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Accumulating evidence suggests that interactions between the brain and gut microbiota significantly impact brain function and mental health. In the present study, we aimed to investigate whether young, healthy adults without psychiatric diagnoses exhibit differences in metabolic stool and microbiota profiles based on depression/anxiety scores and heart rate variability (HRV) parameters. Untargeted nuclear magnetic resonance-based metabolomics was used to identify fecal metabolic profiles. Results were subjected to multivariate analysis through principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA), and the metabolites were identified through VIP score. Metabolites separating asymptomatic and symptomatic groups were acetate, valine, and glutamate, followed by sugar regions, glutamine, acetone, valerate, and acetoacetate. The main metabolites identified in high vagal tone (HVT) and low vagal tone (LVT) groups were acetate, valerate, and glutamate, followed by propionate and butyrate. In addition to the metabolites identified by the PLS-DA test, significant differences in aspartate, sarcosine, malate, and methionine were observed between the groups. Levels of acetoacetate were higher in both symptomatic and LVT groups. Valerate levels were significantly increased in the symptomatic group, while isovalerate, propionate, glutamate, and acetone levels were significantly increased in the LVT group. Furthermore, distinct abundance between groups was only confirmed for the Firmicutes phylum. Differences between participants with high and low vagal tone suggest that certain metabolites are involved in communication between the vagus nerve and the brain.

Hymenaea genus has been used in folk medicine in Brazil, but few studies investigated its toxicity profile. Thus, the aim of this study was to determine toxicological parameters of Hymenaea courbaril stem bark hydroalcoholic extract by utilizing three cell lines including murine macrophages (RAW 264.7), mouse fibroblast cells (L929) and human lung fibroblast (MRC-5), as well as Salmonella/ microsome assay, and in vivo Caenorhabditis elegans model. The predominant detected phytoconstituents in the extract were coumarins, flavonoids, phenolics, tannins and saponins and by HPLC analysis, astilbin (AST) was found to be the main component. The DPPH assay demonstrated that H. courbaril hydroalcoholic extract exhibited potent antioxidant activity, with an IC 50 of 3.12 μg/ml. The extract at concentrations of 400 and 800 μg/ml decreased cell viability 48 hr after treatment in L929 and MRC-5 cell lines. In the Raw 264.7 strain, just the highest concentration (800 μg/ml) lowered cell viability within 48 hr following exposure. The concentration of 100 μg/ml did not markedly affect cell viability in the trypan blue assay. In the alkaline comet assay the extract was found to be non-genotoxic. In the Ames test, the extract exhibited low mutagenic potential without metabolic activation, since only the highest concentrations produced an effect. H. courbaril extract only affected the survival of C. elegans at concentrations of 800 and 1600 μl/ml. These findings demonstrate that H. courbaril extract appears to exert low toxicity as evidenced in vitro and mutagenicity assays; however, the biological relevance of the response of C. elegans survival to safety assessments needs further studies.

Objectives: Bacteroides fragilis is an anaerobic bacterium that is commonly found in the human gut microbiota and an opportunistic pathogen in extra-intestinal infections. B. fragilis displays a robust response to oxidative stress which allows for survival in oxygenated tissues such as the peritoneal cavity and lead to the formation of abscesses. In this study, we investigated the synergy of the oxidative stress response regulators OxyR and BmoR in the ability of B. fragilis to resist oxidative damage and to survive in extra-intestinal infection., Methods: A ΔbmoR ΔoxyR double mutant B. fragilis strain was constructed, and its oxidative stress response was compared to parental and single mutant strains in phenotypical assays and gene expression analysis. The pathogenic potential in an in vivo mouse model of abscess formation was also evaluated., Results: Expression analysis showed a coordinated control of thioredoxin C (trxC) gene expression by BmoR and OxyR during oxygen exposure, with upregulation of trxC in the bmoR mutant strain (4.9-fold increase), downregulation in the oxyR mutant (2.5-fold decrease), and an intermediate level of deregulation (2-fold increase) in the double mutant strain compared to the parent strain. Expression analysis during oxidative stress conditions also showed that BmoR is a major repressor of the CoA-disulfide reductase gene (upregulated 47-fold in the bmoR mutant) while OxyR plays a minor repression role in this gene (upregulated 2.5-fold in the oxyR mutant). Exposure to atmospheric oxygen for up to 72 h revealed that the deletion of bmoR alone had no significant effect in in vitro survival phenotype assays, though it partially abolishes the OxyR sensitivity phenotype in the bmoR/oxyR double mutant strain compared to oxyR mutant. In vivo assays showed that bmoR and oxyR mutants were significantly impaired in the formation and development of abscesses compared to the parent strain in an experimental intra-abdominal infection mouse model., Conclusion: Although the full extent of genes whose expression are modulated by BmoR and OxyR is yet to be defined, we present evidence that these regulators have overlapping functions in B. fragilis response to oxidative stress and ability to form abscess in extra-intestinal tissues., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The aim of this study is to investigate the bioaccessibility and gut metabolism of free and melanoidin-bound phenolic compounds from coffee and bread. Phenolics from coffee were predominantly found in free forms (68%, mainly chlorogenic acids), whereas those from bread were mostly bound to melanoidins (61%, mainly ferulic acid). Bioacessibility of coffee total free phenolics slightly decreased during simulated digestion (87, 86, and 82% after the oral, gastric, and intestinal steps, respectively), with caffeoylquinic acids being isomerized and chlorogenic acids being partially hydrolyzed to the corresponding hydroxycinnamic acids. Bioacessibility of bread total free phenolics decreased during simulated digestion (91, 85, and 67% after the oral, gastric, and intestinal steps, respectively), probably related to complexation with the proteins in simulated gastric and intestinal fluids. Upon gut fermentation, the bioaccessibility of total free phenolics from both coffee and bread decreased, mainly after the first 4 h (56 and 50%, respectively). Caffeic and ferulic acids were the predominant metabolites found during coffee and bread gut fermentation, respectively. Melanoidin-bound phenolics from coffee and bread were progressively released after the gastric and intestinal steps, probably due to hydrolysis caused by the acidic conditions of the stomach and the action of pancreatin from the intestinal fluid. The bioaccessibilities of all phenolics from coffee and bread melanoidins after the gastric and intestinal steps were, on average, 11 and 26%, respectively. During gut fermentation, phenolics bound to both coffee and bread melanoidins were further released by the gut microbiota, whereas those from coffee were also metabolized. This difference could be related to the action of proteases on melanoproteins during gastrointestinal digestion, probably anticipating phenolics release. Nevertheless, bioaccessibilities of melanoidin-bound phenolics reached maximum values after gut fermentation for 24 h (50% for coffee and 51% for bread). In conclusion, the bioaccessibilities of coffee and bread free phenolics during simulated digestion and gut fermentation were remarkably similar, and so were the bioaccessibilities of coffee and bread melanoidin-bound phenolics., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Alves, Lobo, Domingues, Monteiro and Perrone.)

