Unveiling the etiology of peritonsillar abscess using next generation sequencing.

BACKGROUND: Peritonsillar abscess (PTA) is a severe deep neck space infection with an insufficiently characterized bacterial etiology. We aimed to reveal the bacteria associated with PTA applying next generation sequencing (NGS). Tonsil biopsies and pus samples of 91 PTA patients were analysed applying NGS method. RESULTS: Over 400 genera and 800 species belonging to 34 phyla were revealed. The most abundant species in both sample types were Streptococcus pyogenes, Fusobacterium necrophorum and Fusobacterium nucleatum. When present, S. pyogenes was normally a predominant species, although it was recovered as a minor population in some samples dominated by F. nucleatum and occasionally F. necrophorum. S. pyogenes and F. necrophorum were the predominant species (> 10% in a community) in 28 (31%) pus samples, while F. nucleatum in 21 (23%) and S. anginosus in 8 (9%) pus samples. We observed no substantial differences between the microbial findings in pus and tonsil biopsies. CONCLUSIONS: The most probable causative agents of PTA according to our NGS-study include Streptococcus pyogenes, Fusobacterium necrophorum and Fusobacterium nucleatum. Some other streptococci (S. anginosus) and anaerobes (Prevotella, Porphyromonas) may contribute to the infection as well. Pus of the peritonsillar abscess is more representative specimen for microbiological examination than the tonsillar tissue. Our results are important in the context of optimizing the handling of the PTA patients.

Lactobacillus crispatus-dominated vaginal microbiome and Acinetobacter-dominated seminal microbiome support beneficial ART outcome.

INTRODUCTION: Despite the considerable progress made in assisted reproductive technologies (ART), the implantation rate of transferred embryos remains low and in many cases, the reasons for failure remain unclear. We aimed to determine the potential impact of female and male partners' reproductive tract microbiome composition on ART outcome. MATERIAL AND METHODS: The ART couples (n = 97) and healthy couples (n = 12) were recruited into the study. The smaller healthy group underwent a careful selection according to their reproductive and general health criteria. Both vaginal and semen samples were subjected to 16S rDNA sequencing to reveal the bacterial diversity and identify distinct microbial community types. Ethics statement The study was approved by the Ethics Review Committee on Human Research of Tartu University, Tartu, Estonia (protocol no. 193/T-16) on 31 May 2010. Participation in the research was voluntary. Written informed consent was obtained from all study participants. RESULTS: The men with Acinetobacter-associated community who had children in the past, had the highest ART success rate (P < 0.05). The women with bacterial vaginosis vaginal microbiome community and with L. iners-predominant and L. gasseri-predominant microbiome had a lower ART success rate than women with the L. crispatus-predominant or the mixed lactic-acid-bacteria-predominant type (P < 0.05). The 15 couples where both partners had beneficial microbiome types had a superior ART success rate of 53%, when compared with the rest of the couples (25%; P = 0.023). CONCLUSIONS: Microbiome disturbances in the genital tract of both partners tend to be associated with couple's infertility as well as lower ART success levels and may thus need attention before the ART procedure. The incorporation of genitourinary microbial screening as a part of the diagnostic evaluation process may become routine for ART patients if our results are confirmed by other studies.

The complex microbiome from native semen to embryo culture environment in human in vitro fertilization procedure.

BACKGROUND: Only a few microbial studies have conducted in IVF (in vitro fertilization), showing the high-variety bacterial contamination of IVF culture media to cause damage to or even loss of cultured oocytes and embryos. We aimed to determine the prevalence and counts of bacteria in IVF samples, and to associate them with clinical outcome. METHODS: The studied samples from 50 infertile couples included: raw (n = 48), processed (n = 49) and incubated (n = 50) sperm samples, and IVF culture media (n = 50). The full microbiome was analyzed by 454 pyrosequencing and quantitative analysis by real-time quantitative PCR. Descriptive statistics, t-, Mann-Whitney tests and Spearman's correlation were used for comparison of studied groups. RESULTS: The study involved normozoospermic men. Normal vaginal microbiota was present in 72.0% of female partners, while intermediate microbiota and bacterial vaginosis were diagnosed in 12.0 and 16.0%, respectively. The decreasing bacterial loads were found in raw (35.5%), processed (12.0%) and sperm samples used for oocyte insemination (4.0%), and in 8.0% of IVF culture media. The most abundant genera of bacteria in native semen and IVF culture media were Lactobacillus, while in other samples Alphaproteobacteria prevailed. Staphylococcus sp. was found only in semen from patients with inflammation. Phylum Bacteroidetes was in negative correlation with sperm motility and Alphaproteobacteria with high-quality IVF embryos. CONCLUSION: Our study demonstrates that IVF does not occur in a sterile environment. The prevalent bacteria include classes Bacilli in raw semen and IVF culture media, Clostridia in processed and Bacteroidia in sperm samples used for insemination. The presence of Staphylococcus sp. and Alphaproteobacteria associated with clinical outcomes, like sperm and embryo quality.

