Muramidases found in the foregut microbiome of the Tammar wallaby can direct cell aggregation and biofilm formation

We describe here the role of muramidases present in clones of metagenomic DNA that result in cell aggregation and biofilm formation by Escherichia coli. The metagenomic clones were obtained from uncultured Lachnospiraceae-affiliated bacteria resident in the foregut microbiome of the Tammar wallaby. One of these fosmid clones (p49C2) was chosen for more detailed studies and a variety of genetic methods were used to delimit the region responsible for the phenotype to an open reading frame of 1425 bp. Comparative sequence analysis with other fosmid clones giving rise to the same phenotype revealed the presence of muramidase homologues with the same modular composition. Phylogenetic analysis of the fosmid sequence data assigned these fosmid inserts to recently identified, but uncultured, phylogroups of Lachnospiraceae believed to be numerically dominant in the foregut microbiome of the Tammar wallaby. The muramidase is a modular protein containing putative N-acetylmuramoyl--alanine amidase and an endo-β-N-acetylglucosaminidase catalytic module, with a similar organization and functional properties to some Staphylococcal autolysins that also confer adhesive properties and biofilm formation. We also show here that the cloned muramidases result in the production of extracellular DNA, which appears to be the key for biofilm formation and autoaggregation. Collectively, these findings suggest that biofilm formation and cell aggregation in gut microbiomes might occur via the concerted action of carbohydrate-active enzymes and the production of extracellular DNA to serve as a biofilm scaffold.

Brief report: Intestinal dysbiosis in ankylosing spondylitis

Objective Ankylosing spondylitis (AS) is a common, highly heritable immune-mediated arthropathy that occurs in genetically susceptible individuals exposed to an unknown but likely ubiquitous environmental trigger. There is a close relationship between the gut and spondyloarthritis, as exemplified in patients with reactive arthritis, in whom a typically self-limiting arthropathy follows either a gastrointestinal or urogenital infection. Microbial involvement in AS has been suggested; however, no definitive link has been established. The aim of this study was to determine whether the gut in patients with AS carries a distinct microbial signature compared with that in the gut of healthy control subjects. Methods Microbial profiles for terminal ileum biopsy specimens obtained from patients with recent-onset tumor necrosis factor antagonist-naive AS and from healthy control subjects were generated using culture-independent 16S ribosomal RNA gene sequencing and analysis techniques. Results Our results showed that the terminal ileum microbial communities in patients with AS differ significantly (P < 0.001) from those in healthy control subjects, driven by a higher abundance of 5 families of bacteria (Lachnospiraceae [P = 0.001], Ruminococcaceae [P = 0.012], Rikenellaceae [P = 0.004], Porphyromonadaceae [P = 0.001], and Bacteroidaceae [P = 0.001]) and a decrease in the abundance of 2 families of bacteria (Veillonellaceae [P = 0.01] and Prevotellaceae [P = 0.004]). Conclusion We show evidence for a discrete microbial signature in the terminal ileum of patients with AS compared with healthy control subjects. The microbial composition was demonstrated to correlate with disease status, and greater differences were observed between disease groups than within disease groups. These results are consistent with the hypothesis that genes associated with AS act, at least in part, through effects on the gut microbiome.

Shedding Light on the Microbial Community of the Macropod Foregut Using 454-Amplicon Pyrosequencing

Twenty macropods from five locations in Queensland, Australia, grazing on a variety of native pastures were surveyed and the bacterial community of the foregut was examined using 454-amplicon pyrosequencing. Specifically, the V3/V4 region of 16S rRNA gene was examined. A total of 5040 OTUs were identified in the data set (post filtering). Thirty-two OTUs were identified as 'shared' OTUS (i.e. present in all samples) belonging to either Firmicutes or Bacteroidetes (Clostridiales/Bacteroidales). These phyla predominated the general microbial community in all macropods. Genera represented within the shared OTUs included: unclassified Ruminococcaceae, unclassified Lachnospiraceae, unclassified Clostridiales, Peptococcus sp. Coprococcus spp., Streptococcus spp., Blautia sp., Ruminoccocus sp., Eubacterium sp., Dorea sp., Oscillospira sp. and Butyrivibrio sp. The composition of the bacterial community of the foregut samples of each the host species (Macropus rufus, Macropus giganteus and Macropus robustus) was significantly different allowing differentiation between the host species based on alpha and beta diversity measures. Specifically, eleven dominant OTUs that separated the three host species were identified and classified as: unclassified Ruminococcaceae, unclassified Bacteroidales, Prevotella spp. and a Syntrophococcus sucromutans. Putative reductive acetogens and fibrolytic bacteria were also identified in samples. Future work will investigate the presence and role of fibrolytics and acetogens in these ecosystems. Ideally, the isolation and characterization of these organisms will be used for enhanced feed efficiency in cattle, methane mitigation and potentially for other industries such as the biofuel industry.

