Detection of fiber-digesting bacteria in the forestomach contents of llamas (Lama glama) by PCR

AbstractThe high fibrolytic activity and large biomass of strictly-anaerobic bacteria that inhabit the rumen makes them primarily responsible for the degradation of the forage consumed by ruminants. Llamas feed mainly on low quality fibrous roughages that are digested by an active and diverse microflora. The products of this fermentation are volatile fatty acids and microbial biomass, which will be used by the animals. The aim of this study was to detect the three major fiber-digesting anaerobic bacteria in the forestomach contents of llamas by PCR. In this study, we detected Ruminococcus albus, Ruminococcus flavefaciens and Fibrobacter succinogenes in the forestomach contents of eight native llamas from Argentina

YqiC of Salmonella enterica serovar Typhimurium is a membrane fusogenic protein required for mice colonization

<p>Abstract</p> <p>Background</p> <p><it>Salmonella enterica </it>serovar Typhimurium is an intracellular bacterial pathogen which can colonize a variety of hosts, including human, causing syndromes that vary from gastroenteritis and diarrhea to systemic disease.</p> <p>Results</p> <p>In this work we present structural information as well as insights into the <it>in vivo </it>function of YqiC, a 99-residue protein of <it>S</it>. Typhimurium, which belongs to the cluster of the orthologous group 2960 (COG2960). We found that YqiC shares biophysical and biochemical properties with <it>Brucella abortus </it>BMFP, the only previously characterized member of this group, such as a high alpha helix content, a coiled-coil domain involved in trimerization and a membrane fusogenic activity <it>in vitro</it>. In addition, we demonstrated that YqiC localizes at cytoplasmic and membrane subcellular fractions, that a <it>S</it>. Typhimurium <it>yqiC </it>deficient strain had a severe attenuation in virulence in the murine model when inoculated both orally and intraperitoneally, and was impaired to replicate at physiological and high temperatures <it>in vitro</it>, although it was still able to invade and replicate inside epithelial and macrophages cell lines.</p> <p>Conclusion</p> <p>This work firstly demonstrates the importance of a COG2960 member for pathogen-host interaction, and suggests a common function conserved among members of this group.</p

Brucella

La brucelosis también llamada fiebre de Malta, fiebre ondulante o del Mediterráneo es una enfermedad zoonótica, conocida desde fines del siglo XIX, que afecta a especies animales de sangre caliente. Es de difusión mundial y aunque ha sido erradicada en algunos países industrializados continúa siendo un problema difícil de resolver en países del área del Mediterráneo, del oeste de Asia, de África y de América Latina. En nuestra región se la conoce desde principios del siglo XX, se cree que fue introducida con los animales ingresados por los españoles durante la conquista, aunque es difícil precisar dónde apareció por primera vez. Algunos autores afirman que en 1898 fue diagnosticada en Venezuela, también ha sido asociada a una epidemia descrita como fiebre de larga duración, ocurrida en Perú entre 1906 y 1907. Su persistencia se puede explicar por las particularidades geográficas de los países, la práctica de criar distintas especies de animales compartiendo pasturas y abrevaderos y la permanente interacción del medio rural con el urbano para adquirir insumos y comercializar productos. Los animales domésticos, en algunos lugares, son faenados sin control sanitario y sus múltiples derivados son manufacturados de manera artesanal, se expenden en la vecindad y en las rutas de acceso a las ciudades.Fil: Lucero, Nidia E.. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud "Dr. C. G. Malbrán"; ArgentinaFil: Almiron, Marta. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Cravero, Silvio L.. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias; ArgentinaFil: Trangoni, Marcos D.. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias; Argentin

Interplay between Two RND Systems Mediating Antimicrobial Resistance in Brucella suis▿

The RND-type efflux pumps are responsible for the multidrug resistance phenotype observed in many clinically relevant species. Also, RND pumps have been implicated in physiological processes, with roles in the virulence mechanisms of several pathogenic bacteria. We have previously shown that the BepC outer membrane factor of Brucella suis is involved in the efflux of diverse drugs, probably as part of a tripartite complex with an inner membrane translocase. In the present work, we characterize two membrane fusion protein-RND translocases of B. suis encoded by the bepDE and bepFG loci. MIC assays showed that the B. suis ΔbepE mutant was more sensitive to deoxycholate (DOC), ethidium bromide, and crystal violet. Furthermore, multicopy bepDE increased resistance to DOC and crystal violet and also to other drugs, including ampicillin, norfloxacin, ciprofloxacin, tetracycline, and doxycycline. In contrast to the ΔbepE mutant, the resistance profile of B. suis remained unaltered when the other RND gene (bepG) was deleted. However, the ΔbepE ΔbepG double mutant showed a more severe phenotype than the ΔbepE mutant, indicating that BepFG also contributes to drug resistance. An open reading frame (bepR) coding for a putative regulatory protein of the TetR family was found upstream of the bepDE locus. BepR strongly repressed the activity of the bepDE promoter, but DOC released the repression mediated by BepR. A clear induction of the bepFG promoter activity was observed only in the BepDE-defective mutant, indicating a regulatory interplay between the two RND efflux pumps. Although only the BepFG-defective mutant showed a moderate attenuation in model cells, the activities of both bepDE and bepFG promoters were induced in the intracellular environment of HeLa cells. Our results show that B. suis harbors two functional RND efflux pumps that may contribute to virulence