Intestinal barrier is essential for dietary products and microbiota compartmentalization and therefore gut homeostasis. When this barrier is broken, cecal content overflows into the peritoneal cavity, leading to local and systemic robust inflammatory response, characterizing peritonitis and sepsis. It has been shown that IL-1β contributes with inflammatory storm during peritonitis and sepsis and its inhibition has beneficial effects to the host. Therefore, we investigated the mechanisms underlying IL-1β secretion using a widely adopted murine model of experimental peritonitis. The combined injection of sterile cecal content (SCC) and the gut commensal bacteria Bacteroides fragilis leads to IL-1β-dependent peritonitis, which was mitigated in mice deficient in NLRP3 (nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3) inflammasome components. Typically acting as a damage signal, SCC, but not B. fragilis , activates canonical pathway of NLRP3 promoting IL-1β secretion in vitro and in vivo. Strikingly, absence of fiber in the SCC drastically reduces IL-1β production, whereas high-fiber SCC conversely increases this response in an NLRP3-dependent manner. In addition, NLRP3 was also required for IL-1β production induced by purified dietary fiber in primed macrophages. Extending to the in vivo context, IL-1β-dependent peritonitis was worsened in mice injected with B. fragilis and high-fiber SCC, whereas zero-fiber SCC ameliorates the pathology. Corroborating with the proinflammatory role of dietary fiber, IL-1R-deficient mice were protected from peritonitis induced by B. fragilis and particulate bran. Overall, our study highlights a function, previously unknown, for dietary fibers in fueling peritonitis through NLRP3 activation and IL-1β secretion outside the gut., (Copyright © 2021 by The American Association of Immunologists, Inc.)

Humans live in symbiosis with a diverse community of microorganisms, which has evolved to carry out many specific tasks that benefit the host, including protection against invading pathogens. Within the chemical diversity of the gastrointestinal tract, small molecules likely constitute chemical cues for the communication between the microbiota and pathogens. Therefore, we sought to investigate if molecules produced by the human gut microbiota show biological activity against the human pathogen Vibrio cholerae . To probe the effects of the gut metabolome on V. cholerae , we investigated its response to small-molecule extracts from human feces, from a complex bacterial community cultivated in vitro , and from culture supernatants of Enterocloster citroniae, Bacteroides thetaiotaomicron , and Bacteroides vulgatus . Using RNA sequencing, we determined the impact of the human gut metabolome on V. cholerae global gene expression. Among the genes downregulated in the presence of the fecal extract, the most overrepresented functional category was cell motility, which accounted for 39% of repressed genes. Repression of V. cholerae motility by the fecal extract was confirmed phenotypically, and E. citroniae extracts reproduced this phenotype. A complex in vitro microbial community led to increased motility, as did extracts from B. vulgatus , a species present in this community. Accordingly, mucin penetration was also repressed by fecal and E. citroniae extracts, suggesting that the phenotypes observed may have implications for host colonization. Together with previous studies, this work shows that small molecules from the gut metabolome may have a widespread, significant impact on microbe-microbe interactions established in the gut environment.

This study aimed at investigating two strategies to enhance the bioaccessibility of phenolic compounds from whole-wheat breads: enzymatic bioprocessing and addition of green coffee infusion. Although both strategies had a significant effect on increasing the contents of total soluble phenolic compounds in breads, the addition of green coffee infusion was much more relevant (19.1-fold) than enzymatic bioprocessing (1.8-fold). The phenolic compounds present as soluble forms were completely released from all breads' matrix already at the oral phase of digestion. While gastric digestion did not promote the release of insoluble phenolic compounds, intestinal conditions led to a slight release. All bread samples showed maximum phenolic compounds bioaccessibility after 4 h of gut fermentation. Upon the end of in vitro digestion and gut fermentation, the difference between the strategies was that enzymatic bioprocessing accelerated ferulic acid release, while the addition of green coffee infusion increased 10.4-fold the overall phenolic compounds bioaccessibility., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Extracellular matrix (ECM) proteins are highly abundant in the human body and can be found in various tissues, most prominently in connective tissue and basement membrane. For invasive bacterial pathogens, these structures function as physical barriers that block access to underlying tissues. The ability to bind and degrade these barriers is important for the establishment of infections and migration to other body sites. In the oral cavity, the ECM and the basement membrane (BM) are important components of the Junctional epithelium (JE) that closes the gap between the teeth surface and the mucosa. In periodontitis, the JE is breached by invading pathogenic bacteria, particularly strict anaerobic species. In periodontitis, invading microorganisms induce an unregulated and destructive host response through polymicrobial synergism and dysbiosis that attracts immune cells and contributes to the destruction of connective tissue and bone in the periodontal pocket. Colonization of the periodontal pocket is the first step to establish this infection, and binding to ECM is a major advantage in this site. Several species of strict anaerobic bacteria are implicated in acute and chronic periodontitis, and although binding to ECM proteins was studied in these species, few adhesins were identified so far, and the mechanisms involved in adhesion are largely unidentified. This review summarizes the data available on the interaction of strict anaerobic bacteria and components of the ECM.

