Diet Complexity and Estrogen Receptor β Status Affect the Composition of the Murine Intestinal Microbiota

Intestinal microbial dysbiosis contributes to the dysmetabolism of luminal factors, including steroid hormones (sterones) that affect the development of chronic gastrointestinal inflammation and the incidence of sterone-responsive cancers of the breast, prostate, and colon. Little is known, however, about the role of specific host sterone nucleoreceptors, including estrogen receptor β (ERβ), in microbiota maintenance. Herein, we test the hypothesis that ERβ status affects microbiota composition and determine if such compositionally distinct microbiota respond differently to changes in diet complexity that favor Proteobacteria enrichment. To this end, conventionally raised female ERβ^+/+ and ERβ^−/− C57BL/6J mice (mean age of 27 weeks) were initially reared on 8604, a complex diet containing estrogenic isoflavones, and then fed AIN-76, an isoflavone-free semisynthetic diet, for 2 weeks. 16S rRNA gene surveys revealed that the fecal microbiota of 8604-fed mice and AIN-76-fed mice differed, as expected. The relative diversity of Proteobacteria, especially the Alphaproteobacteria and Gammaproteobacteria, increased significantly following the transition to AIN-76. Distinct patterns for beneficial Lactobacillales were exclusive to and highly abundant among 8604-fed mice, whereas several Proteobacteria were exclusive to AIN-76-fed mice. Interestingly, representative orders of the phyla Proteobacteria, Bacteroidetes, and Firmicutes, including the Lactobacillales, also differed as a function of murine ERβ status. Overall, these interactions suggest that sterone nucleoreceptor status and diet complexity may play important roles in microbiota maintenance. Furthermore, we envision that this model for gastrointestinal dysbiosis may be used to identify novel probiotics, prebiotics, nutritional strategies, and pharmaceuticals for the prevention and resolution of Proteobacteria-rich dysbiosis.     

Molecular Characterization of the Fecal Microbiota in Patients with Nonalcoholic Steatohepatitis – A Longitudinal Study

Background

The human gut microbiota has profound influence on host metabolism and immunity. This study characterized the fecal microbiota in patients with nonalcoholic steatohepatitis (NASH). The relationship between microbiota changes and changes in hepatic steatosis was also studied.

Methods

Fecal microbiota of histology-proven NASH patients and healthy controls was analyzed by 16S ribosomal RNA pyrosequencing. NASH patients were from a previously reported randomized trial on probiotic treatment. Proton-magnetic resonance spectroscopy was performed to monitor changes in intrahepatic triglyceride content (IHTG).

Results

A total of 420,344 16S sequences with acceptable quality were obtained from 16 NASH patients and 22 controls. NASH patients had lower fecal abundance of Faecalibacterium and Anaerosporobacter but higher abundance of Parabacteroides and Allisonella. Partial least-square discriminant analysis yielded a model of 10 genera that discriminated NASH patients from controls. At month 6, 6 of 7 patients in the probiotic group and 4 of 9 patients in the usual care group had improvement in IHTG (P = 0.15). Improvement in IHTG was associated with a reduction in the abundance of Firmicutes (R² = 0.4820, P = 0.0028) and increase in Bacteroidetes (R² = 0.4366, P = 0.0053). This was accompanied by corresponding changes at the class, order and genus levels. In contrast, bacterial biodiversity did not differ between NASH patients and controls, and did not change with probiotic treatment.

Conclusions

NASH patients have fecal dysbiosis, and changes in microbiota correlate with improvement in hepatic steatosis. Further studies are required to investigate the mechanism underlying the interaction between gut microbes and the liver.     

