Intrinsic aerobic capacity governs the associations between gut microbiota composition and fat metabolism age-dependently in rat siblings.

Host genetic factors affecting the gut microbiome play an important role in obesity, yet limited attention has been paid on the host genetic factors linked to physical fitness in modifying the microbiome. This study determined whether sibling-matched pairs of rats selectively bred for high (HCR) and low (LCR) aerobic capacity differ in their microbiome age-dependently and which taxa associate with differential in metabolism. Several taxa in young adult rats (hereafter young) linked to inherited aerobic capacity, while in older adult (hereafter old) rats most of the differences between the lines associated with body weight. Despite the absence of weight differential between LCR and HCR when young, the LCR microbiome contained more Actinobacteria, Veillonellaceae, Coriobacteriaceae, Phascolarctobacterium, and Ruminococcus; taxa previously linked to obesity. This raises the question whether the microbiome contributes to the later development of obesity in LCR. Age-related differences were detected in almost all taxa in both rat lines. The young HCR measured higher for serum glycerol and free fatty-acids and lower for cholesterol, HDL, LDL, and triglycerides than LCR. The old HCR differed from the old LCR by lower LDL. Several metabolites, including LDL, are associated age and genetic background-dependently with the microbiome, which might explain the metabolic differences between the lines. While old lines did not differ in visceral adipose tissue gene expression, the young HCR expressed more inflammatory genes than LCR, and several taxa including Proteobacteria associated with these genes. In conclusion, intrinsic aerobic capacity governs the microbiome, which may influence body weight, metabolism, and gene expression.

High-throughput DNA extraction strategy for fecal microbiome studies.

Microbiome studies are becoming larger in size to detect the potentially small effect that environmental factors have on our gut microbiomes, or that the microbiome has on our health. Therefore, fast and reproducible DNA isolation methods are needed to handle thousands of fecal samples. We used the Chemagic 360 chemistry and Magnetic Separation Module I (MSMI) instrument to compare two sample preservatives and four different pre-treatment protocols to find an optimal method for DNA isolation from thousands of fecal samples. The pre-treatments included bead beating, sample handling in tube and plate format, and proteinase K incubation. The optimal method offers a sufficient yield of high-quality DNA without contamination. Three human fecal samples (adult, senior, and infant) with technical replicates were extracted. The extraction included negative controls (OMNIgeneGUT, DNA/RNA shield fluid, and Chemagic Lysis Buffer 1) to detect cross-contamination and ZymoBIOMICS Gut Microbiome Standard as a positive control to mimic the human gut microbiome and assess sensitivity of the extraction method. All samples were extracted using Chemagic DNA Stool 200 H96 kit (PerkinElmer, Finland). The samples were collected in two preservatives, OMNIgeneGUT and DNA/RNA shield fluid. DNA quantity was measured using Qubit-fluorometer, DNA purity and quality using gel electrophoresis, and taxonomic signatures with 16S rRNA gene-based sequencing with V3V4 and V4 regions. Bead beating increased bacterial diversity. The largest increase was detected in gram-positive genera Blautia, Bifidobacterium, and Ruminococcus. Preservatives showed minor differences in bacterial abundances. The profiles between the V3V4 and V4 regions differed considerably with lower diversity samples. Negative controls showed signs from genera abundant in fecal samples. Technical replicates of the Gut Standard and stool samples showed low variation. The selected isolation protocol included recommended steps from manufacturer as well as bead beating. Bead beating was found to be necessary to detect hard-to-lyse bacteria. The protocol was reproducible in terms of DNA yield among different stool replicates and the ZymoBIOMICS Gut Microbiome Standard. The MSM1 instrument and pre-treatment in a 96-format offered the possibility of automation and handling of large sample collections. Both preservatives were feasible in terms of sample handling and had low variation in taxonomic signatures. The 16S rRNA target region had a high impact on the composition of the bacterial profile. IMPORTANCE: Next-generation sequencing (NGS) is a widely used method for determining the composition of the gut microbiota. Due to the differences in the gut microbiota composition between individuals, microbiome studies have expanded into large population studies to maximize detection of small effects on microbe-host interactions. Thus, the demand for a rapid and reliable microbial profiling is continuously increasing, making the optimization of high-throughput 96-format DNA extraction integral for NGS-based downstream applications. However, experimental protocols are prone to bias and errors from sample collection and storage, to DNA extraction, primer selection and sequencing, and bioinformatics analyses. Methodological bias can contribute to differences in microbiome profiles, causing variability across studies and laboratories using different protocols. To improve consistency and confidence of the measurements, the standardization of microbiome analysis methods has been recognized in many fields.