The oral cytotoxicity, antimicrobial and anti-demineralizing effects of a tincture from Bauhinia forficata Link tincture (BFLT) were evaluated in vitro and ex vivo . Susceptibility tests (minimum inhibitory and microbicidal concentrations-MIC and time-kill assay-MMC) were performed against planktonic oral microorganisms. The contents of phenolic compounds were investigated. Cytotoxic potential was evaluated on oral fibroblasts after 1-5 min exposure to BFLT. Blocks of sound bovine enamel ( N  = 60) were inoculated with a saliva pool and sustained in a multiple plaque growth system for 48 h to form a biofilm. Biofilm blocks were randomly divided into groups-G ( n  = 10): G1-Baseline (48 h maturation biofilm), G2-BFLT 23.2 mg/mL, G3-Ethanol 81.20 g/mL, G4-Chlorhexidine 0.12%, G5-Growth control, and G6-Blank control. Treatments (50  μ L/1 min) were performed once a day for a week. Streptococcus spp. (S) and total microorganism (TM) counts were expressed as Log 10 CFU/mL. Biofilm height was evaluated by confocal microscopy analyses (CMA). Final surface hardness was assessed and percentage of microhardness loss (% MHL) was calculated. Results were significant when P  < .05. BFLT inhibited all tested microorganisms (MIC = 1.3-23.2 mg/mL) and promoted optical reduction (0.05-0.22 nm) of all microorganisms after 48-h treatment compared with controls. After 5-min treatment, BFLT showed low values of cell death (3.20%). G2-BFLT reduced S (6.61 ± 0.20) and TM (7.14 ± 0.38) compared with G1-Baseline (S = 7.82 ± 0.28; TM = 8.81 ± 0.67) and G5-Growth control (S = 7.48 ± 0.39; TM = 7.89 ± 0.68); but G4-chlororexidine (S = 6.11 ± 0.48; TM = 6.45 ± 0.16) showed the highest antibiofilm activity. CMA was not different among treatment groups. G2 showed lower % MHL compared with G5, although G4 presented the lowest. Results suggest BFLT is beneficial against dental caries, showing antimicrobial effects against a mature dental biofilm and no cytotoxicity.

Several factors affect the composition of species that inhabit our intestinal tract, including mode of delivery, genetics and nutrition. Antimicrobial peptides and proteins secreted in the gastrointestinal tract are powerful tools against bacteria. Lactoferrin (LF) inhibits the growth of several bacterial species, such as Enterobacteriaceae, but may stimulate probiotic bacteria. Activity of LF against gut symbiotic species of the Bacteroides genus could give us insights on how these species colonize the gut. We investigated the effects of the antimicrobial protein lactoferrin and its derived peptide, lactoferricin B on two species of strict anaerobes, opportunistic pathogens that cause diseases in both adults and children, commonly found in the microbiota of the human gastrointestinal tract, Bacteroides fragilis and B. thetaiotaomicron., In vitro biofilm formation and binding to laminin were strongly inhibited by a low concentration of lactoferrin (12.5 μg/ml). Conversely, the growth of the strains in a micro-dilution assay in minimal media with different iron sources was not affected by physiological concentrations (2 mg/ml) of apo-lactoferrin or holo-lactoferrin. The combination of lactoferrin with antibiotics in synergism assays was also negative. The lactoferricin B fragment was also unable to inhibit growth in a similar test with concentrations of up to 32 μg/ml. Resistance to lactoferrin could confer an advantage to these species, even when high amount of this protein is present in the gastrointestinal tract. However, colonization is hampered by the binding and biofilm inhibitiory effect of lactoferrin, which may explain the low prevalence of Bacteroides in healthy babies. Resistance to this antimicrobial protein may help understand the success of these opportunistic pathogens during infection in the peritoneum., Competing Interests: Declaration of competing interest The authors declare no conflict of interests., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The microbiota influences host health through several mechanisms, including protecting it from pathogen colonization. Staphylococcus epidermidis is one of the most frequently found species in the skin microbiota, and its presence can limit the development of pathogens such as Staphylococcus aureus S. aureus causes diverse types of infections ranging from skin abscesses to bloodstream infections. Given the increasing prevalence of S. aureus drug-resistant strains, it is imperative to search for new strategies for treatment and prevention. Thus, we investigated the activity of molecules produced by a commensal S. epidermidis isolate against S. aureus biofilms. We showed that molecules present in S. epidermidis cell-free conditioned media (CFCM) caused a significant reduction in biofilm formation in most S. aureus clinical isolates, including all 4 agr types and agr -defective strains, without any impact on growth. S. epidermidis molecules also disrupted established S. aureus biofilms and reduced the antibiotic concentration required to eliminate them. Preliminary characterization of the active compound showed that its activity is resistant to heat, protease inhibitors, trypsin, proteinase K, and sodium periodate treatments, suggesting that it is not proteinaceous. RNA sequencing revealed that S. epidermidis -secreted molecules modulate the expression of hundreds of S. aureus genes, some of which are associated with biofilm production. Biofilm formation is one of the main virulence factors of S. aureus and has been associated with chronic infections and antimicrobial resistance. Therefore, molecules that can counteract this virulence factor may be promising alternatives as novel therapeutic agents to control S. aureus infections. IMPORTANCE S. aureus is a leading agent of infections worldwide, and its main virulence characteristic is the ability to produce biofilms on surfaces such as medical devices. Biofilms are known to confer increased resistance to antimicrobials and to the host immune responses, requiring aggressive antibiotic treatment and removal of the infected surface. Here, we investigated a new source of antibiofilm compounds, the skin microbiome. Specifically, we found that a commensal strain of S. epidermidis produces molecules with antibiofilm activity, leading to a significant decrease of S. aureus biofilm formation and to a reduction of previously established biofilms. The molecules potentiated the activity of antibiotics and affected the expression of hundreds of S. aureus genes, including those associated with biofilm formation. Our research highlights the search for compounds that can aid us in the fight against S. aureus infections., (Copyright © 2020 American Society for Microbiology.)

