é um componente do sistema morfogenético das células bacterianas. É uma proteína transmembranar que localiza em bandas ao longo do eixo longitudinal da célula. Em Bacillus subtilis, RodZ consiste numa porção citoplasmática, RodZn, e em uma parte extra-citoplasmática, RodZc. RodZn contém um domínio em helixturn- helix (HTH), enquanto que RodZc pode ser dividido num domínio coiled-coil e num domínio terminal C, de função desconhecida. Um segmento transmembranar (TM) único separa RodZn de RodZc. A eliminação de rodZ causa alongamento do nucleóide e leva à produção de células polares nucleadas. Aqui, mostramos que RodZn é estruturado, estável e em hélice α. Descobrimos que as substituições Y32A e L33A na suposta hélice de reconhecimento (3) do motivo HTH, bem como as substituições Y49A e F53A, fora do motivo HTH (4), causam divisão assimétrica, mas apenas as últimas levam à deslocalização sub-celular de RodZ. Sugerimos que as hélices 3 e 4 são utilizadas para uma interacção proteína-proteína ou proteína- DNA essencial para divisão celular enquanto que 4 deve contactar um componente do citosqueleto, possivelmente MreB, uma vez que a correcta localização sub-celular de RodZ depende desta proteína. Em todos os mutantes as células polares são anucleadas, pelo que concluímos que o alongamento do nucleóide não é um prérequisito para divisão assimétrica. RodZc é largamente não estruturado mas com conteúdo de folha , sendo estabilizado pelo domínio coiled-coil. Mostramos uma relação homóloga entre RodZc e a bomba de transporte Na+/Ca2+ NCX1 e identificámos dois resíduos no domínio C, G265 e N275, essenciais para a manutenção da forma celular. Estes resíduos fazem parte de um motivo em gancho que pode actuar como um local de interacção com um ligando desconhecido. RodZn e RodZc são monoméricos em solução. Contudo, na membrana, RodZ interage consigo própria num sistema de dois híbridos (Split-Ubiquitin) em levedura, sugerindo que possa formar multímeros in vivo.-----------ABSTRACT: RodZ is a transmembrane component of the bacterial core morphogenic apparatus. RodZ localizes in bands long the longitudinal axis of the cell, and it is though to functionally link the cell wall to the actin cytoskeleton. In Bacillus subtilis, RodZ consists of a cytoplasmic moiety, RodZn, and an extracytoplasmic moiety, RodZc. RodZn contains a predicted helix-turn-helix domain, whereas RodZc is thought to contain a coiled-coil region and a terminal C domain of unknown function. A single transmembrane domain separates RodZn from RodZc. Deletion of rodZ causes elongation of the nucleoid and leads to the production of polar minicells containing DNA. Here, we have studied the structure and function of RodZn and RodZc. We show that RodZn is a stable, folded, -helical domain. We discovered that the Y32A and L33A substitutions within the presumptive recognition helix (3) of the HTH motif, as well as the Y49A and F53A substitutions outside of the HTH motif (in 4) cause asymmetric cell division. However, only the substitutions in 4 cause sub-celular delocalization of RodZ. We suggest that 3 and 4 are used for a protein-protein or protein-DNA interaction important for cell division, whereas 4 is likely to contact a cytoskeletal component, presumably MreB. The polar cells formed by all the mutants are anucleate. We conclude that nucleoid elongation is not a prerequisite for asymmetric division. RodZc appears to be a largely unstructured domain, with some -sheet content, and is stabilized by the coiled-coil region. We show a homology relationship between RodZc and the NCX1 Na+/Ca2+ transporter and we found two residues within the C domain, G265 and N275, that are important for cell shape determination. These residues are predicted to be essential determinants of a claw-like motif, which may act as a binding site for an unknown ligand. Both the isolated RodZn and RodZc proteins are monomeric in solution. However, because full-length RodZ interacts with itself in a split-ubiquitin yeast two-hybrid assay, we suggest that it may dimerize or form higher order multimers in vivo.

Characterization of rabies virus isolated from a colony of Eptesicus furinalis bats in Brazil

Some bat species have adapted to the expanding human population by acquiring the ability to roost in urban buildings, increasing the exposure risk for people and domestic animals, and consequently, the likelihood of transmitting rabies. Three dead bats were found in the yard of a house in an urban area of Jundiaí city in the state of São Paulo in southeast Brazil. Two of the three bats tested positive for rabies, using Fluorescent Antibody and Mouse Inoculation techniques. A large colony of Eptesicus furinalis was found in the house's attic, and of the 119 bats captured, four more tested positive for rabies. The objectives of this study were to report the rabies diagnosis, characterize the isolated virus antigenically and genetically, and study the epidemiology of the colony.

Characterization of Vibrio Parahaemolyticus isolated from oysters and mussels in São Paulo, Brazil

Vibrio parahaemolyticus is a marine bacterium, responsible for gastroenteritis in humans. Most of the clinical isolates produce thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) encoded by tdh and trh genes respectively. In this study, twenty-three V. parahaemolyticus, previously isolated from oysters and mussels were analyzed by PCR using specific primers for the 16S rRNA and virulence genes (tdh, trh and tlh) and for resistance to different classes of antibiotics and PFGE. Nineteen isolates were confirmed by PCR as V. parahaemolyticus. The tlh gene was present in 100% of isolates, the tdh gene was identified in two (10.5%) isolates, whereas the gene trh was not detected. Each isolate was resistant to at least one of the nine antimicrobials tested. Additionally, all isolates possessed the blaTEM-116 gene. The presence of this gene in V. parahaemolyticus indicates the possibility of spreading this gene in the environment. Atypical strains of V. parahaemolyticus were also detected in this study.

