Leandro Araujo Lobo

Production of bacteriocin by Bacteroides fragilis and partial characterization

The ability of Bacteroides fragilis strains, isolated from various sources, to produce bacteriocin was evaluated. All strains isolated from intestinal infections were producers in high levels and less susceptible to the others. Strains from other origins were found to produce bacteriocin at a medium level and they were variably susceptible. Some properties of one bacteriocin produced by the Bact. fragilis 079298–3 strain were analysed, providing evidence of its protein nature, with stability over a wide range of pH and retained inhibitory activity after heating. This variability seems to suggest that bacteriocin typing is a good method for this species.

Antimicrobial resistance of strains of the Bacteroides fragilis group isolated from the intestinal tract of children and adults in Brazil

The results of this study show that there is a high frequency of resistant species in the Bacteroides fragilis group in the intestinal tract of children and adults in Brazil. B. fragilis was not studied. Of the 73 strains examined, B. distasonis was the most resistant species to penicillin, cefoxitin, cefotaxime and clindamycin. High rates of multiresistance were found, most commonly to penicillin and clindamycin (18 of 36 strains). High levels of beta-lactamase production were detected in isolates showing high resistance to penicillin and multiresistance to the cephamycins, suggesting a widespread dissemination of such resistance.

The interaction of Bacteroides fragilis with components of the human fibrinolytic system

Bacteroides fragilis is a minor component of the intestinal microbiota and the most frequently isolated from intra-abdominal infections and bacteremia. Previously, our group has shown that molecules involved in laminin-1 (LMN-1) recognition were present in outer membrane protein extracts of B. fragilis MC2 strain. One of these proteins was identified and showed 98% similarity to a putative B. fragilis plasminogen-binding protein precursor, deposited in the public database. Thus, the objective of this work was to overexpress and further characterize this novel adhesin. The ability of B. fragilis MC2 strain and purified protein to convert plasminogen into plasmin was tested. Our results showed that B. fragilis strain MC2 strain adhered to both LMN-1 and plasminogen and this adhesion was inhibited by either LMN-1 or plasminogen. Regarding the plasminogen activation activity, both the whole bacterial cell and the purified protein converted plasminogen into plasmin similar to streptokinase used as a positive control. Bacterial receptors that recognize plasminogen bind to it and enhance its activation, transforming a nonproteolytic bacterium into a proteolytic one. We present in vitro evidence for a pathogenic function of the plasminogen receptor in promoting adherence to laminin and also the formation of plasmin by B. fragilis.

The role of BmoR, a MarR Family Regulator, in the survival of Bacteroides fragilis during oxidative stress.

The intestinal opportunistic pathogen Bacteroides fragilis is among the most aerotolerant species of strict anaerobic bacteria and survives exposure to atmospheric oxygen for up to 72h. Under these circumstances, a strong oxygen stress response (OSR) mechanism is activated and the expression of as much as 45% of B. fragilis genes is altered. One of the most important regulators of this response is the product of the oxyR gene, but other regulation systems are in place during the OSR. The MarR family of transcriptional regulators has been shown to control several physiological events in bacteria, including response to stress conditions. In B. fragilis, at least three homologs of MarR regulators are present, one of which, bmoR, is upregulated during oxidative stress independently of oxyR. In this study, we demonstrate that the inactivation of the bmoR gene in B. fragilis diminishes its ability to withstand oxidative stress caused either by exposure to atmospheric oxygen or hydrogen peroxide. Recovery of growth rate on pre-oxidized media under anaerobiosis is slower than that observed in parental strain. Addition of hydrogen peroxide has a similar effect on the growth rate. Complementation of the mutant strain partially recovered the oxygen resistance phenotype, but the overexpression of the gene in the parental strain was also deleterious to a lesser extent. Our results indicate that BmoR has a role in the OSR in B. fragilis, particularly in the initial stages of oxygen exposure.

Structural analysis of Pla protein from the biological warfare agent Yersinia pestis: docking and molecular dynamics of interactions with the mammalian plasminogen system

Yersinia pestis protein Pla is a plasmid-coded outer membrane protein with aspartic-protease activity. Pla exhibits a plasminogen (Plg) activator activity (PAA) that promotes the cleavage of Plg to the active serine-protease form called plasmin. Exactly how Pla activates Plg into plasmin remains unclear. To investigate this event, we performed the interactions between the predicted Plg and Pla protein structures by rigid-body docking with the HEX program and evaluated the complex stability by molecular dynamics (MD) using the GROMACS package programs. The predicted docked complex of Plg–Pla shows the same interaction site predicted by experimental site-direct mutagenesis in other studies. After a total of 8 ns of MD simulation, we observed the relaxation of the beta-barrel structure of Pla and the progressive approximation and stabilization between the cleavage site of Plg into the extracellular loops of Pla, followed by the increase in the number of H bonds. We also report here the aminoacids that participate in the active site and the sub sites of interaction. The total understanding of these interactions can be an important tool for drug design against bacterial proteases.

