In vitro evaluation of single- and multi-strain probiotics: inter-species inhibition between probiotic strains, and inhibition of pathogens

Many studies comparing the effects of single- and multi-strain probiotics on pathogen inhibition compare treatments with different concentrations. They also do not examine the possibility of inhibition between probiotic strains with a mixture. We tested the ability of 14 single-species probiotics to inhibit each other using a cross-streak assay, and agar spot test. We then tested the ability of 15 single-species probiotics and 5 probiotic mixtures to inhibit C. difficile, E. coli and S. Typhimurium, using the agar spot test. Testing was done with mixtures created in two ways: one group contained component species incubated together, the other group of mixtures was made using component species which had been incubated separately, equalised to equal optical density, and then mixed in equal volumes. Inhibition was observed for all combinations of probiotics, suggesting that when used as such there may be inhibition between probiotics, potentially reducing efficacy of the mixture. Significant inter-species variation was seen against each pathogen. When single species were tested against mixtures, the multi-species preparations displayed significantly (p<0.05 or less) greater inhibition of pathogens in 12 out of 24 cases. Despite evidence that probiotic species will inhibit each other when incubated together in vitro, in many cases a probiotic mixture was more effective at inhibiting pathogens than its component species when tested at approximately equal concentrations of biomass. This suggests that using a probiotic mixture might be more effective at reducing gastrointestinal infections, and that creating a mixture using species with different effects against different pathogens may have a broader spectrum of action that a single provided by a single strain.

The rulB gene of plasmid pWW0 is a hotspot for the site-specific insertion of integron-like elements found in the chromosomes of environmental Pseudomonas fluorescens group bacteria

The rulAB operon of Pseudomonas spp. confers fitness traits on the host and has been suggested to be a hotspot for insertion of mobile elements that carry avirulence genes. Here, for the first time, we show that rulB on plasmid pWW0 is a hotspot for the active site-specific integration of related integron-like elements (ILEs) found in six environmental pseudomonads (strains FH1–FH6). Integration into rulB on pWW0 occurred at position 6488 generating a 3 bp direct repeat. ILEs from FH1 and FH5 were 9403 bp in length and contained eight open reading frames (ORFs), while the ILE from FH4 was 16 233 bp in length and contained 16 ORFs. In all three ILEs, the first 5.1 kb (containing ORFs 1–4) were structurally conserved and contained three predicted site-specific recombinases/integrases and a tetR homologue. Downstream of these resided ORFs of the ‘variable side’ with structural and sequence similarity to those encoding survival traits on the fitness enhancing plasmid pGRT1 (ILEFH1 and ILEFH5) and the NR-II virulence region of genomic island PAGI-5 (ILEFH4). Collectively, these ILEs share features with the previously described type III protein secretion system effector ILEs and are considered important to host survival and transfer of fitness enhancing and (a)virulence genes between bacteria.

Relation of pretreatment sequence diversity in NS5A region of HCV genotype 1 with immune response between pegylated-INF/ribavirin therapy outcomes

Hepatitis C virus (HCV) infection frequently persists despite substantial virus-specific immune responses and the combination of pegylated interferon (INF)-alpha and ribavirin therapy. Major histocompatibility complex class I restricted CD8+ T cells are responsible for the control of viraemia in HCV infection, and several studies suggest protection against viral infection associated with specific HLAs. The reason for low rates of sustained viral response (SVR) in HCV patients remains unknown. Escape mutations in response to cytotoxic T lymphocyte are widely described; however, its influence in the treatment outcome is ill understood. Here, we investigate the differences in CD8 epitopes frequencies from the Los Alamos database between groups of patients that showed distinct response to pegylated alpha-INF with ribavirin therapy and test evidence of natural selection on the virus in those who failed treatment, using five maximum likelihood evolutionary models from PAML package. The group of sustained virological responders showed three epitopes with frequencies higher than Non-responders group, all had statistical support, and we observed evidence of selection pressure in the last group. No escape mutation was observed. Interestingly, the epitope VLSDFKTWL was 100% conserved in SVR group. These results suggest that the response to treatment can be explained by the increase in immune pressure, induced by interferon therapy, and the presence of those epitopes may represent an important factor in determining the outcome of therapy.