ANAMMOX-denitrification biomass in microbial fuel cell to enhance the electricity generation and nitrogen removal efficiency.

The inoculum biomass was collected from a pilot-scale (3 m3 process tank) nitritation-anaerobic ammonium oxidation (ANAMMOX) (deammonification moving bed biofilm (DeaMBBR)) reactor demonstrating the highest total nitrogen removal rate (TNRR) of 0.33 kg N m-3 day-1. This biomass was used for inoculating the anodic chamber of a microbial fuel cell (MFC) to investigate the capacity of DeaMBBR biomass to act as an exo-electrogenic consortia. Performance of MFCs inoculated with ANAMMOX-specific consortia collected from DeaMBBR (MFC-ANA) and another MFC-CON inoculated with a septic tank mixed anaerobic consortium as a control was investigated for electrochemical performance and wastewater treatment efficiency. These MFCs were operated for the total duration of 419 days during which regular feed was given and performance was monitored for first 30 cycles and last 30 cycles, with each cycle of 3 day duration. The MFC-ANA continuously generated bio-energy with higher volumetric power density (9.5 W m-3 and 6.0 W m-3) in comparison to MFC-CON (4.9 and 2.9 W m-3) during the first 30 and last 30 cycles of operational period, respectively. MFC-ANA also achieved 84 ± 2% and 80 ± 2% of COD removal efficiency and 89 ± 4% and 73 ± 2% of total nitrogen removal efficiency during first 30 and last 30 cycles of operational period, respectively. The improvement of nitrogen removal and power production in case of MFC-ANA over MFC-CON could be attributed to the ANAMMOX-denitrifiers populations and Trichococcus (14.92%) as denitrifying exo-electrogenic microbes (4.46%), respectively.

The composition of faecal microbiota is related to the amount and variety of dietary fibres.

For normal gut and body function, the diet should contain variety of dietary fibres. To elucidate the links between food intake, especially the variety of dietary fibres, faecal microbiota, body mass index and content of blood lipids, 59 healthy subjects on common Estonian diet were enrolled. The dietary records were analysed at nutrient level while seven categories of fibres were characterised to differentiate variety of fibre profiles consumed. The data of the high fibre (HF) intake (>15.1 g/1000 kcal) and the low fibre (LF) intake (<9.4 g/1000 kcal) groups were comparatively evaluated. LF diets associated with Collinsella, Coprococcus and Dorea, and higher consumption of meat and white flour products while HF diet with Roseburia, Bacteroides xylanisolvens and Oxalobacter formigenes, and arabinoxylan and pectin rich cereals and vegetables. Based on the results, modulation of the colon microbiota can be suggested by careful variation and enrichment of dietary fibre sources.

Composition and metabolism of fecal microbiota from normal and overweight children are differentially affected by melibiose, raffinose and raffinose-derived fructans.

The aim of the study was to investigate the metabolism of non-digestible oligo- and polysaccharides by fecal microbiota, using isothermal microcalorimetry. The five tested substrates were raffinose, melibiose, a mixture of oligo- and polysaccharides produced from raffinose by levansucrase, levan synthesized from raffinose, and levan from timothy grass. Two inocula were comprised of pooled fecal samples from overweight or normal-weight children, from healthy adult volunteers and a pure culture of Bacteroides thetaiotaomicron as a reference bacterium for colon microbiota. The growth was analyzed based on the heat evolution curves, and the production of organic acids and gases. Taxonomic profiles of the microbiota were assessed by 16S rDNA sequencing. Raffinose and melibiose promoted the growth of bifidobacteria in all fecal pools. Several pool-specific substrate-related responses to raffinose and melibiose were revealed. Lactate-producing bacteria (Streptococcus and Enterococcus) became enriched in the pool of overweight children resulting in lactic acid as the major fermentation product on short saccharides. Acetic and butyric acids were prevalent at fermentation in the normal-weight pool coinciding with the enrichment of Catenibacterium. In the adult pool, the specific promotion of Bacteroides and Lachnospiraceae by levans was disclosed. In the fecal pool of normal-weight children, levans stimulated the growth of Senegalimassilia and Lachnoclostridium and this particular pool also showed the highest maximum heat production rate at levan fermentation. Levans and raffinose-derived oligosaccharides, but not raffinose and melibiose were completely fermented by a pure culture of Bacteroides thetaiotaomicron. The main conclusion from the study is that fecal microbiota of normal and overweight children have different compositions and they respond in specific manners to non-digestible oligo- and polysaccharides: raffinose, melibiose, raffinose-derived oligosaccharides and levans. The potential of the tested saccharides to support a healthy balance of colon microbiota requires further studies.

