Bead-Beat Micro AX Gravity

Methanogenic archaea are a functionally important component of the intestinal microbiota of humans and animals, participating in the utilization of detrimental hydrogen produced during gut fermentation. Despite this, archaeal DNA has rarely been found in intestinal microbiome analyses, which prompts the need to optimize detecting procedures of these microorganisms, including the DNA isolation step. Three commercially available kits for DNA isolation and one extra purification kit that removes PCR inhibitors were evaluated on chicken droppings. In addition, different variants of mechanical lysis and a double elution were tested to ensure the maximum efficiency of DNA isolation from archaea as well as bacteria. A quantitative real-time PCR was used to monitor the optimization progress. As a result, the combination of the selected Genomic Mini AX Bacteria+ kit with a 2-min-long sonication by ultrasonic probe and enzymatic pretreatment gave excellent extraction efficiency rates for DNA of methanogenic archaea (an approximate 50-fold increase compared to the standard enzymatic lysis described by the producer) and, at the same time, provided optimal protection of DNA extracted from bacteria susceptible to enzymatic lysis. The presented results indicate that the optimized protocol allows for highly efficient extraction of total DNA, which is well-suited for quantitative microbial analyses by real-time PCR.

Inflammatory bowel disease (IBD) is a serious global public health concern. Although the pathogenesis of the disease is currently unknown, it has been reported to be associated with both intestinal microbiota and inflammatory mediators. There is evidence suggesting that the feces of the Plateau pika is useful for treating gastrointestinal injuries and pain. Although fecal microbiota transplantation is highly efficacious intervention for IBD prevention, however, potential the transfer of pathogenic microbes or toxic substances is potentially hazardous. Fortunately, micropore filtering of the donor feces can minimize the risk of bacterial infection allowing retention of the therapeutic effects of the residual bacteriophages. Here, we demonstrated that Plateau pika feces not only alleviated the IBD symptoms but also promoted optimal structure and composition of the intestinal microbiota. Additionally, Plateau pika feces transfer also enhanced phenotypic features, such as, body-weight, disease activity index, and histological scores. In conclusion, Plateau pika feces was found to protect mice against colitis induced by dextran sodium sulfate by reducing inflammation and regulating microbial dysbiosis. These findings suggest the potential of Plateau pika feces as an alternative therapy for IBD.

Fungi inhabiting bird nests may pose a serious threat to living organisms. Therefore, the main goal of the study was to identify cultivable fungi in the nest of grey heron (Ardea cinerea) located near the city centre of Wroclaw (Poland). Overall, 10 different fungal species were obtained which were both cosmopolitan and potentially hazardous to humans, homoiothermous animals and plants. The greatest number of fungal species was obtained from the nest fragments with visible fungal growth, and the least from western conifer seed bugs (Leptoglossus occidentalis) inhabiting the nests. The damp chamber allowed isolation of Aspergillus fumigatus, Penicillium coprophilum, and P. griseofulvum as directly related to the occurrence of visible fungal growth on plant fragments of grey heron nests.

Group sizes in an animal study are calculated from estimates on variation, effect, power and significance level. Much of the variation in glucose related parameters of the diet-induced obese (DIO) mouse model is due to inter-individual variation in gut microbiota composition. In addition, standard tandem repeats (STRs) in the non-coding DNA shows that inbred mice are not always homogenic. C57BL/6NTac (B6NTac) mice from Taconic and C57BL/6NRj (B6NRj) mice from Janvier Labs were fed a high calorie diet and treated with liraglutide. The fecal microbiota was sequenced before high-calorie feeding (time 1) and after diet-induced obesity instantly before liraglutide treatment (time 2) and mice were divided into clusters on the basis of their microbiota. Although liraglutide in both sub-strains alleviated glucose intolerance and reduced body weight, in a one-way ANOVA a borderline reduction in glycosylated hemoglobin (HbA1c) could only be shown in B6NTac mice. However, if the microbiota clusters from time 1 or time 2 were incorporated in a two-way ANOVA, the HbA1c effect was significant in B6NTac mice in both analyses, while this did not change anything in B6NRj mice. In a one-way ANOVA the estimated group size needed for a significant HbA1c effect in B6NTac mice was 42, but in two-way ANOVAs based upon microbiota clusters of time 1 or time 2 it was reduced to 21 or 12, respectively. The lowering impact on glucose tolerance was also powered by incorporation of microbiota clusters of both times in both sub-strains. B6NRj had up to six, while B6NTac had maximum three alleles in some of their STRs. In B6NRj mice in 28.8% of the STRs the most prevalent allele had a gene frequency less than 90%, while this was only 6.6% in the B6NTac mice. However, incorporation of the STRs with the highest number of alleles or the most even distribution of frequencies in two-way ANOVAs only had little impact on the outcome of data evaluation. It is concluded that the inclusion of microbiota clusters in a two-way ANOVA in the evaluation of the glucose related effects of an intervention in the DIO mouse model might be an efficient tool for increasing power and reducing group sizes in mouse sub-strains, if these have a microbiota, which influences these parameters.

Many bacteria and archaea harbor the adaptive CRISPR-Cas system, which stores small nucleotide fragments from previous invasions of nucleic acids via viruses or plasmids. This molecular archive blocks further invaders carrying identical or similar nucleotide sequences. However, few of these systems have been confirmed experimentally to be active in gut bacteria. Here, we demonstrate experimentally that the type I-C CRISPR-Cas system of the prevalent gut bacterium Eggerthella lenta can specifically target and cleave foreign DNA in vitro by using a plasmid transformation assay. We also show that the CRISPR-Cas system acquires new immunities (spacers) from the genome of a virulent E. lenta phage using traditional phage assays in vitro but also in vivo using gnotobiotic (GB) mice. Both high phage titer and an increased number of spacer acquisition events were observed when E. lenta was exposed to a low multiplicity of infection in vitro, and three phage genes were found to contain protospacer hotspots. Fewer new spacer acquisitions were detected in vivo than in vitro. Longitudinal analysis of phage-bacteria interactions showed sustained coexistence in the gut of GB mice, with phage abundance being approximately one log higher than the bacteria. Our findings show that while the type I-C CRISPR-Cas system is active in vitro and in vivo, a highly virulent phage in vitro was still able to co-exist with its bacterial host in vivo. Taken altogether, our results suggest that the CRISPR-Cas defense system of E. lenta provides only partial immunity in the gut.