Comparison between Clinical Aspects and Salivary Microbial Profile of Children with and without Early Childhood Caries: A Preliminary Study

PURPOSE To compare the clinical aspects and microbial profile of children with and without early childhood caries (ECC). STUDY DESIGN 14 patients (7 without caries and 7 with ECC) were submitted to anamnesis, clinical exam and saliva collection for microbiological analyses. Counts of Streptococcus mutans, Lactobacillus spp. Candida spp., and total microorganisms were performed by culture methods. Microbial diversity was characterized by PCR-DGGE. Demographic/clinical data and salivary microbial counts were compared between groups. RESULTS Habits of hygiene and breastfeeding presented no association with ECC. Use of pacifiers was associated with absence of caries (p=0.035). Counts of total microorganisms and Candida spp. did not differ between the groups. The ECC group presented larger quantity of S. mutans (p=0.026) and Lactobacillus spp. (p=0.002). There was no correlation between microorganisms and breastfeeding and pacifier use. The dmf-t of ECC Group was 10.5 ± 1.9 and the modified dmf-t was 11.3 ± 3.6. The DGGE demonstrated difference in the pattern of bands between the groups. CONCLUSION Pacifiers usage was associated with the absence of ECC and microorganism number was higher in the caries group. The PCR-DGGE revealed a characteristic microbial diversity in the ECC Group, being an excellent tool for observing the dynamics of the salivary microbial community in these patients.

Production of AI‐2 is mediated by the S‐ribosylhomocystein lyase gene luxS in Bacteroides fragilis and Bacteroides vulgatus

Quorum sensing is a cell–cell signaling mechanism based on cell density and that involves the production of hormone‐like molecules called autoinducers (AI). One of the most studied AIs has been termed AI‐2, and its biosynthesis requires the enzyme encoded by luxS. We have previously described for the first time that Bacteroides species can produce molecules with AI‐2 activity. In this study, we focus on the detection of luxS and its activity as the AI‐2 synthase in Bacteroides species. The strains Bacteroides fragilis B3b and Bacteroides vulgatus ATCC 8482 were selected based on a positive phenotype for AI‐2 production and the presence of a putative luxS in the genome, respectively. In order to identify the luxS gene, cloning and heterologous expression strategies were utilized. We demonstrate that both strains contain functional luxS orthologs that can complement AI‐2 production in Escherichia coli.

The Bfp60 surface adhesin is an extracellular matrix and plasminogen protein interacting in Bacteroides fragilis.

Plasminogen (Plg) is a highly abundant protein found in the plasma component of blood and is necessary for the degradation of fibrin, collagen, and other structural components of tissues. This fibrinolytic system is utilized by several pathogenic species of bacteria to manipulate the host plasminogen system and facilitate invasion of tissues during infection by modifying the activation of this process through the binding of Plg at their surface. Bacteroides fragilis is the most commonly isolated Gram-negative obligate anaerobe from human clinical infections, such as intra-abdominal abscesses and anaerobic bacteraemia. The ability of B. fragilis to convert plasminogen (Plg) into plasmin has been associated with an outer membrane protein named Bfp60. In this study, we characterized the function of Bfp60 protein in B. fragilis 638R by constructing the bfp60 defective strain and comparing its with that of the wild type regarding binding to laminin-1 (LMN-1) and activation of Plg into plasmin. Although the results showed in this study indicate that Bfp60 surface protein of B. fragilis is important for the recognition of LMN-1 and Plg activation, a significant slow activation of Plg into plasmin was observed in the mutant strain. For that reason, the possibility of another unidentified mechanism activating Plg is also present in B. fragilis cannot be discarded. The results demonstrate that Bfp60 protein is responsible for the recognition of laminin and Plg-plasmin activation. Although the importance of this protein is still unclear in the pathogenicity of the species, it is accepted that since other pathogenic bacteria use this mechanism to disseminate through the extracellular matrix during the infection, it should also contribute to the virulence of B. fragilis.

Application of DNA sequence analysis based on five different conserved genes (16S rDNA, rpoB, gdh, est and pgm) for intra-species discrimination of Bacteroides fragilis

In the past few years, many studies revealed a remarkable genetic variability in Bacteroides fragilis species, and the existence of two divisions was proposed according to presence or absence of the cfiA (metallo-β-lactamase/carbapenemase) gene. The aim of this study was to evaluate the use of DNA sequence analysis for glutamate dehydrogenase (gdh), phosphoglucomutase (pgm) and esterase (est) metabolic genes, in comparison to RNA polymerase β subunit (rpoB) and 16S ribosomal RNA (rrs) gene sequencing, to identify the presence of these two groups in seventeen B. fragilis strains. Based on phylogenetic trees, only the est gene sequences generated a classification similar to rrs- and rpoB-genes. On the other hand, the genes pgm and gdh did not allow the discrimination of these divisions. The est gene sequence can be suggested as an additional tool for differentiation of the two groups in B. fragilis, providing highly reproducible and reliable data in B. fragilis taxonomy.

Probiotic treatment during neonatal age provides optimal protection against experimental asthma through the modulation of microbiota and T cells

The incidence of allergic diseases, which increased to epidemic proportions in developed countries over the last few decades, has been correlated with altered gut microbiota colonization. Although probiotics may play a critical role in the restoration of gut homeostasis, their efficiency in the control of allergy is controversial. Here, we aimed to investigate the effects of probiotic treatment initiated at neonatal or adult ages on the suppression of experimental ovalbumin (OVA)-induced asthma. Neonatal or adult mice were orally treated with probiotic bacteria and subjected to OVA-induced allergy. Asthma-like symptoms, microbiota composition and frequencies of the total CD4+ T lymphocytes and CD4+Foxp3+ regulatory T (Treg) cells were evaluated in both groups. Probiotic administration to neonates, but not to adults, was necessary and sufficient for the absolute prevention of experimental allergen-induced sensitization. The neonatally acquired tolerance, transferrable to probiotic-untreated adult recipients by splenic cells from tolerant donors, was associated with modulation of gut bacterial composition, augmented levels of cecum butyrate and selective accumulation of Treg cells in the airways. Our findings reveal that a cross-talk between a healthy microbiota and qualitative features inherent to neonatal T cells, especially in the Treg cell subset, might support the beneficial effect of perinatal exposure to probiotic bacteria on the development of long-term tolerance to allergens.