Characterization of the Fecal Microbiota of Pigs before and after Inoculation with “Brachyspira hampsonii”

“Brachyspira hampsonii” causes disease indistinguishable from swine dysentery, and the structure of the intestinal microbiome likely plays a role in determining susceptibility of individual pigs to infection and development of clinical disease. The objectives of the current study were to determine if the pre-inoculation fecal microbiota differed between inoculated pigs that did (INOC MH) or did not (INOC non-MH) develop mucohaemorrhagic diarrhea following challenge with “B. hampsonii”, and to quantify changes in the structure of the microbiome following development of clinical disease. Fecal microbiota profiles were generated based on amplification and sequencing of the cpn60 universal target sequence from 89 samples from 18 pigs collected at −8, −5, −3 and 0 days post-inoculation, and at termination. No significant differences in richness, diversity or taxonomic composition distinguished the pre-inoculation microbiomes of INOC MH and INOC non-MH pigs. However, the development of bloody diarrhea in inoculated pigs was associated with perturbation of the microbiota relative to INOC non-MH or sham-inoculated control pigs. Specifically, the fecal microbiota of INOC MH pigs was less dense (fewer total 16S rRNA copies per gram of feces), and had a lower Bacteroidetes:Firmicutes ratio. Further investigation of the potential long-term effects of Brachyspira disease on intestinal health and performance is warranted.     

Association of calpastatin with inactive calpain: a novel mechanism to control the activation of the protease?

It is generally accepted that the Ca(2+)-dependent interaction of calpain with calpastatin is the most relevant mechanism involved in the regulation of Ca(2+)-induced proteolysis. We now report that a calpain-calpastatin association can occur also in the absence of Ca(2+) or at very low Ca(2+) concentrations, reflecting the physiological conditions under which calpain retains its inactive conformational state. The calpastatin binding region is localized in the non-inhibitory L-domain containing the amino acid sequences encoded by exons 4-7. This calpastatin region recognizes a calpain sequence located near the end of the DII-domain. Interaction of calpain with calpastatins lacking these sequences becomes strictly Ca(2+)-dependent because, under these conditions, the transition to an active state of the protease is an obligatory requirement. The occurrence of the molecular association between Ca(2+)-free calpain and various recombinant calpastatin forms has been demonstrated by the following experimental results. Addition of calpastatin protected calpain from trypsin digestion. Calpain was coprecipitated when calpastatin was immunoprecipitated. The calpastatin molecular size increased following exposure to calpain. The two proteins comigrated in zymogram analysis. Furthermore, calpain-calpastatin interaction was perturbed by protein kinase C phosphorylation occurring at sites located at the exons involved in the association. At a functional level, calpain-calpastatin interaction at a physiological concentration of Ca(2+) represents a novel mechanism for the control of the amount of the active form of the protease potentially generated in response to an intracellular Ca(2+) influx.     

Composition of the Vaginal Microbiota in Women of Reproductive Age – Sensitive and Specific Molecular Diagnosis of Bacterial Vaginosis Is Possible?

Background and Objective

Bacterial vaginosis (BV) is the most common vaginal disorder, characterized by depletion of the normal lactobacillus-dominant microbiota and overgrowth of commensal anaerobic bacteria. This study aimed to investigate the composition of the vaginal microbiota in women of reproductive age (healthy women and women with BV), with the view of developing molecular criteria for BV diagnosis.

Materials and Methods

Vaginal samples from 163 women (79 control, 73 BV and 11 intermediate (Lactobacillary grade II flora) cases) were analyzed using 454 pyrosequencing of the hypervariable regions V3–V4 of the 16S rRNA gene and 16 quantitative bacterial species/genus-specific real-time PCR assays. Sensitivities and specificities of potential BV markers were computed using the Amsel criteria as reference standard for BV. The use of quantitative thresholds for prediction of BV, determined for both relative abundance measured with 454 pyrosequencing and bacterial load measured with qPCR, was evaluated.

Results

Relative to the healthy women, the BV patients had in their vaginal microbiota significantly higher prevalence, loads and relative abundances of the majority of BV associated bacteria. However, only Gardnerella vaginalis, Atopobium vaginae, Eggerthella, Prevotella, BVAB2 and Megasphaera type 1 detected at or above optimal thresholds were highly predictable for BV, with the best diagnostic accuracy shown for A. vaginae. The depletion of Lactobacillus species combined with the presence of either G. vaginalis or A. vaginae at diagnostic levels was a highly accurate BV predictor.

Conclusions

Quantitative determination of the presence of G. vaginalis, A. vaginae, Eggerthella, Prevotella, BVAB2 and Megasphaera type 1 as well as the depletion of Lactobacillus was highly accurate for BV diagnosis. Measurements of abundance of normal and BV microbiota relative to total bacteria in vaginal fluid may provide more accurate BV diagnosis, and be used for test-of-cure, rather than qualitative detection or absolute counts of BV related microorganisms.     

