Chris McSweeney

Mucolytic bacteria with increased prevalence in IBD mucosa augment in vitro utilization of mucin by other bacteria.

OBJECTIVES:Mucosa-associated bacteria are increased in inflammatory bowel disease (IBD), which suggests the possibility of an increased source of digestible endogenous mucus substrate. We hypothesized that mucolytic bacteria are increased in IBD, providing increased substrate to sustain nonmucolytic mucosa-associated bacteria.METHODS:Mucolytic bacteria were characterized by the ability to degrade human secretory mucin (MUC2) in pure and mixed anaerobic cultures. Real-time PCR was used to enumerate mucosa-associated mucolytic bacteria in 46 IBD and 20 control patients. Bacterial mucolytic activity was tested in vitro using purified human MUC2.RESULTS:We confirm increased total mucosa-associated bacteria 16S rRNA gene in macroscopically and histologically normal intestinal epithelium of both Crohns disease (CD) (mean 1.9-fold) and ulcerative colitis (UC) (mean 1.3-fold). We found a disproportionate increase in some mucolytic bacteria. Mean Ruminococcus gnavus were increased >4-fold and Ruminococcus torques ∼100-fold in macroscopically and histologically normal intestinal epithelium of both CD and UC. The most abundantly detected mucolytic bacterium in controls, Akkermansia muciniphila, was reduced many fold in CD and in UC. Coculture of A. muciniphila with MUC2 as the sole carbon source led to reduction in its abundance while it augmented growth of other bacteria.CONCLUSIONS:Mucolytic bacteria are present in healthy humans, where they are an integral part of the mucosa-associated bacterial consortium. The disproportionate increase in R. gnavus and R. torques could explain increased total mucosa-associated bacteria in IBD.

Dysbiosis of fecal microbiota in Crohn's disease patients as revealed by a custom phylogenetic microarray.

Background: A custom phylogenetic microarray composed of small subunit ribosomal RNA probes, representing ≈500 bacterial species from the human and animal gut, was developed and evaluated for analysis of gut microbial diversity using fecal samples from healthy subjects and Crohns disease (CD) patients. Methods: Oligonucleotide probes (≈40 mer) used on the microarray were selected from published articles or designed with the “GoArray” microarray probe design program using selected bacterial 16S rRNA sequences. Fecal 16S rDNA from individual samples of six healthy subjects and six CD patients were used as template to generate fluorescently labeled cRNA that was hybridized to the microarray. Differences revealed by the microarray in relative abundance of microbial populations between healthy and diseased patients were verified using quantitative real‐time polymerase chain reaction (PCR) with species‐specific primer sets. Results: The microarray analyses showed that Eubacterium rectale, Bacteroides fragilis group, B. vulgatus, Ruminococcus albus, R. callidus, R. bromii, and Faecalibacterium prausnitzii were 5–10‐fold more abundant in the healthy subjects than in the CD patients, while Enterococcus sp., Clostridium difficile, Escherichia coli, Shigella flexneri, and Listeria sp. were more abundant in the CD group. Conclusions: The microarray detected differences in abundance of bacterial populations within the phylum Firmicutes that had been reported previously for the same samples based on phylogenetic analysis of metagenomic clone libraries. In addition, the microarray showed that Enterococcus sp. was in higher abundance in the CD patients. This microarray should be another useful tool to examine the diversity and abundance of human intestinal microbiota. (Inflamm Bowel Dis 2010)

Characterization of the gastrointestinal microbiota in health and inflammatory bowel disease

&NA; The enteric bacterial flora play a key role in maintaining health. Inflammatory bowel disease is associated with quantitative and qualitative alterations in the microbiota. Early characterization of the microbiota involved culture‐dependent techniques. The advent of metagenomic techniques, however, allows for structural and functional characterization using culture‐independent methods. Changes in diversity, together with quantitative alterations in specific bacterial species, have been identified. The functional significance of these changes, and their pathogenic role, remain to be elucidated. (Inflamm Bowel Dis 2011;)

Association between specific mucosa‐associated microbiota in Crohn's disease at the time of resection and subsequent disease recurrence: A pilot study

Crohns disease pathogenesis involves alterations in the gut microbiota. We characterized the mucosa‐associated microbiota at the time of surgical resection and 6 months later to identify bacterial profiles associated with recurrence and remission.

SPRING: an RCT study of probiotics in the prevention of gestational diabetes mellitus in overweight and obese women

BackgroundObesity is increasing in the child-bearing population as are the rates of gestational diabetes. Gestational diabetes is associated with higher rates of Cesarean Section for the mother and increased risks of macrosomia, higher body fat mass, respiratory distress and hypoglycemia for the infant. Prevention of gestational diabetes through life style intervention has proven to be difficult. A Finnish study showed that ingestion of specific probiotics altered the composition of the gut microbiome and thereby metabolism from early gestation and decreased rates of gestational diabetes in normal weight women. In SPRING (the Study of Probiotics IN the prevention of Gestational diabetes), the effectiveness of probiotics ingestion for the prevention of gestational diabetes will be assessed in overweight and obese women.Methods/designSPRING is a multi-center, prospective, double-blind randomized controlled trial run at two tertiary maternity hospitals in Brisbane, Australia. Five hundred and forty (540) women with a BMI > 25.0 kg/m2 will be recruited over 2 years and receive either probiotics or placebo capsules from 16 weeks gestation until delivery. The probiotics capsules contain > 1x109 cfu each of Lactobacillus rhamnosus GG and Bifidobacterium lactis BB-12 per capsule. The primary outcome is diagnosis of gestational diabetes at 28 weeks gestation. Secondary outcomes include rates of other pregnancy complications, gestational weight gain, mode of delivery, change in gut microbiome, preterm birth, macrosomia, and infant body composition. The trial has 80% power at a 5% 2-sided significance level to detect a >50% change in the rates of gestational diabetes in this high-risk group of pregnant women.DiscussionSPRING will show if probiotics can be used as an easily implementable method of preventing gestational diabetes in the high-risk group of overweight and obese pregnant women.

