Comparative composition of bacteria in the human intestinal microflora during remission and active ulcerative colitis

Ulcerative colitis is a severe, relapsing and remitting disease of the human large intestine characterised by inflammation of the mucosa and submucosa. The main site of disease is the sigmoid/rectal region of the large bowel but the aetiology remains unknown. There is considerable evidence to indicate that the components of the resident colonic microflora can play an important role in initiation of the disease. The present study was aimed at characterising the faecal microflora of ulcerative colitis patients in remission and active phases to determine profile differences. Faecal samples were obtained from 12 patients, 6 with active colitis and 6 in remission. The samples were analysed for populations of lactobacilli, bifidobacteria, clostridia, bacteroides, sulphate-reducing bacteria (SRB) and total bacteria using culture independent fluorescence in situ hybridisation (FISH). Lactobacillus-specific denaturing gradient gel electrophoresis (DGGE) was then performed to compare the species present. Numbers of lactobacilli were significantly lower (p<0.05) during the active phase of the disease but the other populations tested did not differ. DGGE analysis revealed that Lactobacillus salivarus, Lactobacillus manihotivorans and Pediococcus acidilactici were present in remission, but not during active inflammation. These results imply that a reduction in intestinal Lactobacillus species may be important in the initiation of ulcerative colitis.

Effects of bovine alpha-lactalbumin and casein glycomacropeptide-enriched infant formulae on faecal microbiota in healthy term infants

Objective: Certain milk factors may promote the growth of a host-friendly gastrointestinal microbiota, for example, one that is predominated by bifidobacteria, a perceived healthpromoting genus. This may explain why breast-fed infants experience fewer intestinal infections than their formula-fed counterparts who are believed to have a more diverse microbiota, which is similar to that of adults. The effects of formulas supplemented with 2 such ingredients from bovine milk, a-lactalbumin (alpha-lac) and casein glycomacropeptide (GMP), on gut flora were investigated in this study. Patients and Methods: Six-week-old (4-8 wk), healthy term infants were randomised to a standard infant formula or 1 of 2 test formulae enriched in alpha-Jac with higher or lower GMP until 6 months. Faecal bacteriology was determined by the culture-independent procedure fluorescence in situ hybridisation. Results: There was a large fluctuation of bacterial counts within groups with no statistically significant differences between groups. Although all groups showed a. predominance of bifidobacteria, breast-fed infants had a small temporary increase in counts. Other bacterial levels varied in formula-fed groups, which overall showed an adult-like faecal microflora. Conclusions: It can be speculated that a prebiotic effect for alpha-lac and GMP is achieved only with low starting populations of beneficial microbiota (eg, infants not initially breast-fed.

rRNA probes used to quantify the effects of glycomacropeptide and alpha-lactalbumin supplementation on the predominant groups of intestinal bacteria of infant rhesus monkeys challenged with enteropathogenic Escherichia coli

Objectives: Certain milk factors may help to promote the growth of a host-friendly colonic microflora (e.g. bifidobacteria, lactobacilli) and explain why breast-fed infants experience fewer and milder intestinal infections than those who are formula-fed. The effects of supplementation of formula with two such milk factors was investigated in this study. Materials and Methods: Infant rhesus macaques were breastfed, fed control formula, or formula supplemented with glycomacropeptide (GMP) or alpha-lactalburnin (alpha-LA) from birth to 5 months of age. Blood was drawn monthly and rectal swabs were collected weekly. At 4.5 months of age, 10(8) colonyforming units of enteropathogenic E.coli O127, strain 2349/68 (EPEC) was given orally and the response to infection assessed. The bacteriology of rectal swabs pre- and post-infection was determined by culture independent fluorescence in situ hybridization. Results: Post-challenge, breast-fed infants and infants fed alpha-LA-supplemented formula had no diarrhea, whilst those infants fed GMP-supplemented formula had intermittent diarrhea. In infants fed control formula the diarrhea was acute. Conclusions: Supplementation of infant formula with appropriate milk proteins may be useful for improving the infant's ability to resist acute infection caused by E.coli.

In vitro evaluation of the microbiota modulation abilities of different sized whole oat grain flakes.