Association of the FCRL3 gene with rheumatoid arthritis: a further example of population specificity?

Association of a functional promoter polymorphism mapping to the Fc receptor-like 3 (FCRL3) gene has recently been reported and replicated with rheumatoid arthritis (RA) in Japanese populations. The aim of this study was to investigate association of the FCRL3 gene with RA in UK subjects. DNA was available from 1065 patients with RA and 2073 population controls from the UK. Four single nucleotide polymorphism (SNP) markers (FCRL3-169*C/T (fclr3_3, rs7528684), fclr3_4 (rs11264799), fclr3_5 (rs945635), fclr3_6 (rs3761959)) all previously associated with RA in a Japanese population were genotyped in 761 RA samples and 484 controls. In the remaining samples, only the putative disease causal polymorphism, FCRL3-169*C/T, was tested. Genotyping was performed using either the Sequenom MassArray iPlex platform or a 5' Allelic discrimination assay (Taqman, ABI). Extensive linkage disequilibrium was present across the promoter SNPs genotyped (r² values = 0.60-0.98). Allele frequencies did not differ between RA cases and controls either for the putative disease causal polymorphism (odds ratio FCRL3-169*C allele = 0.97 (0.87-1.07), p = 0.51) or for the other SNPs tested. Similarly, no association was detected with RA using haplotype analysis or when stratification by shared epitope carriage or by presence of rheumatoid factor was undertaken. This study was powered to detect an effect size of 1.24 or greater for the FCRL3-169*C/T functional promoter polymorphism but no evidence for association was detected, suggesting that this gene will not have a substantial effect in determining susceptibility to RA in populations of Northern European descent.     

Inter Individual Variations of the Fish Skin Microbiota: Host Genetics Basis of Mutualism?

The commensal microbiota of fish skin is suspected to provide a protection against opportunist infections. The skin of fish harbors a complex and diverse microbiota that closely interacts with the surrounding water microbial communities. Up to now there is no clear evidence as to whether the host regulates the recruitment of environmental bacteria to build a specific skin microbiota. To address this question, we detected Quantitative Trait Loci (QTL) associated with the abundance of specific skin microbiota bacterial strains in brook charr (Salvelinus fontinalis), combining 16S RNA tagged-amplicon 454 pyrosequencing with genetic linkage analysis. Skin microbiota analysis revealed high inter-individual variation among 86 F2 fish progeny based upon the relative abundance of bacterial operational taxonomic units (OTUs). Out of those OTUs, the pathogenic strain Flavobacterium psychrophilum and the non-pathogenic strain Methylobacterium rhodesianum explained the majority of inter-individual distances. Furthermore, a strong negative correlation was found between Flavobacterium and Methylobacterium, suggesting a mutually competitive relationship. Finally, after considering a total of 266 markers, genetic linkage analysis highlighted three major QTL associated with the abundance of Lysobacter, Rheinheimera and Methylobacterium. All these three genera are known for their beneficial antibacterial activity. Overall, our results provide evidence that host genotype may regulate the abundance of specific genera among their surface microbiota. They also indicate that Lysobacter, Rheinheimera and Methylobacterium are potentially important genera in providing protection against pathogens.     

Stabilization of Soil Organic Matter: Association with Minerals or Chemical Recalcitrance?