The inhibitory activity of a Bauhinia forficata tincture (TBF) was investigated against oral microorganism's strains and against a mature oral biofilm. The viability of planktonic cells was analyzed by Minimal Inhibitory and Microbicidal concentrations of TBF. Salivary samples from health volunteers were collected and mixed to form a saliva pool. An aliquot from this pool were seeded on membranes, which were incubated to form biofilm (48 h). The biofilm was treated according to the groups: G1-Chlorhexidine 0.12%; G2-TBF at the highest MMC; G3-Ethanol at the TBF highest MMC. G4 was the growth control. Streptococcus spp. (S) and total microorganisms (TM) from biofilm were counted. TBF was microbicidal against all oral pathogens. G2 was able to reduce the counts of S and TM from biofilm compared to G3 and G4, but less than G1 ( p  < 0.05). TBF is able to reduce the microbial levels from a mature oral biofilm.

During the last decades it has become increasingly clear that the microbes that live on and in humans are critical for health. The communities they form, termed microbiomes, are involved in fundamental processes such as the maturation and constant regulation of the immune system. Additionally, they constitute a strong defense barrier to invading pathogens, and are also intricately linked to nutrition. The parameters that affect the establishment and maintenance of these microbial communities are diverse, and include the genetic background, mode of birth, nutrition, hygiene, and host lifestyle in general. Here, we describe the characterization of the gut microbiome of individuals living in the Amazon, and the comparison of these microbial communities to those found in individuals from an urban, industrialized setting. Our results showed striking differences in microbial communities from these two types of populations. Additionally, we used high-throughput metabolomics to study the chemical ecology of the gut environment and found significant metabolic changes between the two populations. Although we cannot point out a single cause for the microbial and metabolic changes observed between Amazonian and urban individuals, they are likely to include dietary differences as well as diverse patterns of environmental exposure. To our knowledge, this is the first description of gut microbial and metabolic profiles in Amazonian populations, and it provides a starting point for thorough characterizations of the impact of individual environmental conditions on the human microbiome and metabolome.

Despite the broad assessment of sponge bacterial diversity through cultivation-independent and dependent strategies, the knowledge focusing on cultivable anaerobes from this holobiont is still incipient. Plakina is a genus with the highest number of described species from the smallest of poriferan classes, Homoscleromorpha. The Brazilian Atlantic coast has been presenting itself as a hotspot for the discovery of new plakinidae species, with initial surveys just now concerning to characterize their microbiome. The current study aimed to isolate and identify strict anaerobes from recently described species of Plakina collected at the coast of Cabo Frio, RJ. Samples of four sympatric morphotypes of Plakina cyanorosea and Plakina cabofriense were collected on the coast of Cabo Frio, RJ. Using five different culture media, a total of 93 bacterial isolates were recovered, among which 60 were strict anaerobes and, ultimately, 34 remaining viable. A total of 76.5% from these strains were mostly identified as Clostridium bifermentans by mass spectrometry and 82.4% identified by 16S rRNA sequencing, almost all of them affiliated to the genus Paraclostridium, and with one isolate identified as Clostridium butyricum by both techniques. None of the anaerobic bacteria exhibited antimicrobial activity by the adopted screening test. The present work highlights not only the need for cultivation and characterization of the anaerobic microbiota from marine sponges but also adds the existing scarce knowledge of culturable bacterial communities from Homoscleromorph sponges from Brazilian coast.

Bacteroides fragilis, an opportunistic pathogen and commensal bacterium in the gut, is one the most aerotolerant species among strict anaerobes. However, the mechanisms that control gene regulation in response to oxidative stress are not completely understood. In this study, we show that the MarR type regulator, BmoR, regulates the expression of genes involved in the homeostasis of intracellular redox state. Transcriptome analysis showed that absence of BmoR leads to altered expression in total of 167 genes. Sixteen of these genes had a 2-fold or greater change in their expression. Most of these genes are related to LPS biosynthesis and carbohydrates metabolism, but there was a significant increase in the expression of genes related to the redox balance inside the cell. A pyridine nucleotide-disulfide oxidoreductase located directly upstream of bmoR was shown to be repressed by direct binding of BmoR to the promoter region. The expression of two other genes, coding for a thiosulphate:quinone-oxidoreductase and a thioredoxin, are indirectly affected by bmoR mutation during oxygen exposure. Phenotypic assays showed that BmoR is important to maintain the thiol/disulfide balance in the cell, confirming its relevance to B. fragilis response to oxidative stress.