Epidemiological studies and healthy eating guidelines suggest a positive correlation between ingestion of whole grain cereal and food rich in fibre with protection from chronic diseases. The prebiotic potential of whole grains may be related, however, little is known about the microbiota modulatory capability of oat grain or the impact processing has on this ability. In this study the fermentation profile of whole grain oat flakes, processed to produce two different sized flakes (small and large), by human faecal microbiota was investigated in vitro. Simulated digestion and subsequent fermentation by gut bacteria was investigated using pH controlled faecal batch cultures inoculated with human faecal slurry. The different sized oat flakes, Oat 23’s (0.53–0.63 mm) and Oat 25’s/26’s (0.85–1.0 mm) were compared to oligofructose, a confirmed prebiotic, and cellulose, a poorly fermented carbohydrate. Bacterial enumeration was carried out using the culture independent technique, fluorescent in situ hybridisation, and short chain fatty acid (SCFA) production monitored by gas chromatography. Significant changes in total bacterial populations were observed after 24 h incubation for all substrates except Oat 23’s and cellulose. Oats 23’s fermentation resulted in a significant increase in the Bacteroides–Prevotella group. Oligofructose and Oats 25’s/26’s produced significant increases in Bifidobacterium in the latter stages of fermentation while numbers declined for Oats 23’s between 5 h and 24 h. This is possibly due to the smaller surface area of the larger flakes inhibiting the simulated digestion, which may have resulted in increased levels of resistant starch (Bifidobacterium are known to ferment this dietary fibre). Fermentation of Oat 25’s/26’s resulted in a propionate rich SCFA profile and a significant increase in butyrate, which have both been linked to benefiting host health. The smaller sized oats did not produce a significant increase in butyrate concentration. This study shows for the first time the impact of oat grain on the microbial ecology of the human gut and its potential to beneficially modulate the gut microbiota through increasing Bifidobacterium population.

Konjac glucomannan hydrolysate beneficially modulates bacterial composition and activity within the faecal microbiota

The prebiotic potential of a konjac glucomannan hydrolysate (GMH) was investigated in vitro using batch cultures inoculated with human faeces. Bacterial enumeration was carried out using the culture independent technique, fluorescent in situ hybridisation (FISH), and short chain fatty acid (SCFA) production was monitored by gas chromatography. The populations of Bifidobacterium genus, Lactobacillus–Enterococcus group and the Atopobium group all significantly increased after GMH and inulin fermentation. The Bacteroides–Prevotella group had a lower end population after GMH fermentation while inulin gave an increase, although these differences were not significant. No significant differences in SCFA concentrations were observed between inulin and GMH. As with inulin, GMH produced selective stimulation of beneficial gut microbiota and a favourable SCFA profile. In order to confirm a beneficial effect of GMH further in vivo studies involving healthy human volunteers should be considered.

Detection and identification of species-specific bacteria associated with synanthropic mites

Internal bacterial communities of synanthropic mites Acarus siro, Dermatophagoides farinae, Lepidoglyphus destructor, and Tyrophagus putrescentiae (Acari: Astigmata) were analyzed by culturing and culture-independent approaches from specimens obtained from laboratory colonies. Homogenates of surface-sterilized mites were used for cultivation on non-selective agar and DNA extraction. Isolated bacteria were identified by sequencing of the 16S rRNA gene. PCR amplified 16S rRNA genes were analyzed by terminal restriction fragment length polymorphism analysis (T-RFLP) and cloning sequencing. Fluorescence in situ hybridization using universal bacterial probes was used for direct bacterial localization. T-RFLP analysis of 16S rRNA gene revealed distinct species-specific bacterial communities. The results were further confirmed by cloning and sequencing (284 clones). L. destructor and D. farinae showed more diverse communities then A. siro and T. putrescentiae. In the cultivated part of the community, the mean CFUs from four mite species ranged from 5.2 × 102 to 1.4 × 103 per mite. D. farinae had significantly higher CFUs than the other species. Bacteria were located in the digestive and reproductive tract, parenchymatical tissue, and in bacteriocytes. Among the clones, Bartonella-like bacteria occurring in A. siro and T. putresecentiae represented a distinct group related to Bartonellaceae and to Bartonella-like symbionts of ants. The clones of high similarity to Xenorhabdus cabanillasii were found in L. destructor and D. farinae, and one clone related to Photorhabdus temperata in A. siro. Members of Sphingobacteriales cloned from D. farinae and A. siro clustered with the sequences of “Candidatus Cardinium hertigii” and as a separate novel cluster.