Molecular characterization of Aeromonas spp. and Vibrio cholerae O1 isolated during a diarrhea outbreak

This work aimed to assess pathogenic potential and clonal relatedness of Aeromonas sp. and Vibrio cholerae isolates recovered during a diarrhea outbreak in Brazil. Clinical and environmental isolates were investigated for the presence of known pathogenic genes and clonal relatedness was assessed by intergenic spacer region (ISR) 16S-23S amplification. Four Aeromonas genes (lip, exu, gcat, flaA/B) were found at high overall frequency in both clinical and environmental isolates although the lip gene was specifically absent from selected species. A fifth gene, aerA, was rarely found in A. caviae, the most abundant species. The ISR profile revealed high heterogeneity among the Aeromonas isolates and no correlation with species identification. In contrast, in all the V. cholerae isolates the four genes investigated (ctxA, tcpA, zot and ace) were amplified and revealed homogeneous ISR and RAPD profiles. Although Aeromonas isolates were the major enteric pathogen recovered, their ISR profiles are not compatible with a unique cause for the diarrhea events, while the clonal relationship clearly implicates V. cholerae in those cases from which it was isolated. These results reinforce the need for a better definition of the role of aeromonads in diarrhea and whether they benefit from co-infection with V. cholerae.

Toxicity of saponin isolated from Gymnema sylvestre R. Br. (Asclepiadaceae) against Culex tritaeniorhynchus Giles (Diptera: Culicidae) Japanese encephalitis vector mosquito in India

To determine the larvicidal activity of various extracts of Gymnema sylvestre against the Japanese Encephalitis vector, Culex tritaeniorynchus in Tamilnadu, India. To identify the active principle present in the promising fraction obtained in Chlorofom:Methanol extract of Fraction 2. The G. sylvestre leaf extracts were tested, employing WHO procedure against fourth instar larvae of C. tritaeniorhynchus and the larval mortalities were recorded at various concentrations (6.25, 12.5, 25.0, 50 and 100 µg/mL); the 24h LC50 values of the G. Sylvestre leaf extracts were determined following Probit analysis. It was noteworthy that treatment level 100 µg/mL exhibited highest mortality rates for the three different crude extracts and was significantly different from the mean mortalities recorded for the other concentrations. The LC50 values of 34.756 µg/mL (24.475-51.41), 31.351 µg/mL (20.634-47.043) and 28.577 µg/mL (25.159-32.308) were calculated for acetone, chloroform and methanol extract with the chi-square values of 10.301, 31.351 and 4.093 respectively. The present investigation proved that G. Sylvestre could be possibly utilized as an important component in the Vector Control Program.

gvSIG An Open Source GIS solution for the Android Platform

Dissertation submitted in partial fulfilment of the requirements for the Degree of Master of Science in Geospatial Technologies

RESISTANCE TO AMOXICILLIN, CLARITHROMYCIN AND CIPROFLOXACIN OF Helicobacter pylori ISOLATED FROM SOUTHERN BRAZIL PATIENTS

Introduction: Helicobacter pylori is a bacteria which infects half the world population and is an important cause of gastric cancer. The eradication therapy is not always effective because resistance to antimicrobials may occur. The aim of this study was to determine the susceptibility profile of H. pylori to amoxicillin, clarithromycin and ciprofloxacin in the population of Southern Brazil. Material and methods: Fifty four samples of H. pylori were evaluated. The antibiotics susceptibility was determined according to the guidelines of the British Society for Antimicrobial Chemotherapy and the Comité de l'Antibiogramme de la Société Française de Microbiologie. Results: Six (11.1%) H. pylori isolates were resistant to clarithromycin, one (1.9%) to amoxicillin and three (5.5%) to ciprofloxacin. These indices of resistance are considered satisfactory and show that all of these antibiotics can be used in the empirical therapy. Conclusion: The antibiotics amoxicillin and clarithromycin are still a good option for first line anti-H. pylori treatment in the population of Southern Brazil.

ANTIMICROBIAL DRUG RESISTANCE IN STRAINS OF Escherichia coli ISOLATED FROM FOOD SOURCES

A variety of foods and environmental sources harbor bacteria that are resistant to one or more antimicrobial drugs used in medicine and agriculture. Antibiotic resistance in Escherichia coli is of particular concern because it is the most common Gram-negative pathogen in humans. Hence this study was conducted to determine the antibiotic sensitivity pattern of E. coli isolated from different types of food items collected randomly from twelve localities of Hyderabad, India. A total of 150 samples comprising; vegetable salad, raw egg-surface, raw chicken, unpasteurized milk, and raw meat were processed microbiologically to isolate E. coli and to study their antibiotic susceptibility pattern by the Kirby-Bauer method. The highest percentages of drug resistance in isolates of E. coli were detected from raw chicken (23.3%) followed by vegetable salad (20%), raw meat (13.3%), raw egg-surface (10%) and unpasteurized milk (6.7%). The overall incidence of drug resistant E. coli was 14.7%. A total of six (4%) Extended Spectrum β-Lactamase (ESBL) producers were detected, two each from vegetable salads and raw chicken, and one each from raw egg-surface and raw meat. Multidrug resistant strains of E. coli are a matter of concern as resistance genes are easily transferable to other strains. Pathogen cycling through food is very common and might pose a potential health risk to the consumer. Therefore, in order to avoid this, good hygienic practices are necessary in the abattoirs to prevent contamination of cattle and poultry products with intestinal content as well as forbidding the use of untreated sewage in irrigating vegetables.