Comparison of five assays for DNA extraction from bacterial cells in human faecal samples

Aim To determine the most effective DNA extraction method for bacteria in faecal samples. Materials and Results This study assessed five commercial methods, that is, NucliSens easyMag, QIAamp DNA Stool Mini kit, PureLink Microbiome DNA purification kit, QIAamp PowerFecal DNA kit and RNeasy PowerMicrobiome kit, of which the latter has been optimized for DNA extraction. The DNA quantity and quality were determined using Nanodrop, Qubit and qPCR. The PowerMicrobiome kit recovered the highest DNA concentration, whereby this kit also recovered the highest gene copy number of Gram positives, Gram negatives and total bacteria. Furthermore, the PowerMicrobiome kit in combination with mechanical pre-treatment (bead beating) and with combined enzymatic and mechanical pre-treatment (proteinase K+mutanolysin+bead beating) was more effective than without pre-treatment. Conclusion From the five DNA extraction methods that were compared, the PowerMicrobiome kit, preceded by bead beating, which is standard included, was found to be the most effective DNA extraction method for bacteria in faecal samples. Significance and Impact of the Study The quantity and quality of DNA extracted from human faecal samples is a first important step to optimize molecular methods. Here we have shown that the PowerMicrobiome kit is an effective DNA extraction method for bacterial cells in faecal samples for downstream qPCR purpose

p-Cresyl sulfate

If chronic kidney disease (CKD) is associated with an impairment of kidney function, several uremic solutes are retained. Some of these exert toxic effects, which are called uremic toxins. p-Cresyl sulfate (pCS) is a prototype protein-bound uremic toxin to which many biological and biochemical (toxic) effects have been attributed. In addition, increased levels of pCS have been associated with worsening outcomes in CKD patients. pCS finds its origin in the intestine where gut bacteria metabolize aromatic amino acids, such as tyrosine and phenylalanine, leading to phenolic end products, of which pCS is one of the components. In this review we summarize the biological effects of pCS and its metabolic origin in the intestine. It appears that, according to in vitro studies, the intestinal bacteria generating phenolic compounds mainly belong to the families Bacteroidaceae, Bifidobacteriaceae, Clostridiaceae, Enterobacteriaceae, Enterococcaceae, Eubacteriaceae, Fusobacteriaceae, Lachnospiraceae, Lactobacillaceae, Porphyromonadaceae, Staphylococcaceae, Ruminococcaceae, and Veillonellaceae. Since pCS remains difficult to remove by dialysis, the gut microbiota could be a future target to decrease pCS levels and its toxicity, even at earlier stages of CKD, aiming at slowing down the progression of the disease and decreasing the cardiovascular burden

Influence of chronic azithromycin treatment on the composition of the oropharyngeal microbial community in patients with severe asthma

Background: This study of the oropharyngeal microbiome complements the previously published AZIthromycin in Severe ASThma (AZISAST) clinical trial, where the use of azithromycin was assessed in subjects with exacerbationprone severe asthma. Here, we determined the composition of the oropharyngeal microbial community by means of deep sequencing of the amplified 16S rRNA gene in oropharyngeal swabs from patients with exacerbationprone severe asthma, at baseline and during and after 6 months treatment with azithromycin or placebo. Results: A total of 1429 OTUs were observed, of which only 59 were represented by more than 0.02% of the reads. Firmicutes, Bacteroidetes, Fusobacteria, Proteobacteria and Actinobacteria were the most abundant phyla and Streptococcus and Prevotella were the most abundant genera in all the samples. Thirteen species only accounted for two thirds of the reads and two species only, i.e. Prevotella melaninogenica and Streptococcus mitis/pneumoniae, accounted for one fourth of the reads. We found that the overall composition of the oropharyngeal microbiome in patients with severe asthma is comparable to that of the healthy population, confirming the results of previous studies. Long term treatment (6 months) with azithromycin increased the species Streptococcus salivarius approximately 5-fold and decreased the species Leptotrichia wadei approximately 5-fold. This was confirmed by Boruta feature selection, which also indicated a significant decrease of L. buccalis/L. hofstadtii and of Fusobacterium nucleatum. Four of the 8 treated patients regained their initial microbial composition within one month after cessation of treatment. Conclusions: Despite large diversity of the oropharyngeal microbiome, only a few species predominate. We confirm the absence of significant differences between the oropharyngeal microbiomes of people with and without severe asthma. Possibly, long term azithromycin treatment may have long term effects on the composition of the oropharygeal microbiome in half of the patients