The effect of carbonyl group in the asymmetry Of (3,4)J(CH) coupling constants in norbornanones

A rationalization of the known difference between the (3,4)J(C4H1) and (3,4)J(C1H4) couplings transmitted mainly through the 7-bridge in norbornanone is presented in terms of the effects of hyperconjugative interactions involving the carbonyl group. Theoretical and experimental studies Of (3,4)J(CH) couplings were carried out in 3-endo- and 3-exo-X-2-norbornanone derivatives (X = Cl, Br) and in exo- and endo-2-noborneol compounds. Hyperconjugative interactions were studied with the natural bond orbital (NBO) method. Hyperconjugative interactions involving the carbonyl pi*c(2) =o and sigma*c(2) =o antibonding orbitals produce a decrease of three-bond contribution to both (3,4) J(C4H1) and (3,4)J(C1H4) couplings. However, the latter antibonding orbital also undergoes a strong sigma c(3)-c(4) ->sigma*c(2) =o interaction, which defines an additional coupling pathway for (3,4)J(C4H1) but not for (3,4)J(C1H4). This pathway is similar to that known for homoallylic couplings, the only difference being the nature of the intermediate antibonding orbital; i.e. for (3,4)J(C4H1) it is of sigma*-type, while in homoallylic couplings it is of pi*-type. Copyright (c) 2007 John Wiley & Sons, Ltd.

Disparity between Multilocus Enzyme Electrophoresis, Microsatellite Markers and Pulsed-Field Gel Electrophoresis in epidemiological tracking of Candida albicans

Various molecular systems are available for epidemiological, genetic, evolutionary, taxonomic and systematic studies of innumerable fungal infections, especially those caused by the opportunistic pathogen C. albicans. A total of 75 independent oral isolates were selected in order to compare Multilocus Enzyme Electrophoresis (MLEE), Electrophoretic Karyotyping (EK) and Microsatellite Markers (Simple Sequence Repeats - SSRs), in their abilities to differentiate and group C. albicans isolates (discriminatory power), and also, to evaluate the concordance and similarity of the groups of strains determined by cluster analysis for each fingerprinting method. Isoenzyme typing was performed using eleven enzyme systems: Adh, Sdh, M1p, Mdh, Idh, Gdh, G6pdh, Asd, Cat, Po, and Lap (data previously published). The EK method consisted of chromosomal DNA separation by pulsed-field gel electrophoresis using a CHEF system. The microsatellite markers were investigated by PCR using three polymorphic loci: EF3, CDC3, and HIS3. Dendrograms were generated by the SAHN method and UPGMA algorithm based on similarity matrices (S(SM)). The discriminatory power of the three methods was over 95%, however a paired analysis among them showed a parity of 19.7-22.4% in the identification of strains. Weak correlation was also observed among the genetic similarity matrices (S(SM)(MLEE) x S(SM)(EK) x S(SM)(SSRs)). Clustering analyses showed a mean of 9 +/- 12.4 isolates per cluster (3.8 +/- 8 isolates/taxon) for MLEE, 6.2 +/- 4.9 isolates per cluster (4 +/- 4.5 isolates/taxon) for SSRs, and 4.1 +/- 2.3 isolates per cluster (2.6 +/- 2.3 isolates/taxon) for EK. A total of 45 (13%), 39(11.2%), 5 (1.4%) and 3 (0.9%) clusters pairs from 347 showed similarity (Si) of 0.1-10%, 10.1-20%, 20.1-30% and 30.1-40%, respectively. Clinical and molecular epidemiological correlation involving the opportunistic pathogen C. albicans may be attributed dependently of each method of genotyping (i.e., MLEE, EK, and SSRs) supplemented with similarity and grouping analysis. Therefore, the use of genotyping systems that give results which offer minimum disparity, or the combination of the results of these systems, can provide greater security and consistency in the determination of strains and their genetic relationships. (C) 2010 Elsevier B.V. All rights reserved.