Soil organic matter (OM) can be stabilized against decomposition by association with minerals, by its inherent recalcitrance and by occlusion in aggregates. However, the relative contribution of these factors to OM stabilization is yet unknown. We analyzed pool size and isotopic composition (¹⁴C, ¹³C) of mineral-protected and recalcitrant OM in 12 subsurface horizons from 10 acidic forest soils. The results were related to properties of the mineral phase and to OM composition as revealed by CPMAS ¹³C-NMR and CuO oxidation. Stable OM was defined as that material which survived treatment of soils with 6 wt% sodium hypochlorite (NaOCl). Mineral-protected OM was extracted by subsequent dissolution of minerals by 10% hydrofluoric acid (HF). Organic matter resistant against NaOCl and insoluble in HF was considered as recalcitrant OM. Hypochlorite removed primarily ¹⁴C-modern OM. Of the stable organic carbon (OC), amounting to 2.4–20.6 g kg⁻¹ soil, mineral dissolution released on average 73%. Poorly crystalline Fe and Al phases (Fe_o, Al_o) and crystalline Fe oxides (Fe_d−o) explained 86% of the variability of mineral-protected OC. Atomic C_p/(Fe+Al)_p ratios of 1.3–6.5 suggest that a portion of stable OM was associated with polymeric Fe and Al species. Recalcitrant OC (0.4–6.5 g kg⁻¹ soil) contributed on average 27% to stable OC and the amount was not correlated with any mineralogical property. Recalcitrant OC had lower Δ¹⁴C and δ¹³C values than mineral-protected OC and was mainly composed of aliphatic (56%) and O-alkyl (13%) C moieties. Lignin phenols were only present in small amounts in either mineral-protected or recalcitrant OM (mean 4.3 and 0.2 g kg⁻¹ OC). The results confirm that stabilization of OM by interaction with poorly crystalline minerals and polymeric metal species is the most important mechanism for preservation of OM in these acid subsoil horizons.     

Projection of Young-Old and Old-Old with Functional Disability: Does Accounting for the Changing Educational Composition of the Elderly Population Make a Difference?

This study compares projections, up to year 2040, of young-old (aged 60-79) and old-old (aged 80+) with functional disability in Singapore with and without accounting for the changing educational composition of the Singaporean elderly. Two multi-state population models, with and without accounting for educational composition respectively, were developed, parameterized with age-gender-(education)-specific transition probabilities (between active, functional disability and death states) estimated from two waves (2009 and 2011) of a nationally representative survey of community-dwelling Singaporeans aged ≥60 years (N=4,990). Probabilistic sensitivity analysis with the bootstrap method was used to obtain the 95% confidence interval of the transition probabilities. Not accounting for educational composition overestimated the young-old with functional disability by 65 percent and underestimated the old-old by 20 percent in 2040. Accounting for educational composition, the proportion of old-old with functional disability increased from 40.8 percent in 2000 to 64.4 percent by 2040; not accounting for educational composition, the proportion in 2040 was 49.4 percent. Since the health profiles, and hence care needs, of the old-old differ from those of the young-old, health care service utilization and expenditure and the demand for formal and informal caregiving will be affected, impacting health and long-term care policy.     

Does the Arthropod Microbiota Impact the Establishment of Vector-Borne Diseases in Mammalian Hosts?

The impact of the microbiota on the immune status of its host is a source of intense research and publicity. In comparison, the effect of arthropod microbiota on vector-borne infectious diseases has received little attention. A better understanding of the vector microbiota in relation to mammalian host immune responses is vital, as it can lead to strategies that affect transmission and improve vaccine design in a field of research where few vaccines exist and effective treatment is rare. Recent demonstrations of how microbiota decrease pathogen development in arthropods, and thus alter vector permissiveness to vector-borne diseases (VBDs), have led to renewed interest. However, hypotheses on the interactions between the arthropod-derived microbiota and the mammalian hosts have yet to be addressed. Advances in DNA sequencing technology, increased yield and falling costs, mean that these studies are now feasible for many microbiologists and entomologists. Here, we distill current knowledge and put forward key questions and experimental designs to shed light on this burgeoning research topic.     

Can Immune Response Mechanisms Explain the Fecal Shedding Patterns of Cattle Infected with Mycobacterium avium Subspecies paratuberculosis?

Johne’s disease (JD) is a chronic disease in ruminants and is caused by infection with Mycobacterium avium subspecies paratuberculosis (MAP). At late stages of the disease, MAP bacilli are shed via feces excretion and in turn create the potential for oral-fecal transmission. The role of the host immune response in MAP bacteria shedding patterns at different stages of JD is still unclear. We employed mathematical modeling to predict if the variation in MAP shedding could be correlated to the immune response in infected animals. We used a novel inverse modeling approach that assumed biological interactions among the antigen-specific lymphocyte proliferation response (cell-mediated response), antibody/humoral immune responses, and MAP bacteria. The modeling framework was used to predict and test possible biological interactions between the measured variables and returns only the essential interactions that are relevant in explaining the observed cattle MAP experimental infection data. Through confronting the models with data, we predicted observed effects (enhancement or suppression) and extents of interactions among the three variables. This analysis enabled classification of the infected cattle into three different groups that correspond to the unique predicted immune responses that are essential to explain the data from cattle within these groups. Our analysis highlights the strong and weak points of the modeling approach, as well as the key immune mechanisms predicted to be expressed in all animals and those that were different between animals, hence giving insight into how animals exhibit different disease dynamics and bacteria shedding patterns.     