Isolation and biochemical characterization of two lipases from a metagenomic library of China Holstein cow rumen.

Two novel lipase genes RlipE1 and RlipE2 which encoded 361- and 265-amino acid peptides, respectively, were recovered from a metagenomic library of the rumen microbiota of Chinese Holstein cows. A BLAST search revealed a high similarity (90%) between RlipE2 and a carboxylesterase from Thermosinus carboxydivorans Nor1, while there was a low similarity (below 50%) between RlipE1 and other lipases. Phylogenetic analysis indicated that RlipE2 clustered with the lipolytic enzymes from family V while RlipE1 clustered with six other putative bacterial lipases which might constitute a new subfamily. The recombinant lipases were thermally unstable and retained 60% activity over a pH range of 6.5-8.5. Substrate specificity assay indicated that both enzymes had higher hydrolytic activity toward laurate (C(12)), palmitate (C(16)) and stearate (C(18)). The novel phylogenetic affiliation and high specificity of both enzymes for long-chain fatty acid make them interesting targets for manipulation of rumen lipid metabolism.

Intestinal Gas Capsules: A Proof-of-Concept Demonstration

School of Electrical and Computer Engineering, RMIT University, Department of Gastroenterology, The Alfred Hospital, Monash University, Monash Ageing Research Centre, Monash University, Melbourne, School of Applied Sciences, RMIT University, Bundoora, Department of Agriculture and Food Systems, The University of Melbourne, Parkville, and Commonwealth Scientific and Industrial Research Organisation (CSIRO), Australia

Ruminal bacterial community shifts in grain-, sugar-, and histidine-challenged dairy heifers

Ruminal bacterial community composition (BCC) and its associations with ruminal fermentation measures were studied in dairy heifers challenged with combinations of grain, fructose, and histidine in a partial factorial study. Holstein-Friesian heifers (n=30) were randomly allocated to 5 triticale grain-based treatment groups: (1) control (no grain), (2) grain [fed at a dry matter intake (DMI) of 1.2% of body weight (BW)], (3) grain (0.8% of BW DMI) + fructose (0.4% of BW DMI), (4) grain (1.2% of BW DMI) + histidine (6g/head), and (5) grain (0.8% of BW DMI) + fructose (0.4% of BW DMI) + histidine (6g/head). Ruminal fluid was collected using a stomach tube 5, 115, and 215min after consumption of the rations and bacterial 16S ribosomal DNA sequence data was analyzed to characterize bacteria. Large variation among heifers and distinct BCC were evident in a between-group constrained principal components analysis. Bacterial composition in the fructose-fed heifers was positively related to total lactate and butyrate concentrations. Bacterial composition was positively associated with ruminal ammonia, valerate, and histamine concentrations in the grain-fed heifers. The predominant phyla were the Firmicutes (57.6% of total recovered sequences), Bacteroidetes (32.0%), and candidate phylum TM7 (4.0%). Prevotella was the dominant genus. In general, grain or histidine or their interactions with time had minimal effects on the relative abundance of bacterial phyla and families. Fructose increased and decreased the relative abundance of the Firmicutes and Proteobacteria phyla over time, respectively, and decreased the abundance of the Prevotellaceae family over time. The relative abundance of the Streptococcaceae and Veillonellaceae families was increased in the fructose-fed heifers and these heifers over time. A total of 31 operational taxonomic units differed among treatment groups in the 3.6h sampling period, Streptococcus bovis was observed in fructose fed animals. The TM7 candidate phylum had an increased abundance of sequence reads by over 2.5 fold due to the introduction of histidine into the diet. Rapid changes in BCC can occur in a short period after a single substrate challenge and the nature of these changes may influence ruminal acidosis risk and differ from those in cattle exposed to substrate challenges over a longer time period.

Identification of metabolically active proteobacterial and archaeal communities in the rumen by DNA- and RNA-derived 16S rRNA gene

To gain new insights into the metabolic contribution of bacterial group in the rumen.

Isolation and survey of novel fluoroacetate-degrading bacteria belonging to the phylum Synergistetes

Microbial dehalogenation of chlorinated compounds in anaerobic environments is well known, but the degradation of fluorinated compounds under similar conditions has rarely been described. Here, we report on the isolation of a bovine rumen bacterium that metabolizes fluoroacetate under anaerobic conditions, the mode of degradation and its presence in gut ecosystems. The bacterium was identified using 16S rRNA gene sequence analysis as belonging to the phylum Synergistetes and was designated strain MFA1. Growth was stimulated by amino acids with greater quantities of amino acids metabolized in the presence of fluoroacetate, but sugars were not fermented. Acetate, formate, propionate, isobutryate, isovalerate, ornithine and H(2) were end products of amino acid metabolism. Acetate was the primary end product of fluoroacetate dehalogenation, and the amount produced correlated with the stoichiometric release of fluoride which was confirmed using fluorine nuclear magnetic resonance ((19) F NMR) spectroscopy. Hydrogen and formate produced in situ were consumed during dehalogenation. The growth characteristics of strain MFA1 indicated that the bacterium may gain energy via reductive dehalogenation. This is the first study to identify a bacterium that can anaerobically dehalogenate fluoroacetate. Nested 16S rRNA gene-specific PCR assays detected the bacterium at low numbers in the gut of several herbivore species.