Development and validation of a qPCR-based method for quantifying Shiga toxin-encoding and other lambdoid bacteriophages

P>To address whether seasonal variability exists among Shiga toxin-encoding bacteriophage (Stx phage) numbers on a cattle farm, conventional plaque assay was performed on water samples collected over a 17 month period. Distinct seasonal variation in bacteriophage numbers was evident, peaking between June and August. Removal of cattle from the pasture precipitated a reduction in bacteriophage numbers, and during the winter months, no bacteriophage infecting Escherichia coli were detected, a surprising occurrence considering that 1031 tailed-bacteriophages are estimated to populate the globe. To address this discrepancy a culture-independent method based on quantitative PCR was developed. Primers targeting the Q gene and stx genes were designed that accurately and discriminately quantified artificial mixed lambdoid bacteriophage populations. Application of these primer sets to water samples possessing no detectable phages by plaque assay, demonstrated that the number of lambdoid bacteriophage ranged from 4.7 x 104 to 6.5 x 106 ml-1, with one in 103 free lambdoid bacteriophages carrying a Shiga toxin operon (stx). Specific molecular biological tools and discriminatory gene targets have enabled virus populations in the natural environment to be enumerated and similar strategies could replace existing propagation-dependent techniques, which grossly underestimate the abundance of viral entities.