Molecular Identification of Candida Species in the Oral Microbiota of Individuals with Down Syndrome: A Case–Control Study

Mycopathologia - Candida species are common in the human oral microbiota and may cause oral candidiasis (OC) when the microbiota equilibrium is disturbed. Immunosuppressed individuals are...     

Intestinal microflora and homeostasis of the mucosal immune response: implications for probiotic bacteria?

The intestinal microflora can be considered a postnatally acquired organ that is composed of a large diversity of bacteria that perform important functions for the host and can be modulated by environmental factors, such as nutrition. Specific components of the intestinal microflora, including lactobacilli and bifidobacteria, have been associated with beneficial effects on the host, such as promotion of gut maturation and integrity, antagonisms against pathogens and immune modulation. Beyond this, the microflora seems to play a significant role in the maintenance of intestinal immune homeostasis and prevention of inflammation. The contribution of the intestinal epithelial cell in the first line of defense against pathogenic bacteria and microbial antigens has been recognized. However, the interactions of intestinal epithelial cells with indigenous bacteria are less well understood. This review will summarize the increasing scientific attention to mechanisms of the innate immune response of the host towards different components of the microflora, and suggest a potential role for selected probiotic bacteria in the regulation of intestinal inflammation.     

Intestinal schistosomiasis: Can a urine sample decide the infection?

Intestinal schistosomiasis, one of the neglected tropical diseases whose control depends on accurate diagnosis of the disease prevalence. The use of low sensitive Kato Katz (KK) fecal egg detection method as a reference gold standard is not an accurate indication especially in low transmission areas. Latent class analysis frameworks especially the Bayesian could be used instead to compare between different diagnostic tests without the use of a gold standard method as a reference. Thus, this study compared two urine-based tests for the detection of circulating antigen and cell free DNA of Schistosoma mansoni versus KK method using the Bayesian latent class analytical framework and in two models where the trace results of point of contact - assay of circulating cathodic antigen (POC-CCA) were once estimated as positive, and as negative in the other model. The Bayesian framework in the trace CCA positive model showed an estimate of disease prevalence of 26% (95% BCI:0 to 60%). POC-CCA showed the highest sensitivity (74% with BCI: 9 to 91%) and lowest specificity for (20% with BCI: 0% to 37%) and the reverse for KK. For POC-CCA with traces considered negative, it was found that results between the three tests were moderated where the positivity for infection by Schistosoma antigen detection and PCR for cell free DNA approached that estimated by the Bayesian framework (44%), and the specificity for point of contact assay(81%; 95%BCI: 59% to 100%) rose in hand with its sensitivity(77%, 95% BCI:53% to 100%) and with results for PCR test (sensitivity = 80%; 95% BCI: 61% to 100%, specificity = 69%; 95% BIC: 47% to 100%). KK remains with the highest specificity while its sensitivity in the two models never exceeded 22%. Thus, we conclude that the use of a single urine sample could be very sensitive and highly specific in the diagnosis of intestinal schistosomiasis using either the trace negative model of point of contact assay, or conventional PCR, when compared to the fecal egg detection using duplicate KK. However, the use of a single tool restricts the management of the disease in areas of low endemicity.     

Does the Initial Floristic Composition model of succession really work?

Harvey & Holzman, in this issue of the Journal of Vegetation Science, describe divergent successional pathways after fire in Pinus muricata forest. Fourteen years after fire, the shrub Ceanothus thyrsiflorus had occupied flat parts of the area. They predicted it would continue to prevent succession to P. muricata forest, as in the initial floristic composition concepts of Clements and Egler.