LAMP technology: Rapid identification of Brucella and Mycobacterium avium subsp. paratuberculosis

In this study, we developed new sets of primers to detect Brucella spp. and M. avium subsp. paratuberculosis (MAP) through isothermal amplification. We selected a previously well-characterized target gene, bscp31, specific for Brucella spp. and IS900 for MAP. The limits of detection using the loop-mediated isothermal amplification (LAMP) protocols described herein were similar to those of conventional PCR targeting the same sequences. Hydroxynaphtol blue and SYBR GreenTM allowed direct naked-eye detection with identical sensitivity as agarose gel electrophoresis. We included the LAMP-based protocol in a rapid identification scheme of the respective pathogens, and all tested isolates were correctly identified within 2 to 3 h. In addition, both protocols were suitable for specifically identifying the respective pathogens; in the case of Brucella, it also allowed the identification of all the biovars tested. We conclude that LAMP is a suitable rapid molecular typing tool that could help to shorten the time required to identify insidious bacteria in low-complexity laboratories, mainly in developing countries

Identification and Characterization of a Brucella abortus ATP-Binding Cassette Transporter Homolog to Rhizobium meliloti ExsA and Its Role in Virulence and Protection in Mice

Brucella abortus is a facultative intracellular bacterial pathogen that causes abortion in domestic animals and undulant fever in humans. The mechanism of virulence of Brucella spp. is not fully understood yet. Furthermore, genes that allow Brucella to reach the intracellular niche and to interact with host cells need to be identified. Using the genomic survey sequence (GSS) approach, we identified the gene encoding an ATP-binding cassette (ABC) transporter of B. abortus strain S2308. The deduced amino acid sequence encoded by this gene exhibited 69 and 67% identity with the sequences of the ABC transporters encoded by the exsA genes of Rhizobium meliloti and Mesorhizobium loti, respectively. Additionally, B. abortus ExsA, like R. meliloti and M. loti ExsA, possesses ATP-binding motifs and the ABC signature domain features of a typical ABC transporter. Furthermore, ortholog group analysis placed B. abortus ExsA in ortholog group 6 of ABC transporters more likely to be involved in bacterial pathogenesis. In R. meliloti, ExsA is an exopolysaccharide transporter essential for alfalfa root nodule invasion and establishment of infection. To test the role of ExsA in Brucella pathogenesis, an exsA deletion mutant was constructed. Replacement of the wild-type exsA by recombination was demonstrated by Southern blot analysis of Brucella genomic DNA. Decreased survival in mice of the Brucella ΔexsA mutant compared to the survival of parental strain S2308 demonstrated that ExsA is critical for full bacterial virulence. Additionally, the B. abortus exsA deletion mutant was used as a live vaccine. Challenge experiments revealed that the exsA mutant strain induced superior protective immunity in BALB/c mice compared to the protective immunity induced by strain S19 or RB51

The TolC Homologue of Brucella suis Is Involved in Resistance to Antimicrobial Compounds and Virulence

Brucella spp., like other pathogens, must cope with the environment of diverse host niches during the infection process. In doing this, pathogens evolved different type of transport systems to help them survive and disseminate within the host. Members of the TolC family have been shown to be involved in the export of chemically diverse molecules ranging from large protein toxins to small toxic compounds. The role of proteins from the TolC family in Brucella and other α-2-proteobacteria has been explored little. The gene encoding the unique member of the TolC family from Brucella suis (BepC) was cloned and expressed in an Escherichia coli mutant disrupted in the gene encoding TolC, which has the peculiarity of being involved in diverse transport functions. BepC fully complemented the resistance to drugs such as chloramphenicol and acriflavine but was incapable of restoring hemolysin secretion in the tolC mutant of E. coli. An insertional mutation in the bepC gene strongly affected the resistance phenotype of B. suis to bile salts and toxic chemicals such as ethidium bromide and rhodamine and significantly decreased the resistance to antibiotics such as erythromycin, ampicillin, tetracycline, and norfloxacin. Moreover, the B. suis bepC mutant was attenuated in the mouse model of infection. Taken together, these results suggest that BepC-dependent efflux processes of toxic compounds contribute to B. suis survival inside the host