Nucleic acids enrichment of fungal pathogens to study host-pathogen interactions

Fungal infections, ranging from superficial to life-threatening infections, represent a major public health problem that affects 25% of the worldwide population. In this context, the study of host-pathogen interactions within the host is crucial to advance antifungal therapy. However, since fungal cells are usually outnumbered by host cells, the fungal transcriptome frequently remains uncovered. We compared three different methods to selectively lyse human cells from in vitro mixes, composed of Candida cells and peripheral blood mononuclear cells. In order to prevent transcriptional modification, the mixes were stored in RNAlater. We evaluated the enrichment of fungal cells through cell counting using microscopy and aimed to further enrich fungal nucleic acids by centrifugation and by reducing contaminant nucleic acids from the host. We verified the enrichment of fungal DNA and RNA through qPCR and RT-qPCR respectively and confirmed that the resulting RNA has high integrity scores, suitable for downstream applications. The enrichment method provided here, i.e., lysis with Buffer RLT followed by centrifugation, may contribute to increase the proportion of nucleic acids from fungi in clinical samples, thus promoting more comprehensive analysis of fungal transcriptional profiles. Although we focused on C. albicans, the enrichment may be applicable to other fungal pathogens

TF2Network : predicting transcription factor regulators and gene regulatory networks in Arabidopsis using publicly available binding site information

A gene regulatory network (GRN) is a collection of regulatory interactions between transcription factors (TFs) and their target genes. GRNs control different biological processes and have been instrumental to understand the organization and complexity of gene regulation. Although various experimental methods have been used to map GRNs in Arabidop-sis thaliana, their limited throughput combined with the large number of TFs makes that for many genes our knowledge about regulating TFs is incomplete. We introduce TF2Network, a tool that exploits the vast amount of TF binding site information and enables the delineation of GRNs by detecting potential regulators for a set of co-expressed or functionally related genes. Validation using two experimental benchmarks reveals that TF2Network predicts the correct regulator in 75-92% of the test sets. Furthermore, our tool is robust to noise in the input gene sets, has a low false discovery rate, and shows a better performance to recover correct regulators compared to other plant tools. TF2Network is accessible through a web interface where GRNs are interactively visualized and annotated with various types of experimental functional information. TF2Network was used to perform systematic functional and regulatory gene annotations, identifying new TFs involved in circadian rhythm and stress response

High prevalence of curable sexually transmitted infections among pregnant women in a rural county hospital in Kilifi, Kenya

Background : Women attending antenatal care (ANC) in resource-limited countries are frequently screened for syphilis and HIV, but rarely for other sexually transmitted infections (STIs). We assessed the prevalence of curable STIs, defined as infection with either Chlamydia trachomatis or Neisseria gonorrhoeae or Trichomonas vaginalis, from July to September 2015. Methods : In a cross-sectional study, women attending ANC at the Kilifi County Hospital, Kenya, had a urine sample tested for C. trachomatis/N. gonorrhoeae by GeneXpert and a vaginal swab for T. vaginalis by culture. Bacterial vaginosis (BV) was defined as a Nugent score of 7-10 of the Gram stain of a vaginal smear in combination with self-reported vaginal discharge. Genital ulcers were observed during collection of vaginal swabs. All women responded to questions on socio-demographics and sexual health and clinical symptoms of STIs. Predictors for curable STIs were assessed in multivariable logistic regression. Results : A total of 42/202 (20.8%, 95% confidence interval (CI):15.4-27.0) women had a curable STI. The prevalence was 14.9% for C. trachomatis (95% Cl:10.2-20.5), 1.0% for N. gonorrhoeae (95% CI: 0.1-3.5), 7.4% for T. vaginalis (95% CI:4.2-12.0), 19.3% for BV (95% CI: 14.1-25.4) and 2.5% for genital ulcers (95% CI: 0.8-5.7). Predictors for infection with curable STIs included women with a genital ulcer (adjusted odds ratio (AOR) = 35.0, 95% CI: 2.7-461.6) compared to women without a genital ulcer, women who used water for cleaning after visiting the toilet compared to those who used toilet paper or other solid means (AOR = 4.1, 95% CI:1.5-11.3), women who reported having sexual debut = 18 years (AOR = 2.7, 95% Cl:1.1-6.6), and BV-positive women (AOR = 2.7, 95% Cl:1.1-6.6) compared to BV-negative women. Conclusion : One in five women attending ANC had a curable STI. These infections were associated with genital ulcers, hygiene practices, early sexual debut and bacterial vaginosis