Violacein is a violet pigment produced by Chromobacterium violaceum that possesses several functions such as antibacterial, antiviral, antifungal, and antioxidant activities. The search for potential compounds and therapies that may interfere with and modulate the gut microbial consortia without causing severe damage and increased resistance is important for the treatment of inflammatory, allergic, and metabolic diseases. The aim of the present work was to evaluate the ability of violacein to change microbial patterns in the mammalian gut by favoring certain groups over the others in order to be used as a therapy for diseases associated with changes in the intestinal microflora. To do this, we used male Wistar rats, and administered violacein orally, in low (50 μg/ml) and high (500 μg/ml) doses for a month. Initially, the changes in the microbial diversity were observed by DGGE analyses that showed that the violacein significantly affects the gut microbiota of the rats. Pyrosequencing of 16S rDNA was then employed using a 454 GS Titanium platform, and the results demonstrated that higher taxonomic richness was observed with the low violacein treatment group, followed by the control group and high violacein treatment group. Modulation of the microbiota at the class level was observed in the low violacein dose, where Bacilli and Clostridia (Firmicutes) were found as dominant. For the high violacein dose, Bacilli followed by Clostridia and Actinobacteria were present as the major components. Further analyses are crucial for a better understanding of how violacein affects the gut microbiome and whether this change would be beneficial to the host, providing a framework for the development of alternative treatment strategies for intestinal diseases using this compound., Competing Interests: The authors have declared that no competing interests exist.

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.

Bacteroides fragilis is the strict anaerobic bacteria most commonly found in human infections, and has a high mortality rate. Among other virulence factors, the remarkable ability to acquire resistance to a variety of antimicrobial agents and to tolerate nanomolar concentrations of oxygen explains in part their success in causing infection and colonizing the mucosa. Much attention has been given to genes related to multiple drug resistance derived from plasmids, integrons or transposon, but such genes are also detected in chromosomal systems, like the mar (multiple antibiotic resistance) locus, that confer resistance to a range of drugs. Regulators like MarR, that control expression of the locus mar, also regulate resistance to organic solvents, disinfectants and oxygen reactive species are important players in these events. Strains derived from the parental strain 638R, with mutations in the genes hereby known as marRI (BF638R&#95;3159) and marRII (BF638R&#95;3706) were constructed by gene disruption using a suicide plasmid. Phenotypic response of the mutant strains to hydrogen peroxide, cell survival assay against exposure to oxygen, biofilm formation, resistance to bile salts and resistance to antibiotics was evaluated. The results showed that the mutant strains exhibit statistically significant differences in their response to oxygen stress, but no changes were observed in survival when exposed to bile salts. Biofilm formation was not affected by either gene disruption. Both mutant strains however, became more sensitive to multiple antimicrobial drugs tested. This indicates that as observed in other bacterial species, MarR are an important resistance mechanism in B. fragilis., (Copyright © 2017 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.)

The human microbiome is a collection of microorganisms that inhabit every surface of the body that is exposed to the environment, generally coexisting peacefully with their host. These microbes have important functions, such as producing vitamins, aiding in maturation of the immune system, and protecting against pathogens. We have previously shown that a small-molecule extract from the human fecal microbiome has a strong repressive effect on Salmonella enterica serovar Typhimurium host cell invasion by modulating the expression of genes involved in this process. Here, we describe the characterization of this biological activity. Using a series of purification methods, we obtained fractions with biological activity and characterized them by mass spectrometry. These experiments revealed an abundance of aromatic compounds in the bioactive fraction. Selected compounds were obtained from commercial sources and tested with respect to their ability to repress the expression of hilA , the gene encoding the master regulator of invasion genes in Salmonella We found that the aromatic compound 3,4-dimethylbenzoic acid acts as a strong inhibitor of hilA expression and of invasion of cultured host cells by Salmonella Future studies should reveal the molecular details of this phenomenon, such as the signaling cascades involved in sensing this bioactive molecule. IMPORTANCE Microbes constantly sense and adapt to their environment. Often, this is achieved through the production and sensing of small extracellular molecules. The human body is colonized by complex communities of microbes, and, given their biological and chemical diversity, these ecosystems represent a platform where the production and sensing of molecules occur. In previous work, we showed that small molecules produced by microbes from the human gut can significantly impair the virulence of the enteric pathogen Salmonella enterica Here, we describe a specific compound from the human gut that produces this same effect. The results from this work not only shed light on an important biological phenomenon occurring in our bodies but also may represent an opportunity to develop drugs that can target these small-molecule interactions to protect us from enteric infections and other diseases., (Copyright © 2017 American Society for Microbiology.)

The Antarctic soil microbial community has a crucial role in the growth and stabilization of higher organisms, such as vascular plants. Analysis of the soil microbiota composition in that extreme environmental condition is crucial to understand the ecological importance and biotechnological potential. We evaluated the efficiency of isolation and abundance of strict anaerobes in the vascular plant Deschampsia antarctica rhizosphere collected in the Antarctic's Admiralty Bay and associated biodiversity to metabolic perspective and enzymatic activity. Using anaerobic cultivation methods, we identified and isolated a range of microbial taxa whose abundance was associated with Plant Growth-Promoting Bacteria (PGPB) and presences were exclusively endemic to the Antarctic continent. Firmicutes was the most abundant phylum (73 %), with the genus Clostridium found as the most isolated taxa. Here, we describe two soil treatments (oxygen gradient and heat shock) and 27 physicochemical culture conditions were able to increase the diversity of anaerobic bacteria isolates. Heat shock treatment allowed to isolate a high percentage of new species (63.63 %), as well as isolation of species with high enzymatic activity (80.77 %), which would have potential industry application. Our findings contribute to the understanding of the role of anaerobic microbes regarding ecology, evolutionary, and biotechnological features essential to the Antarctic ecosystem.

