Heteroantagonismo entre estirpes de Enterobacter agglomerans isoladas de urubu (Coragyps atratus) como produtoras e Pseudomonas aeruginosa como reveladoras / Evaluation of the heteroantagonism between Enterobacter agglomerans strains isolated from vulture (Coragyps atratus) for production and Pseudomonas aeruginosa as developed

Avaliou-se o heteroantagonismo entre Enterobacter agglomerans isolada do trato gastrintestinal de urubu (Coragyps atratus) e Pseudomonas aeruginosa isoladas do ambiente hospitalar. Foram utilizados o método de sobrecamada ou lento e a técnica direta ou de poços. Pelo método da sobrecamada, de 196 testes realizados para pesquisa da atividade antagonista, foi detectada a presença de halos de inibição relacionados ao fenômeno de heteroantagonismo em 118 deles (60,2 por cento). Pelo método de poços, obtiveram-se resultados semelhantes. As sete amostras de E. agglomerans foram capazes de realizar heteroantagonismo nas condições testadas, que foram detectados pela formação de halos claros de inibição. O extrato de levedura adicionado a 1 por cento no meio de cultura foi um suplemento adequado para a demonstração do antagonismo.

The heteroantagonism between Enterobacter agglomerans, isolated from the gastrointestinal tract of American vulture Coragyps atratus, and Pseudomonas aeruginosa isolated from a hospital environment was evaluated. The slow (layer) and the wells (direct) techniques were tested, using agar and soy tryptone broth pH 7.3 at 37ºC. Through the slow method from 196 tests, inhibition growth halos, related heteroantagonism phenomenon observed in 118, corresponding to 60.2 percent positive results. Equivalent positive results were detected using wells (direct) methodology. The seven samples of E. agglomerans tested were capable of revealing heteroantagonism in the experimental conditions; antagonism reveled by the presence of a clear growth inhibition halo. The added 1 percent yeast extract to media was adequate for revealing antagonisms best.

Heteroduplex mobility assay for rapid, sensitive and specific detection of mycobacteria.

We report an improved method for the detection and identification of mycobacteria using PCR and the heteroduplex mobility shift assay (HMA). The HMA for detection of mycobacteria was based on the microheterogeneity within the DNA coding sequences for 16S rRNA. A remarkable shift between single-stranded, heteroduplex and homoduplex bands in PAGE was observed among the Mycobacterium spp. tested. The Mycobacteria HMA (MHMA) of amplified PCR products from mycobacteria DNA coding for 16S rDNA derived from culture showed a specific heteroduplexes formed among different Mycobacterium species. Other bacterium species were distinguished from Mycobaterium due to slow migrating heteroduplexes mobility bands observed when M. bovis (BCG), M. avium, or M. fortuitum were used as a standard. The specific heteroduplexes were detected when as little as 1 etag of DNA template was used, although better results were obtained with 5 etag and when PCR products of sample test and mycobacterium standard were mixed at a ratio of 1.8. To correctly evaluate the feasibility of using MHMA to detect and identify mycobacteria, 15 clinical sample patients were tested. All MTB-positive clinical samples were identified by MHMA as well as the negative samples. In addition, MHMA will, in principle, be applicable to the detection and classification of any microorganism showing differences within the 16S rRNA as well as to the identification of new and unrecognized bacterial species.

Organization and structure of an Onchocerca beta-tubulin gene.

In filarial worms, as in other eukaryotes, microtubules are essential multifunctional components. The major protein of microtubules is tubulin, a heterodimer of two distinct polypeptides, alpha and beta. Tubulin is particularly important in helminthic parasites as a target for anthelminthic benzimidazoles, which bind to it and inhibit microtubule assembly. Two genomic Onchocerca gibsoni libraries were constructed in lambda NM1149 (EcoRI and HindIII). Three clones accounted for the entire gene: one from the EcoRI library (using a Plasmodium falciparum probe) containing the central part of the gene, and two from the HindIII library (using as probes PCR amplified fragments from the ends of the EcoRI clone) which, respectively, contained the 5'- and 3'-ends of the gene. The sequencing procedure for the EcoRI clone relied on the construction of a double-digested DraI/HindIII shotgun library. A number of recombinants were sequenced and aligned with each other for comparison. The sequencing of the overlapping 5'- and 3'-end clones was done by using a series of oligonucleotides. The sequence of the O. gibsoni beta-tubulin gene was completely determined, revealing an exceptionally complex structure as compared to the known beta-tubulin genes: 5,797 base pairs containing 12 exons and 11 introns. The deduced polypeptide is 444 amino acids long, and its sequence is highly conserved. The position of some introns appear to demarcate functional domains within the protein.

Organization and structure of an Onchocerca ß-tubulin gene

In filarial worms, as in other eukaryotes, microtubules are essential multifunctional components. The major protein of microtubules is tubulin, a heterodimer of two distinct polypeptides, Ó e ß.Tubulin is particulary important in helminthic parasites as a target for anthelminthic benzimidazoles, wich bind to it and inhibit microtubule assembly. Two genomic Onchocerca gibsoni libraries were constructed in NM1149(EcoRi and HindIII). Three clones accounted for the entire gene: one from the EcoRi library (using a Plasmodium falciparum probe) containing the central part of the gene, and two from the HindIII library (using as probes PCR amplified fragments from the ends of the EcoRI clone) which, respectively, contained the 5'- and 3' -ends of the gene. The sequencing procedure for the EcoRI clone relied on the construction of a double-digested DraI/HindIII shotgun library. A number of recombinants were sequenced and aligned with each other for comparison. The sequencing of the overlapping 5' - and 3'-end clones was done by ...