Comparative Analysis of Gut Microbiota in Centenarians and Young People: Impact of Eating Habits and Childhood Living Environment.

The composition of centenarians' gut microbiota has consistently been used as a model for healthy aging studies. However, there is an incomplete understanding of how childhood living conditions and eating habits affect the development and composition of gastrointestinal microbiota in centenarians with good cognitive functions. We compared the gut microbiota as well as the living and eating habits of the oldest-old group and the young people group. The richness and diversity of microbiota and the abundance of hereditary and environmental microbes were higher in people with longevity than young people. People with longevity ate more potatoes and cereal products. In their childhood, they had more exposure to farm animals and did not have sewers compared with young people. Young people's gut microbiota contained more butyrate-producing bacteria and bacteria that characterized an animal-based Western diet. These results expand our understanding of the effects of childhood environment and diet on the development and stability of the microbiota in people with longevity.

Low-carbohydrate high-fat weight reduction diet induces changes in human gut microbiota.

Obesity has become a major public health problem in recent decades. More effective interventions may result from a better understanding of microbiota alterations caused by weight loss and diet. Our objectives were (a) to calculate the fiber composition of a specially designed low-calorie weight loss diet (WLD), and (b) to evaluate changes in the composition of gut microbiota and improvements in health characteristics during WLD. A total of 19 overweight/obese participants were assigned to 20%-40% reduced calories low-carbohydrate high-fat diet for four weeks. Protein and fat content in the composed diet was 1.5 times higher compared to that in the average diet of the normal weight reference group, while carbohydrate content was 2 times lower. Food consumption data were obtained from the assigned meals. Microbial composition was analyzed before and after WLD intervention from two sequential samples by 16S rRNA gene sequencing. During WLD, body mass index (BMI) was reduced on average 2.5 ± 0.6 kg/m2 and stool frequency was normalized. The assigned diet induced significant changes in fecal microbiota. The abundance of bile-resistant bacteria (Alistipes, Odoribacter splanchnicus), Ruminococcus bicirculans, Butyricimonas, and Enterobacteriaceae increased. Importantly, abundance of bacteria often associated with inflammation such as Collinsella and Dorea decreased in parallel with a decrease in BMI. Also, we observed a reduction in bifidobacteria, which can be attributed to the relatively low consumption of grains. In conclusion, weight loss results in significant alteration of the microbial community structure.

Mainstream-sidestream wastewater switching promotes anammox nitrogen removal rate in organic-rich, low-temperature streams.

The main issues with mainstream anammox application are loss of bacterial activity by low temperatures and by a high organic content of wastewater. We demonstrate a novel switching method between sidestream and mainstream wastewater. The wastewater flow was switched between sidestream (reject water at >22°C) and mainstream (municipal wastewater at 16.5°C), so that the anammox biomass activity and biomass growth could benefit from sidestream conditions. Real sidestream wastewater (biogas plant effluent) (≈1000 mg NH+ 4-N L-1) and synthetic mainstream (municipal wastewater-like source) (≈100 mg NH+ 4-N) wastewater were used for 20 L biofilm reactor feeding. The highest total nitrogen removal rate (TNRR) of 527 g N m-3 d-1 (average TNRR 180 (±140) g N m-3 d-1) was achieved with sidestream wastewater at a low chemical oxygen demand (COD)/TN ratio of 1.1/1. For reactor feeding with mainstream, the highest TNRR achieved was 61 g N m-3 d-1. Average TNRR for mainstream of 20 (±15) g N m-3 d-1 was low due to a higher COD/N ratio of 3.2/1. The highest TNRR in a batch test was achieved at the COD concentration of 480 mg L-1, reflecting a TNRR of ≈5 mg N g-1 TSS h-1. With a high COD concentration of 2600 mg L-1 (TOC/TN = 8/1), TNRR decreased similarly in both feeds to 1.6 mg N g-1 TSS h-1. The anammox microorganism's genus Candidatus Brocadia enrichment in deammonification biofilm reactor was higher in the mainstream operation (7.6% of all bacteria) than in sidestream operation period (<0.7% of all bacteria).

Ameliorating effect of nitrate on nitrite inhibition for denitrifying P-accumulating organisms.