Mammals harbor a complex gut-associated microbiota, comprising bacteria that provide immunological, metabolic and neurological benefits to the host, and contribute to their well-being. However, dysregulation of the microbiota composition, known as dysbiosis, along with the associated mucosal immune response have a key role in the pathogenesis of many inflammatory diseases, including inflammatory bowel diseases (IBDs), type 1 and type 2 diabetes, asthma, multiple sclerosis, among others. In addition, outside the gut lumen, bacteria from microbiota are the causative agent of peritoneal inflammation, abdominal sepsis and systemic sepsis. Critical care interventions during sepsis by antibiotics induce dysbiosis and present acute and long-term poor prognosis. In this review, we discuss immunomodulatory effects of the microbial molecules and products, highlighting the role of Bacteroides fragilis, a human commensal with ambiguous interactions with the host. Moreover, we also address the impact of antibiotic treatment in sepsis outcome and discuss new insights for microbiota modulation.

Bacteroides fragilis is the most commonly isolated anaerobic bacteria from infectious processes. Several virulence traits contribute to the pathogenic nature of this bacterium, including the ability to tolerate the high concentrations of bile found in the gastrointestinal tract (GIT). The activity of bile salts is similar to detergents and may lead to membrane permeabilization and cell death. Modulation of outer membrane proteins (OMPs) is considered a crucial event to bile salts resistance. The primary objective of the current work was to identify B. fragilis proteins associated with the stress induced by high concentration of bile salts. The outer membrane of B. fragilis strain 638R was isolated after growth either in the presence of 2% conjugated bile salts or without bile salts. The membrane fractions were separated on SDS-PAGE and analyzed by ESI-Q/TOF tandem mass spectrometry. A total of 37 proteins were identified; among them nine were found to be expressed exclusively in the absence of bile salts whereas eight proteins were expressed only in the presence of bile salts. These proteins are related to cellular functions such as transport through membrane, nutrient uptake, and protein-protein interactions. This study demonstrates the alteration of OMPs composition in B. fragilis during bile salts stress resistance and adaptation to environmental changes. Proteomics of OMPs was also shown to be a useful approach in the identification of new targets for functional analyses., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Purpose: This study investigated the adherence of oral microorganisms to different types of suture threads., Methods: Pieces of thread were distributed on 24-well plates, according to the following groups: (G1) nylon, (G2) silk, (G3) polyglactin 910, (G4) polyglactin 910 with triclosan. Blank control (G5) consisted of one thread from each group. Adherence to thread tests was performed to observe adhesion of total microorganisms from saliva or two isolates of Prevotella intermedia (ATCC49046) and Fusobacterium nucleatum (ATCC51190). Brain-heart infusion (BHI) medium with or without bacterial inoculum (1.8 × 10(7) CFU/mL) was added to each well of microplates. The microplates were incubated in an anaerobic chamber at 37 °C, for 5 days for biofilm formation., Results: There was no difference between the groups as regard to adhesion of F. nucleatum (p > 0.05). For P. intermedia, the threads in G1 and G4 showed a lower level of adhesion (p < 0.05), with no difference between them. Against total microorganisms, G1 presented a lower level of adherence (p < 0.05), followed by G4; and no difference was observed between G2 and G3., Conclusions: Total microorganisms and P. intermedia have different affinities to the tested suture threads, whereas F. nucleatum presented a similar adherence level. Among the threads, nylon (G1), followed by polyglactin 910 with triclosan (G4) presented the lowest microbial adherence level.

Clostridium difficile is a Gram-positive spore forming anaerobic bacterium, often associated with nosocomial diarrhea and pseudomembranous colitis. The acquisition of this organism occurs primarily in hospitals through accidental ingestion of spores, and its establishment and proliferation in the colon results from the removal of members of the normal intestinal flora during or after antibiotic therapy. In this study, stool samples from patients admitted to the University Hospital Clementino Fraga Filho (HUCCF/UFRJ) were screened for C. difficile toxins with an ELISA test and cultured with standard techniques for C. difficile isolation. A total of 74 stool samples were collected from patients undergoing antibiotic therapy between August 2009 and November 2010, only two (2.7%) were positive in the ELISA test and culture. A third isolate was obtained from a negative ELISA test sample. All cases of CDI were identified in patients with acute lymphoid or myeloid leukemia. Genotypic and phenotypic characterization showed that all strains carried toxins A and B genes, and belonged to PCR-ribotypes 014, 043 and 046. The isolated strains were sensitive to metronidazole and vancomycin, and resistant to ciprofloxacin and levofloxacin. Resistance to moxifloxacin, was present in the strain from PCR-ribotype 014, that showed an amino acid substitution in gyrB gene (Asp 426 → Asn). This is the first time that this mutation in a PCR-ribotype 014 strain has been described in Brazil., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

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., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Objectives: This in situ study aimed to investigate the effect of a sugar-free antibiotic suspension containing amoxicillin and clavulanic acid on enamel hardness of human primary teeth simulating different conditions of cariogenic challenge., Materials and Methods: A crossover, partially double-blind study was conducted in three phases of 14 days each, during which 11 volunteers wore palatal devices containing six dental enamel blocks covered with plastic meshes to allow biofilm formation. Dental blocks were extraorally submitted to treatment with a 20 % sucrose solution at three different daily frequencies of exposure (0, 3, and 8 times/day), and to the antibiotic suspension or its excipients at an 8-h time interval application regimen. On the 14th day of each phase, the blocks were removed for enamel analysis (surface and cross-sectional microhardness--SMH and CSMH)., Results: The antibiotic suspension showed significant higher SMH and CSMH values than the excipients (p < 0.05; Wilcoxon), regardless of the frequency of sucrose exposure. Sucrose exposure did not account for further enamel demineralization both for antibiotic and excipients (p > 0.05; Friedman)., Conclusions: A protective effect of the antibiotic suspension on enamel demineralization was verified because its excipients alone promoted more pronounced surface and subsurface enamel demineralization, even in the absence of sucrose exposure., Clinical Relevance: The use of a sugar-free amoxicillin/clavulanic acid suspension may promote a protective effect on primary enamel demineralization probably due to its topical effect on dental biofilm.