Differences in gastroprotective and mutagenic actions between polar and apolar extracts of Ananas ananassoides

Several plants are used in folk medicine to treat gastrointestinal disorders. Ananas ananassoides (Baker) L. B. Smith (Family Bromeliaceae) is a medicinal plant commonly used in the central region of Brazil against gastric pain. We evaluated two extracts (methanol [MeOH] and dichloromethane [DCM]) obtained from the leaves of A. ananassoides for their ability to protect the gastric mucosa against injuries caused by necrotizing agents (0.3 M HCl/60% ethanol, absolute ethanol, non-steroidal anti-inflammatory drugs, and pylorus ligation) in mice and rats. The best results were obtained after pretreatment with the DCM extract, whereas the MeOH extract did not show any significant anti-ulcerogenic activity but presented mutagenic action. The mechanism of action of the DCM extract suggested the effective participation of endogenous sulfhydryl group in the gastroprotective action. The data, taken together with the absence of acute toxicity and mutagenicity, indicate the apolar extract, instead of the polar, extract of A. ananassoides as a safe and potential new anti-ulcerogenic drug.

Diversity and evolution of macrohabitat use, body size and morphology in a monophyletic group of Neotropical pitvipers (Bothrops)

The Neotropical pitviper genus Bothrops comprises about 40 species, which occur in all main ecosystems of cis-Andean South America. We explored the relationships of body size and form (tail length and stoutness) with macrohabitat use in 20 forms of Bothrops. Sen-ii-arboreal habits appeared only in forest forms. Semi-arboreals are significantly more slender and have longer tails than terrestrials; body size is not significantly different between terrestrials and semi-arboreals. Within Bothrops, independent contrasts for macrohabitat use were significantly correlated with contrasts of tail size (positively) and stoutness (negatively); thus, the more arboreal the species, the longer its tail and the more slender its body. Contrasts of adult body size seems to remain constant over the lower range of macrohabitat use, but to decrease in species of Bothrops which are more arboreal. Reconstructions of character states indicate that: (1) the ancestor of Bothrops was a small, stout, terrestrial species; (2) semi-arboreal habits appeared one to three times in the genus; (3) a decrease in stoutness and an increase in tail length occurred along with an increase in arboreality in some clades. Although macrohabitat use seems to be important in determining body form in Bothrops, our results also indicate that tail size, stoutness and body size may also be affected by selective agents other than macrohabitat use. The selective agents responsible for the shifts in macrohabitat use in Bothrops are still uncertain, although they may have included prey availability and/or predation pressure. The plasticity of macrohabitat use, morphology and body size described in this study may have been key features that facilitated the highly successful ecological diversification of Bothrops in South America.

Gene and species trees of a Neotropical group of treefrogs: Genetic diversification in the Brazilian Atlantic Forest and the origin of a polyploid species

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Relation of pretreatment sequence diversity in NS5A region of HCV genotype 1 with immune response between pegylated-INF/ribavirin therapy outcomes

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Duffy blood group gene polymorphisms among malaria vivax patients in four areas of the Brazilian Amazon region

Background: Duffy blood group polymorphisms are important in areas where Plasmodium vivax predominates, because this molecule acts as a receptor for this protozoan. In the present study, Duffy blood group genotyping in P. vivax malaria patients from four different Brazilian endemic areas is reported, exploring significant associations between blood group variants and susceptibility or resistance to malaria.Methods: the P. vivax identification was determined by non-genotypic and genotypic screening tests. The Duffy blood group was genotyped by PCR/RFLP in 330 blood donors and 312 malaria patients from four Brazilian Amazon areas. In order to assess the variables significance and to obtain independence among the proportions, the Fisher's exact test was used.Results: the data show a high frequency of the FYA/FYB genotype, followed by FYB/FYB, FYA/FYA, FYA/FYB-33 and FYB/FYB-33. Low frequencies were detected for the FYA/FY(X), FYB/FY(X), FYX/FY(X) and FYB-33/FYB-33 genotypes. Negative Duffy genotype (FYB-33/FYB-33) was found in both groups: individuals infected and non-infected (blood donors). No individual carried the FY(X)/FYB-33 genotype. Some of the Duffy genotypes frequencies showed significant differences between donors and malaria patients.Conclusion: the obtained data suggest that individuals with the FYA/FYB genotype have higher susceptibility to malaria. The presence of the FYB-33 allele may be a selective advantage in the population, reducing the rate of infection by P. vivax in this region. Additional efforts may contribute to better elucidate the physiopathologic differences in this parasite/host relationship in regions endemic for P. vivax malaria, in particular the Brazilian Amazon region.

