Cultivation of stable, reproducible microbial communities from different fecal donors using minibioreactor arrays (MBRAs)

Background: Continuous-flow culture models are one tool for studying complex interactions between members of human fecal microbiotas because they allow studies to be completed during an extended period of time under conditions where pH, nutrient availability, and washout of waste products and dead cells can be controlled. Because many of the existing well-validated continuous-flow models are large and complex, we were interested in developing a simpler continuous-flow system that would allow microbial community dynamics to be examined in higher throughput while still maintaining complex microbial communities. To this end, we developed minibioreactor arrays (MBRAs), small volume bioreactors (15 ml) that allow simultaneous cultivation of up to 48 microbial communities in a single anaerobic chamber. Results: We used MBRA to characterize the microbial community dynamics of replicate reactors inoculated from three different human fecal donors and reactors seeded with feces pooled from these three donors. We found that MBRA could be used to efficiently cultivate complex microbial communities that were a subset of the initial fecal inoculum (15–25 % of fecal OTUs initially observed). After an initial acclimation period of approximately 1 week, communities in each reactor stabilized and exhibited day-to-day variation similar to that observed in stable mouse fecal communities. Replicate reactors were predominately populated by shared core microbial communities; variation between replicate reactors was primarily driven by shifts in abundance of shared operational taxonomic units (OTUs). Consistent with differences between fecal donors, MBRA communities present in reactors seeded with different fecal samples had distinct composition and structure. Conclusions: From these analyses, we conclude that MBRAs can be used to cultivate communities that recapitulate key features of human fecal communities and are a useful tool to facilitate higher-throughput studies of the dynamics of these communities

Humanized microbiota mice as a model of recurrent \u3ci\u3eClostridium difficile\u3c/i\u3e disease

Background: Clostridium difficile disease is the leading antibiotic-associated cause of diarrhea and nosocomial acquired infection in the western world. The per annum burden in the USA alone amounts to 250,000 cases with 14,000 ascribed deaths and medical costs in excess of a billion dollars. Novel models for the study of C. difficile infection are therefore pertinent. Results: Germ free C57BL/6 mice gavaged with a healthy human fecal microbiota maintained a stable “humanized” microbiota over multiple generations when housed under specific pathogen-free (SPF) conditions. As with mice containing a conventional microbiota, treatment with a five-antibiotic cocktail followed by a single dose of clindamycin renders the animals susceptible to C. difficile infection (CDI). Interestingly, after recovery from the initial CDI infection, a single intraperitoneal injection of clindamycin is sufficient to induce CDI relapse. Relapse of CDI can be induced up to 35 days postinfection after recovery from the initial infection, and multiple episodes of relapse can be induced. Conclusions: This model enables the study of recurrent C. difficile disease in a host containing a human-derived microbiota. Probiotic treatments using human-derived microbes, either prophylactic or curative, can be tested within the model. The identification and testing of human-derived microbial communities within a humanized microbiota mouse model may enable a higher rate of successful transfer of bacteria-based treatments from the lab to human patients due to the microbes involved initiating from, and being adapted to, the human GI tract

Inter-tissue variability in the stable isotope values of European perch (Perca fluviatilis) and pumpkinseed (Lepomis gibbosus)

Ecological studies on native and invasive populations of European perch Perca fluviatilis and pumpkinseed Lepomis gibbosus are often based on stable isotope (SI) analysis based on dorsal muscle, where samples are usually taken from sacrificed fishes. However, other tissues, such as scale and fin tissue, can be used as non-lethal alternatives, where their SI values can be standardised to dorsal muscle values for comparative purposes. In both perch and pumpkinseed, there was a pattern of δ13C enrichment and δ15N depletion from muscle to fin and scale. As comparative studies must account for these inter-tissue differences prior to analyses, conversion equations for SI data from scale and fin tissue to standardised muscle values are provided

Lactobacillus reuteri 6475 increases bone density in intact females only under an inflammatory setting

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Fluorescence spectroscopy and imaging of myocardial apoptosis

Fluorometry is used to detect intrinsic flavoprotein (FP) and nicotinamide adenine dinucleotide NADH signals in an open-chest rabbit model of myocardial ischemia-reperfusion injury. Myocyte apoptosis has been shown clinically to contribute to infarct size following reperfusion of ischemic myocardium. A noninvasive means of assessing apoptosis in this setting would aid in the treatment of subsequent ventricular remodeling. We show that in vivo fluorometry can be useful in apoptosis detection in open-chest surgeries. Specific changes in myocardial redox states have been shown to indicate the presence of apoptosis. Two main mitochondrial intrinsic fluorophores, NADH and FP signals, were measured during normoxia, ischemia, and reperfusion experimental protocol. Ischemia was induced by occlusion of the largest branch of the circumflex coronary artery and fluorescence signals are collected by applying two different fluorescence techniques: in vivo fluorometry and postmortem cryoimaging. The first technique was employed to detect FP and NADH signals in vivo and the latter technique uses freeze trapping and lowtemperature fluorescence imaging. The heart is snap frozen while still in the chest cavity to make a snapshot of the metabolic state of the tissue. After freezing, the ischemic area and its surrounding border zone were excised and the sample was embedded in a frozen buffer for cryoscanning. These two data sets, in vivo fluorometry and low temperature redox scanning, show consistent extreme oxidation of the mitochondrial redox states (higher redox ratio) suggesting the initiation of apoptosis following reperfusion. This represents the first attempt to assess myocyte apoptosis in the beating heart

Microbiota Reconstitution Does Not Cause Bone Loss in Germ-Free Mice

We thank Jenny Auchtung, James Collins, and Laura Schaefer for technical support. James Collins, and Laura Schaefer provided expertise in tissue collection for the animal experiments, and Jenny Auchtung provided expertise in bioinformatics and microbial community analysis. This work was supported by NIH grant NCCIH R01AT007695-05 to N.P., L.M., and R.A.B., as well as seed funding from Baylor College of Medicine to R.A.B. The authors report that they have no competing interests.Peer reviewedPublisher PD