Appendiceal microbiome in uncomplicated and complicated acute appendicitis: A prospective cohort study.

BACKGROUND: Uncomplicated and complicated acute appendicitis seem to be two different forms of this common abdominal emergency. The contribution of appendiceal microbiota to appendicitis pathogenesis has been suggested, but differences between uncomplicated and complicated appendicitis are largely unknown. We compared the appendiceal microbiota in uncomplicated and complicated acute appendicitis. METHODS: This prospective single-center clinical cohort study was conducted as part of larger multicenter MAPPAC trial enrolling adult patients with computed tomography or clinically confirmed uncomplicated or complicated acute appendicitis. The microbial composition of the appendiceal lumen was determined using 16S rRNA gene amplicon sequencing. RESULTS: Between April 11, 2017, and March 29, 2019, 118 samples (41 uncomplicated and 77 complicated appendicitis) were available. After adjusting for age, sex, and BMI, alpha diversity in complicated appendicitis was higher (Shannon p = 0.011, Chao1 p = 0.006) compared to uncomplicated appendicitis. Microbial compositions were different between uncomplicated and complicated appendicitis (Bray-Curtis distance, P = 0.002). Species poor appendiceal microbiota composition with specific predominant bacteria was present in some patients regardless of appendicitis severity. CONCLUSION: Uncomplicated and complicated acute appendicitis have different appendiceal microbiome profiles further supporting the disconnection between these two different forms of acute appendicitis. STUDY REGISTRATION: ClinicalTrials.gov NCT03257423.

Prospective multicentre cohort trial on acute appendicitis and microbiota, aetiology and effects of antimicrobial treatment: study protocol for the MAPPAC (Microbiology APPendicitis ACuta) trial.

INTRODUCTION: Based on the epidemiological and clinical data, acute appendicitis can present either as uncomplicated or complicated. The aetiology of these different appendicitis forms remains unknown. Antibiotic therapy has been shown to be safe, efficient and cost-effective for CT-confirmed uncomplicated acute appendicitis. Despite appendicitis being one of the most common surgical emergencies, there are very few reports on appendicitis aetiology and pathophysiology focusing on the differences between uncomplicated and complicated appendicitis. Microbiology APPendicitis ACuta (MAPPAC) trial aims to evaluate these microbiological and immunological aspects including immune response in the aetiology of these different forms also assessing both antibiotics non-responders and appendicitis recurrence. In addition, MAPPAC aims to determine antibiotic and placebo effects on gut microbiota composition and antimicrobial resistance. METHODS AND ANALYSIS: MAPPAC is a prospective clinical trial with both single-centre and multicentre arm conducted in close synergy with concurrent trials APPendicitis ACuta II (APPAC II) (per oral (p.o.) vs intravenous+p.o. antibiotics, NCT03236961) and APPAC III (double-blind trial placebo vs antibiotics, NCT03234296) randomised clinical trials. Based on the enrolment for these trials, patients with CT-confirmed uncomplicated acute appendicitis are recruited also to the MAPPAC study. In addition to these conservatively treated randomised patients with uncomplicated acute appendicitis, MAPPAC will recruit patients with uncomplicated and complicated appendicitis undergoing appendectomy. Rectal and appendiceal swabs, appendicolith, faecal and serum samples, appendiceal biopsies and clinical data are collected during the hospital stay for microbiological and immunological analyses in both study arms with the longitudinal study arm collecting faecal samples also during follow-up up to 12 months after appendicitis treatment. ETHICS AND DISSEMINATION: This study has been approved by the Ethics Committee of the Hospital District of Southwest Finland (Turku University Hospital, approval number ATMK:142/1800/2016) and the Finnish Medicines Agency. Results of the trial will be published in peer-reviewed journals. TRIAL REGISTRATION NUMBER: NCT03257423.