Human microbiota in health and disease

Each human body plays host to a microbial population which is both numerically vast (at around 1014 microbial cells) and phenomenally diverse (over 1,000 species). The majority of the microbial species in the gut have not been cultured but the application of culture-independent approaches for high throughput diversity and functionality analysis has allowed characterisation of the diverse microbial phylotypes present in health and disease. Studies in monozygotic twins, showing that these retain highly similar microbiota decades after birth and initial colonisation, are strongly indicative that diversity of the microbiome is host-specific and affected by the genotype. Microbial diversity in the human body is reflected in both richness and evenness. Diversity increases steeply from birth reaching its highest point in early adulthood, before declining in older age. However, in healthy subjects there appears to be a core of microbial phylotypes which remains relatively stable over time. Studies of individuals from diverse geopraphies suggest that clusters of intestinal bacterial groups tend to occur together, constituting ‘enterotypes’. So variation in intestinal microbiota is stratified rather than continuous and there may be a limited number of host/microbial states which respond differently to environmental influences. Exploration of enterotypes and functional groups may provide biomarkers for disease and insights into the potential for new treatments based on manipulation of the microbiome. In health, the microbiota interact with host defences and exist in harmonious homeostasis which can then be disturbed by invading organisms or when ‘carpet bombing’ by antibiotics occurs. In a portion of individuals with infections, the disease will resolve itself without the need for antibiotics and microbial homeostasis with the host’s defences is restored. The administration of probiotics (live microorganisms which when administered in adequate amounts confer a health benefit on the host) represents an artificial way to enhance or stimulate these natural processes. The study of innate mechanisms of antimicrobial defence on the skin, including the production of numerous antimicrobial peptides (AMPs), has shown an important role for skin commensal organisms. These organisms may produce AMPs, and also amplify the innate immune responses to pathogens by activating signalling pathways and processing host produced AMPs. Research continues into how to enhance and manipulate the role of commensal organisms on the skin. The challenges of skin infection (including diseases caused by multiply resistant organisms) and infestations remain considerable. The potential to re-colonise the skin to replace or reduce pathogens, and exploring the relationship between microbiota elsewhere and skin diseases are among a growing list of research targets. Lactobacillus species are among the best known ‘beneficial’ bacterial members of the human microbiota. Of the approximately 120 species known, about 15 are known to occur in the human vagina. These organisms have multiple properties, including the production of lactic acid, hydrogen peroxide and bacteriocins, which render the vagina inhospitable to potential pathogens. Depletion of the of the normal Lactobacillus population and overgrowth of vaginal anaerobes, accompanied by the loss of normal vaginal acidity can lead to bacterial vaginosis – the commonest cause of abnormal vaginal discharge in women. Some vaginal anaerobes are associated with the formation of vaginal biofilms which serve to act as a reservoir of organisms which persists after standard antibiotic therapy of bacterial vaginosis and may help to account for the characteristically high relapse rate in the condition. Administration of Lactobacillus species both vaginally and orally have shown beneficial effects in the treatment of bacterial vaginosis and such treatments have an excellent overall safety record. Candida albicans is a frequent coloniser of human skin and mucosal membranes, and is a normal part of the microbiota in the mouth, gut and vagina. Nevertheless Candida albicans is the most common fungal pathogen worldwide and is a leading cause of serious and often fatal nosocomial infections. What turns this organism from a commensal to a pathogen is a combination of increasing virulence in the organism and predisposing host factors that compromise immunity. There has been considerable research into the use of probiotic Lactobacillus spp. in vaginal candidiasis. Studies in reconstituted human epithelium and monolayer cell cultures have shown that L. rhamnosus GG can protect mucosa from damage caused by Candida albicans, and enhance the immune responses of mucosal surfaces. Such findings offer the promise that the use of such probiotic bacteria could provide new options for antifungal therapy. Studies of changes of the human intestinal microbiota in health and disease are complicated by its size and diversity. The Alimentary Pharmabiotic Centre in Cork (Republic of Ireland) has the mission to ‘mine microbes for mankind’ and its work illustrates the potential benefits of understanding the gut microbiota. Work undertaken at the centre includes: mapping changes in the microbiota with age; studies of the interaction between the microbiota and the gut; potential interactions between the gut microbiota and the central nervous system; the potential for probiotics to act as anti-infectives including through the production of bacteriocins; and the characterisation of interactions between gut microbiota and bile acids which have important roles as signalling molecules and in immunity. The important disease entity where the role of the gut microbiota appears to be central is the Irritable Bowel Syndrome (IBS). IBS patients show evidence of immune activation, impaired gut barrier function and abnormal gut microbiota. Studies with probiotics have shown that these organisms can exert anti-inflammatory effects in inflammatory bowel disease and may strengthen the gut barrier in IBS of the diarrhoea-predominant type. Formal randomised trials of probiotics in IBS show mixed results with limited benefit for some but not all. Studies confirm that administered probiotics can survive and temporarily colonise the gut. They can also stimulate the numbers of other lactic acid bacilli in the gut, and reduce the numbers of pathogens. However consuming live organisms is not the only way to influence gut microbiota. Dietary prebiotics are selectively fermented ingredients that can change the composition and/or activity of the gastrointestinal microbiota in beneficial ways. Dietary components that reach the colon, and are available to influence the microbiota include poorly digestible carbohydrates, such as non-starch polysaccharides, resistant starch, non-digestible oligosaccharides (NDOs) and polyphenols. Mixtures of probiotic and prebiotic ingredients that can selectively stimulate growth or activity of health promoting bacteria have been termed ‘synbiotics’. All of these approaches can influence gut microbial ecology, mainly to increase bifidobacteria and lactobacilli, but metagenomic approaches may reveal wider effects. Characterising how these changes produce physiological benefits may enable broader use of these tactics in health and disease in the future. The current status of probiotic products commercially available worldwide is less than ideal. Prevalent problems include misidentification of ingredient organisms and poor viability of probiotic microorganisms leading to inadequate shelf life. On occasions these problems mean that some commercially available products cannot be considered to meet the definition of a probiotic product. Given the potential benefits of manipulating the human microbiota for beneficial effects, there is a clear need for improved regulation of probiotics. The potential importance of the human microbiota cannot be overstated. ‘We feed our microbes, they talk to us and we benefit. We just have to understand and then exploit this.’ (Willem de Vos).