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., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

Bacteroides fragilis is the most frequent opportunistic pathogen isolated from anaerobic infections. However, there is a paucity of information regarding the genetic and molecular aspects of gene expression of its virulence factors during extra-intestinal infections. A potential virulence factor that has received little attention is the ability of B. fragilis to produce hemolysins. In this study, an implanted perforated table tennis "ping-pong" ball was used as an intra-abdominal artificial abscess model in the rat. This procedure provided sufficient infected exudate for gene expression studies in vivo. Real-time reverse transcription polymerase chain reaction (RT-PCR) was used to quantify the relative expression of hlyA, hlyB, hlyC, hlyD, hlyE, hlyF, hlyG, and hlyIII mRNAs. The hlyA mRNA was induced approximately sixfold after 4 days postinfection compared with the mRNA levels in the inoculum culture prior to infection. The hlyB mRNA increased approximately sixfold after 4 days and 12-fold after 8 days postinfection. Expression of hlyC mRNA increased sixfold after 1 day, 45-fold after 4 days, and 16-fold after 8 days postinfection, respectively. The hlyD and hlyE mRNAs were induced approximately 40-fold and 30-fold, respectively, after 4-days postinfection. The hlyF expression increased approximately threefold after 4-days postinfection. hlyG was induced approximately fivefold after 4 and 8 days postinfection. The hlyIII mRNA levels had a steady increase of approximately four-, eight-, and 12-fold following 1, 4, and 8 days postinfection, respectively. These findings suggest that B. fragilis hemolysins are induced and differentially regulated in vivo. Both parent and hlyBA mutant strains reached levels of approximately 3-8 × 10(9) cfu/mL after 1 day postinfection. However, the hlyBA mutant strain lost 2 logs in viable cell counts compared with the parent strain after 8 days postinfection. This is the first study showing HlyBA is a virulence factor which plays a role in B. fragilis survival in an intra-abdominal abscess model., (© 2013 The Authors. Published by Blackwell Publishing Ltd.)

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., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

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.

In this study, we show the expression of flavin mononucleotide-based fluorescent protein (FbFP) BS2 as a marker for gene expression in the opportunistic human anaerobic pathogen Bacteroides fragilis. Bacteroides fragilis 638R strain carrying osu∷bs2 constructs showed inducible fluorescence following addition of maltose anaerobically compared with nonfluorescent cells under glucose-repressed conditions. Bacteria carrying ahpC∷bs2 or dps∷bs2 constructs were fluorescent following induction by oxygen compared with nonfluorescent cells from the anaerobic control cultures. In addition, when these transcriptional fusion constructs were mobilized into B. fragilis IB263, a constitutive peroxide response strain, fluorescent BS2, was detected in both anaerobic and aerobic cultures, confirming the unique properties of the FbFP BS2 to yield fluorescent signal in B. fragilis in the presence and in the absence of oxygen. Moreover, intracellular expression of BS2 was also detected when cell culture monolayers of J774.1 macrophages were incubated with B. fragilis ahpC∷bs2 or dps∷bs2 strains within an anaerobic chamber. This suggests that ahpC and dps are induced following internalization by macrophages. Thus, we show that BS2 is a suitable tool for the detection of gene expression in obligate anaerobic bacteria in in vivo studies., (© 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

The Pla surface protease of Yersinia pestis activates human plasminogen and is a central virulence factor in bubonic and pneumonic plague. Pla is a transmembrane beta-barrel protein and member of the omptin family of outer membrane proteases which require bound lipopolysaccharide (LPS) to be proteolytically active. Plasminogen activation and autoprocessing of Pla were dramatically higher in Y. pestis cells grown at 37 degrees C than in cells grown at 20 degrees C; the difference in enzymatic activity by far exceeded the increase in the cellular content of the Pla protein. Y. pestis modifies its LPS structure in response to growth temperature. We purified His(6)-Pla under denaturing conditions and compared various LPS types for their capacity to enhance plasmin formation by His(6)-Pla solubilized in detergent. Reactivation of His(6)-Pla was higher with Y. pestis LPSs isolated from bacteria grown at 37 degrees C than with LPSs from cells grown at 25 degrees C. Lack of O antigens and the presence of the outer core region as well as a lowered level of acylation in LPS were found to enhance the Pla-LPS interaction. Genetic substitution of arginine 138, which is part of a three-dimensional protein motif for binding to lipid A phosphates, decreased both the enzymatic activity of His(6)-Pla and the amount of Pla in Y. pestis cells, suggesting the importance of the Pla-lipid A phosphate interaction. The temperature-induced changes in LPS are known to help Y. pestis to avoid innate immune responses, and our results strongly suggest that they also potentiate Pla-mediated proteolysis.

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.