Lower Gastrointestinal Microbiota in Health and Irritable Bowel Syndrome : Characterisation and Effect of Probiotic Intervention

The human gastrointestinal (GI) microbiota is a complex ecosystem that lives in symbiosis with its host. The growing awareness of the importance of the microbiota to the host as well as the development of culture-free laboratory techniques and computational methods has enormously expanded our knowledge of this microbial community. Irritable bowel syndrome (IBS) is a common functional bowel disorder affecting up to a fifth of the Western population. To date, IBS diagnosis has been based on GI symptoms and the exclusion of organic diseases. The GI microbiota has been found to be altered in this syndrome and probiotics can alleviate the symptoms, although clear links between the symptoms and the microbiota have not been demonstrated. The aim of the present work was to characterise IBS related alterations in the intestinal microbiota, their relation to IBS symptoms and their responsiveness to probiotic theraphy. In this thesis research, the healthy human microbiota was characterised by cloning and sequencing 16S rRNA genes from a faecal microbial community DNA pool that was first profiled and fractionated according to its guanine and cytosine content (%G+C). The most noticeable finding was that the high G+C Gram-positive bacteria (the phylum Actinobacteria) were more abundant compared to a corresponding library constructed from the unfractionated DNA pool sample. Previous molecular analyses of the gut microbiota have also shown comparatively low amounts of high G+C bacteria. Furthermore, the %G+C profiling approach was applied to a sample constructed of faecal DNA from diarrhea-predominant IBS (IBS-D) subjects. The phylogenetic microbial community comparison performed for healthy and IBS-D sequence libraries revealed that the IBS-D sample was rich in representatives of the phyla Firmicutes and Proteobacteria whereas Actinobacteria and Bacteroidetes were abundant in the healthy subjects. The family Lachnospiraceae within the Firmicutes was especially prevalent in the IBS-D sample. Moreover, associations of the GI microbiota with intestinal symptoms and the quality of life (QOL) were investigated, as well as the effect of probiotics on these factors. The microbial targets that were analysed with the quantitative real-time polymerase chain reaction (qPCR) in this study were phylotypes (species definition according to 16S rRNA gene sequence similarity) previously associated with either health or IBS. With a set of samples, the presence or abundance of a phylotype that had 94% 16S rRNA gene sequence similarity to Ruminococcus torques (R. torques 94%) was shown to be associated with the severity of IBS symptoms. The qPCR analyses for selected phylotypes were also applied to samples from a six-month probiotic intervention with a mixture of Lactobacillus rhamnosus GG, L. rhamnosus Lc705, Propionibacterium freudenreichii ssp. shermanii JS and Bifidobacterium breve Bb99. The intervention had been previously reported to alleviate IBS symptoms, but no associations with the analysed microbiota representatives were shown. However, with the phylotype-specific assays applied here, the abundance of the R. torques 94% -phylotype was shown to be lowered in the probiotic-receiving group during the probiotic supplementation, whereas a Clostridium thermosuccinogenes 85% phylotype, previously associated with a healthy microbiota, was found to be increased compared to the placebo group. To conclude, with the combination of methods applied, higher abundance of Actinobacteria was detected in the healthy gut than found in previous studies, and significant phylum-level microbiota alterations could be shown in IBS-D. Thus, the results of this study provide a detailed overview of the human GI microbiota in healthy subjects and in subjects with IBS. Furthermore, the IBS symptoms were linked to a particular clostridial phylotype, and probiotic supplementation was demonstrated to alter the GI microbiota towards a healthier state with regard to this and an additional bacterial phylotype. For the first time, distinct phylotype-level alterations in the microbiota were linked to IBS symptoms and shown to respond to probiotic therapy.

Investigation of a new acetogen isolated from an enrichment of the tammar wallaby forestomach

Background: Forestomach fermentation in Australian marsupials such as wallabies and kangaroos, though analogous to rumen fermentation, results in lower methane emissions. Insights into hydrogenotrophy in these systems could help in devising strategies to reduce ruminal methanogenesis. Reductive acetogenesis may be a significant hydrogen sink in these systems and previous molecular analyses have revealed a novel diversity of putative acetogens in the tammar wallaby forestomach.Results: Methanogen-inhibited enrichment cultures prepared from tammar wallaby forestomach contents consumed hydrogen and produced primarily acetate. Functional gene (formyltetrahydrofolate synthetase and acetyl-CoA synthase) analyses revealed a restricted diversity of Clostridiales species as the putative acetogens in the cultures. A new acetogen (growth on H-2/CO2 with acetate as primary end product) designated isolate TWA4, was obtained from the cultures. Isolate TWA4 classified within the Lachnospiraceae and demonstrated > 97% rrs identity to previously isolated kangaroo acetogens. Isolate TWA4 was a potent hydrogenotroph and demonstrated excellent mixotrophic growth (concomitant consumption of hydrogen during heterotrophic growth) with glycerol. Mixotrophic growth of isolate TWA4 on glycerol resulted in increased cell densities and acetate production compared to autotrophic growth. Co-cultures with an autotrophic methanogen Methanobrevibacter smithii revealed that isolate TWA4 performed reductive acetogenesis under high hydrogen concentration (> 5 mM), but not at low concentrations. Under heterotrophic growth conditions, isolate TWA4 did not significantly stimulate methanogenesis in a co-culture with M. smithii contrary to the expectation for organisms growing fermentatively.Conclusions: The unique properties of tammar wallaby acetogens might be contributing factors to reduced methanogen numbers and methane emissions from tammar wallaby forestomach fermentation, compared to ruminal fermentation. The macropod forestomach may be a useful source of acetogens for future strategies to reduce methane emissions from ruminants, particularly if these strategies also include some level of methane suppression and/or acetogen stimulation, for example by harnessing mixotrophic growth capabilities