Characterization of para-Nitrophenol-degrading bacterial communities in river water by using functional markers and stable isotope probing

Microbial degradation is a major determinant of the fate of pollutants in the environment. para-Nitrophenol (PNP) is an EPA listed priority pollutant with a wide environmental distribution, but little is known about the microorganisms that degrade it in the environment. We studied the diversity of active PNP-degrading bacterial populations in river water using a novel functional marker approach coupled with [13C6]PNP stable isotope probing (SIP). Culturing together with culture-independent terminal restriction fragment length polymorphism analysis of 16S rRNA gene amplicons identified Pseudomonas syringae to be the major driver of PNP degradation in river water microcosms. This was confirmed by SIP-pyrosequencing of amplified 16S rRNA. Similarly, functional gene analysis showed that degradation followed the Gram-negative bacterial pathway and involved pnpA from Pseudomonas spp. However, analysis of maleylacetate reductase (encoded by mar), an enzyme common to late stages of both Gram-negative and Gram-positive bacterial PNP degradation pathways, identified a diverse assemblage of bacteria associated with PNP degradation, suggesting that mar has limited use as a specific marker of PNP biodegradation. Both the pnpA and mar genes were detected in a PNP-degrading isolate, P. syringae AKHD2, which was isolated from river water. Our results suggest that PNP-degrading cultures of Pseudomonas spp. are representative of environmental PNP-degrading populations.

Microbiological diversity of generalized aggressive periodontitis by 16S rRNA clonal analysis

Background/aim: The purpose of this study was to determine the bacterial diversity in the subgingival plaque of subjects with generalized aggressive periodontitis by using culture-independent molecular methods based on 16S ribosomal DNA cloning. Methods: Samples from 10 subjects with generalized aggressive periodontitis were selected. DNA was extracted and the 16S rRNA gene was amplified with the universal primer pairs 9F and 1525R. Amplified genes were cloned, sequenced, and identified by comparison with known 16S rRNA sequences. Results: One hundred and ten species were identified from 10 subjects and 1007 clones were sequenced. Of these, 70 species were most prevalent. Fifty-seven percent of the clone (40 taxa) sequences represented phylotypes for which no cultivated isolates have been reported. Several species of Selenomonas and Streptococcus were found at high prevalence and proportion in all subjects. Overall, 50% of the clone libraries were formed by these two genera. Selenomonas sputigena, the species most commonly detected, was found in nine of 10 subjects. Other species of Selenomonas were often present at high levels, including S. noxia, Selenomonas sp. EW084, Selenomonas sp. EW076, Selenomonas FT050, Selenomonas sp. P2PA_80, and Selenomonas sp. strain GAA14. The classical putative periodontal pathogens, such as, Aggregatibacter actinomycetemcomitans, was below the limit of detection and was not detected. Conclusion: These data suggest that other species, notably species of Selenomonas, may be associated with disease in generalized aggressive periodontitis subjects.

COEVOLUTION BETWEEN ATTINE ANTS and ACTINOMYCETE BACTERIA: A REEVALUATION

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Effects of eating disorders on oral fungal diversity

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

Biofilm microbial communities of denture stomatitis

Introduction: Denture stomatitis is a common lesion that affects denture wearers. Its multifactorial etiology seems to depend on a complex and poorly characterized biofilm. The purpose of this study was to assess the composition of the microbial biofilm obtained from complete denture wearers with and without denture stomatitis using culture-independent methods.Methods: Samples were collected from healthy denture wearers and from patients with denture stomatitis. Libraries comprising about 600 cloned 16S ribosomal DNA (rDNA) bacterial sequences and 192 cloned eukaryotic internal transcribed spacer (ITS) region sequences, obtained by polymerase chain reactions, were analyzed.Results: The partial 16S rDNA sequences revealed a total of 82 bacterial species identified in healthy subjects and patients with denture stomatitis. Twenty-seven bacterial species were detected in both biofilms, 29 species were exclusively present in patients with denture stomatitis, and 26 were found only in healthy subjects. Analysis of the ITS region revealed the presence of Candida sp. in both biofilms.Conclusion: The results revealed the extent of the microbial flora, suggesting the existence of distinct biofilms in healthy subjects and in patients with denture stomatitis.

Diversidade funcional, genética e estrutural de micro-organismos do solo após 13 anos de aplicações anuais de lodo de esgoto sob cultura do milho

Pós-graduação em Agronomia (Ciência do Solo) - FCAV

Diversidade e evolução na simbiose entre bactérias e formigas Attini

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