The Serido Group of NE Brazil, a late Neoproterozoic pre- to syn-collisional basin in West Gondwana: insights from SHRIMP U-Pb detrital zircon ages and Sm-Nd crustal residence (T-DM) ages

The Serido Group is a deformed and metamorphosed metasedimentary sequence that overlies early Paleoproterozoic to Archean basement of the Rio Grande do Norte domain in the Borborema Province of NE Brazil. The age of the Serido Group has been disputed over the past two decades, with preferred sedimentation ages being either Paleoproterozoic or Neoproterozoic. Most samples of the Serido Formation, the upper part of the Serido Group, have Sm-Nd T-DM ages between 1200 and 1600 Ma. Most samples of the Jucurutu Formation, the lower part of the Serido Group, have T-DM ages ranging from 1500 to 1600 Ma; some basal units have T-DM ages as old as 2600 Ma, reflecting proximal basement. Thus, based on Sm-Nd data, most, if not all, of the Serido Group was deposited after 1600 Ma and upper parts must be younger than 1200 Ma.Cathodoluminescence photos of detrital zircons show very small to no overgrowths produced during ca. 600 Ma Brasiliano deformation and metamorphism, so that SHRIMP and isotope dilution U-Pb ages must represent crystallization ages of the detrital zircons. Zircons from meta-arkose near the base of the Jucurutu Formation yield two groups of ages: ca. 2200 Ma and ca. 1800 Ma. In contrast, zircons from a metasedimentary gneiss higher in the Jucurutu Formation yield much younger ages, with clusters at ca. 1000 Ma and ca. 650 Ma. Zircons from metasedimentary and metatuffaceous units in the Serido Formation also yield ages primarily between 1000 and 650 Ma, with clusters at 950-1000, 800, 750, and 650 Ma. Thus, most, if not all, of the Serido Group must be younger than 650 Ma. Because these units were deformed and metamorphosed in the ca. 600 Ma Brasiliano fold belt during assembly of West Gondwana, deposition probably occurred ca. 610-650 Ma, soon after crystallization of the youngest population of zircons and before or during the onset of Brasiliano deformation.The Serido Group was deposited upon Paleoproterozoic basement in a basin receiving detritus from a variety of sources. The Jucurutu Formation includes some basal volcanic rocks and initially received detritus from proximal 2.2-2.0 Ga (Transamazonian) to late Paleoproterozoic (1.8-1.7 Ga) basement. Provenance for the upper Jucurutu Formation and all of the Serido Formation was dominated by more distal and younger sources ranging in age from 1000 to 650 Ma. We suggest that the Serido basin may have developed as the result of late Neoproterozoic extension of a pre-existing continental basement, with formation of small marine basins that were largely floored by cratonic basement (subjacent oceanic crust has not yet been found). Immature sediment was initially derived from surrounding land; as the basin evolved much of the detritus probably came from highlands to the south (present coordinates). Alternatively, if the Patos shear zone is a major terrane boundary, the basin may have formed as an early collisional foredeep associated with south-dipping subduction. In any case, within 30 million years the region was compressed, deformed, and metamorphosed during final assembly of West Gondwana and formation of the Brasiliano-Pan African fold belts. (C) 2003 Elsevier B.V. All rights reserved.