Mammalian matrix metalloproteinases (MMPs) degrade collagen networks in extracellular matrices by cleaving collagen and its denatured form gelatin, and thus enhance migration of mammalian cells. The gastrointestinal pathogen Salmonella enterica survives and grows within host macrophages and dendritic cells, and can disseminate in the host by travelling within infected host cells. Here, we report that S. enterica serovar Typhimurium activates proMMP-9 (gelatinase B) secreted by human primary macrophages, and degrades gelatin after growth within J774A.1 murine macrophage-like cells. Both proMMP-9 activation and gelatin degradation were due to expression of the Salmonella surface protease PgtE. Following intraperitoneal infection in BALB/c mice, the amount of a pgtE deletion derivative was nearly ten-fold lower in the livers and spleens of mice than the amount of wild-type S. enterica, suggesting that PgtE contributes to dissemination of Salmonella in the host. PgtE belongs to the omptin family of bacterial beta-barrel transmembrane proteases. The ortholog of PgtE in Yersinia pestis, Pla, which is central for bacterial virulence in plague, was poor in proMMP-9 activation and in gelatin degradation. To model the evolution of these activities in the omptin barrel, we performed a substitution analysis in Pla and genetically modified it into a PgtE-like gelatinase. Our results indicate that PgtE and Pla have diverged in substrate specificity, and suggest that Salmonella PgtE has evolved to functionally mimic mammalian MMPs.

Ebselen has a wide spectrum of interesting therapeutic actions including antioxidant, cytoprotective, neuroprotective and anti-inflammatory activities. Since its antioxidant effect is very well known, this paper links the effects of ebselen in redox cellular status to its possible involvement in the maintenance of the integrity of genomic information by using Saccharomyces cerevisiae strains proficient and deficient in antioxidant defences and the mammalian V79 cell line. Using the alkaline comet assay, we showed that 5-10 microM ebselen does not induce DNA damage in V79 cells. Similarly, these same concentrations diminished the extent of the DNA damage induced by hydrogen peroxide (H(2)O(2)). The modified comet assay using DNA glycosylases (formamidopyrimidine-DNA glycosylase and endonuclease II) showed that after pre-treatment with ebselen followed by exposure to H(2)O(2), oxidative damage as recognized by these enzymes was significantly lower. In the same way, ebselen showed strong activity against H(2)O(2)-induced oxidative damage in the anti-mutagenic assay using S. cerevisiae N123 strain and in the antioxidative assay by using S. cerevisiae strains lacking antioxidant defences. This antioxidant effect was more pronounced for the gpx3 delta mutant, which indicated that ebselen acts by mimicking the GPx3 catalytic activity. The results confirm that ebselen is involved in antioxidant defence and that its antioxidant ability contributes to its anti-mutagenic and anti-genotoxic action.

The beta-barrel outer membrane protease Pla from Yersinia pestis is an important virulence factor in plague and enables initiation of the bubonic plague. Pla is a multifunctional protease whose expression also enhances bacterial adherence to extracellular matrix. It has remained uncertain whether the increase in cellular adhesiveness results from modification of the bacterial surface by Pla, or whether the Pla molecule is an adhesin. Pla was purified as a His6-fusion protein from Escherichia coli and reconstituted with lipopolysaccharide to an enzymatically active form. Purified His6-Pla was coated onto fluorescent micro-particles (FMPs) that expressed plasminogen activity. Pla-coated FMPs also bound to laminin and to reconstituted basement membrane (Matrigel) immobilized on permanox slides, whereas only poor activity was seen with lipopolysaccharide-coated FMPs or bovine serum albumin-coated FMPs. The results show that the Pla molecule has intrinsic adhesive properties and that purified transmembrane proteins coated onto FMPs can be used for functional assays.

Authors present a case report of a 51 years old man, with a past history of peptic ulcer, arterial hypertension and dislipidemia. Three weeks before hospitalization he did a upper endoscopy that revealed a duodenal ulcer and oesophageal varices (grade II/III). During etiologic investigation, hepatic disease and portal hypertension were excluded. He had a goiter that invaded the anterior mediastinum. After thyroidectomy the oesophageal varices disappeared.

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 epidemiology of antimicrobial resistance of clinical isolates and human intestinal strains of Bacteroides fragilis has assumed great importance in the last few years since this microorganism, like other members of the B. fragilis group, can be responsible for the spread of resistance determinants. It is possible that the presence of B. fragilis in polluted aquatic environments might contribute to the spread of resistance. The antimicrobial resistance profile of 44 clinical B. fragilis strains isolated from 1981-1988 and 1991-1998 from the University hospital of Rio de Janeiro, and of 17 faecal and 17 polluted aquatic environmental B. fragilis strains isolated between 1991 and 1998 was determined. The susceptibility tests against penicillin, cefoxitin, imipenem, meropenem, clindamycin, chloramphenicol and metronidazole were performed by Etest in Wilkins-Chalgren agar enriched with 5% sheep blood. Motivated by some high MIC values for cefoxitin and meropenem, the cfiA gene, which codes for a metallo-beta-lactamase, was investigated among all strains, using PCR amplification. The resistance to penicillin was high in the samples from 1981 to 1988 (92.9%) and also in those from 1991 to 1998 (100%), although the MIC90 decreased from 256 mg/L to 24 mg/L. An increase in the resistance level to clindamycin and cefoxitin was seen from one decade to the other, the MIC90 values changing from 4 mg/L to 12 mg/L and from 8 mg/L to 32 mg/L, respectively. The susceptibility profile for metronidazole, chloramphenicol, imipenem and meropenem remained stable, although two clinical strains showed MICs of 6 mg/L and 8 mg/L against meropenem. Almost all human intestinal strains were resistant to penicillin and all of them were susceptible to imipenem, meropenem, chloramphenicol and metronidazole. The MICs of meropenem against two strains isolated from a polluted aquatic environment were 6 mg/L and 32 mg/L. The cfiA gene was detected in five strains, two of which were isolated from clinical specimens against which the MIC values of cefoxitin were high and three from an aquatic environment, whose susceptibility to both cefoxitin and meropenem ranged from sensitive to resistant.

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.