Sequence heterogeneity and phylogenetic relationships between the copia retrotransposon in Drosophila species of the repleta and melanogaster groups

Although the retrotransposon copia has been studied in the melanogaster group of Drosophila species, very little is known about copia dynamism and evolution in other groups. We analyzed the occurrence and heterogeneity of the copia 5' LTR-ULR partial sequence and their phylogenetic relationships in 24 species of the repleta group of Drosophila. PCR showed that copia occurs in 18 out of the 24 species evaluated. Sequencing was possible in only eight species. The sequences showed a low nucleotide diversity, which suggests selective constraints maintaining this regulatory region over evolutionary time. on the contrary, the low nucleotide divergence and the phylogenetic relationships between the D. willistoni/Zaprionus tuberculatus/melanogaster species subgroup suggest horizontal transfer. Sixteen transcription factor binding sites were identified in the LTR-ULR repleta and melanogaster consensus sequences. However, these motifs are not homologous, neither according to their position in the LTR-ULR sequences, nor according to their sequences. Taken together, the low motif homologies, the phylogenetic relationship and the great nucleotide divergence between the melanogaster and repleta copia sequences reinforce the hypothesis that there are two copia families.

Trypanosoma vivax infection dynamics in a cattle herd maintained in a transition area between Pantanal lowlands and highlands of Mato Grosso do Sul, Brazil

Trypanosoma vivax outbreaks in beef cattle in the Pantanal region of Mato Grosso do Sul state, Brazil, causes relevant economical impact due to weight loss, abortion and mortality. Cattle moved from the Pantanal to adjacent areas of this ecosystem for breeding and fattening is a common feature. Therefore an epidemiological study on breeding cows in the transition area between Pantanal lowland and adjacent highlands of Mato Grosso do Sul was performed to determine the T. vivax infection dynamics and outbreak risk. Three experimental groups were formed: Group 1 consisted of cows parasitologically negative by the Woo test and in the enzyme-linked immunosorbent assay for T. vivax antibody detection (Tv-ELISA-Ab); Group 2 parasitologically negative and positive in the Tv-ELISA-Ab; and in Group 3 cows were parasitologically positive and with positive reactions in the Tv-ELISA-Ab. During 24 months, the cows' dislodgment between the above established groups was monitored by Woo test and Tv-ELISA-Ab exams. The tabanid population was also monitored and the highest number occurred during the rainy season. Although parasitemias were detected only in the first four samplings of the experimental period, the cows could be considered as trypanotolerant, because no clinical signs were observed. Despite the higher T. vivax incidence during the dry season, no disease symptoms were seen. Even though T. vivax epidemiological situation in the herd was characterized as endemic with seasonal variation, the probability of outbreaks was null within the conditions of the study.

Variability of esterase patterns in adult flies of the saltans species group of Drosophila (subgenus Sophophora)

Esterases are known for their involvement in several physiological processes and high degree of polymorphism, in many organisms. Such polymorphism has been used to characterize species and species groups and to study genetic changes occurred in their evolutionary history. In the present study, the esterase patterns of 19 strains from 10 species representative of the five subgroups of the saltans species group were analyzed using polyacrylamide gel electrophoresis and alpha- and beta- naphthyl acetates as substrates. Fifty-one esterase bands were detected and classified as 31 alpha-esterases, 18 beta-esterases and two alpha/beta-esterases. on the basis of the inhibition patterns using Malathion and eserine sulfate, 34 bands were classified as carboxylesterases, 14 as acethylesterases and three as cholinesterases. Ten gene loci were tentatively established on the basis of data on band position in the gel, substrate preference and inhibition pattern. Twenty bands were species-specific, the remaining being shared by species from the same or different subgroups. Bands detected exclusively in males and bands with a different frequency or degree of expression between sexes were also detected. In the gels prepared for analysis of gene expression in the body parts (head, thorax and abdomen), the degree of expression of the beta-esterases was higher in the thorax, while the alpha-esterases were expressed predominantly in the abdomen and thorax. A global view of the data available at present on the esterases of the species from the saltans group and their degree of polymorphism are presented, as well as the possibility of using some beta-esterases, because of their characteristics in the gels, as markers for species identification.

Escherichia coli phylogenetic group determination and its application in the identification of the major animal source of fecal contamination

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)