Lowered air supply and organic carbon need are the key factors to reduce wastewater treatment costs and thereby, avoid eutrophication. Denitrifying PO43-- removal (DPR) process using nitrate instead of oxygen for PO43- uptake was started up in the sequencing batch reactor (SBR) at a nitrate dosing rate of 20-25 mg N L-1 d-1. Operation with a real municipal wastewater supplied with CH3COONa, K2HPO4 and KNO3 succeeded in the cultivation of biomass containing denitrifying polyphosphate accumulating organisms (DPAOs). The durations of SBR process anaerobic/anoxic/oxic cycles were 1.5 h, 3.5 h and 1 h, respectively. SBR operation resulted in a maximum PO43--P uptake of 17 mg PO43--P g-1 MLSS. The highest TN and PO43- removal efficiencies were observed during the first half of reactor operation at 77 (±10) % and 71 (±5) %, respectively. An average COD removal rate of 172 (±98) mg g-1 MLSS and a high average removal efficiency of 89 (±4) % were achieved. Nitrite effect with/without nitrate as DPR electron acceptor was investigated in batch-scale to show possibilities to use high nitrite and nitrate contents simultaneously as electron acceptors for the anoxic phosphate uptake. Nitrate attenuation against nitrite toxicity can be economically justified in full-scale treatment applications in which wastewater has a high nitrogen content. Nitrate attenuated nitrite toxicity (caused by nitrite content at 5-100 mg NO2--N L-1) when using supplemental additions of nitrate (at concentrations of 45-200 mg NO3--N L-1) in batch tests. Illumina sequencing emphasized that during biomass adaption microbial community changed by lowered aerobic cycle length and by lowered nitrate dosing towards representation of key DPAO/PAO- organisms, such as Candidatus Accumulibacter, Xanthomonadaceae, Comomonadaceae, Saprospiraceae and Rhodocyclaceae. This study showed that DPAO biomass adaption to nitrate maintained an efficient COD, nitrogen and phosphorus removal and the biomass can be applied for treatment of wastewater containing high nitrite and nitrate content.

Influence of gemcitabine chemotherapy on the microbiota of pancreatic cancer xenografted mice.

BACKGROUND AND AIMS: Pancreatic ductal adenocarcinoma (PDAC) represents the fourth cause of cancer-related death. We aimed to evaluate whether gemcitabine treatment shapes the gut microbiota in a model of PDAC xenografted mice. MATERIALS AND METHODS: Pancreatic cancer xenograft mice were subjected to gemcitabine injection once per week for 3 weeks to assess the tumor volume as compared to control mice injected with normal saline solution. The composition of fecal microbiota, the activation of NF-kB pathway in cancer tissues and the serum metabolomics were further analyzed. RESULTS: Gemcitabine considerably decreases the proportion of Gram- positive Firmicutes (from about 39 to 17%) and the Gram- negative Bacteroidetes (from 38 to 17%) which are the two dominant phyla in the gut of tumor-bearing control mice. This downshift was replaced by an increase of Proteobacteria (Escherichia coli and Aeromonas hydrophila) from 15 up to 32% and Verrucomicrobia (Akkermansia muciniphila) from 5 to 33% in the gut of drug-receiving mice. An overall increase in inflammation-associated bacteria was observed upon gemcitabine. Consistently, activation of the NF-kB canonical pathway was found in cancer tissues from gemcitabine-treated mice. Serum metabolomics revealed a significant decrease of the purine compounds inosine and xanthine, and a decreasing trend for their metabolically-related molecule hypoxanthine. DISCUSSION: Understanding chemotherapy side effects may explain the lack of activity or the chemoresistant processes and it may help to set up strategies to improve the effectiveness of therapy.

Engineered Resistant-Starch (ERS) Diet Shapes Colon Microbiota Profile in Parallel with the Retardation of Tumor Growth in In Vitro and In Vivo Pancreatic Cancer Models.

BACKGROUND/AIMS: Pancreatic cancer (PC) is ranked as the fourth leading cause of cancer-related deaths worldwide. Despite recent advances in treatment options, a modest impact on the outcome of the disease is observed so far. We have previously demonstrated that short-term fasting cycles have the potential to improve the efficacy of chemotherapy against PC. The aim of this study was to assess the effect of an engineered resistant-starch (ERS) mimicking diet on the growth of cancer cell lines in vitro, on the composition of fecal microbiota, and on tumor growth in an in vivo pancreatic cancer mouse xenograft model. MATERIALS AND METHODS: BxPC-3, MIA PaCa-2 and PANC-1 cells were cultured in the control, and in the ERS-mimicking diet culturing condition, to evaluate tumor growth and proliferation pathways. Pancreatic cancer xenograft mice were subjected to an ERS diet to assess tumor volume and weight as compared to mice fed with a control diet. The composition and activity of fecal microbiota were further analyzed in growth experiments by isothermal microcalorimetry. RESULTS: Pancreatic cancer cells cultured in an ERS diet-mimicking medium showed decreased levels of phospho-ERK1/2 (extracellular signal-regulated kinase proteins) and phospho-mTOR (mammalian target of rapamycin) levels, as compared to those cultured in standard medium. Consistently, xenograft pancreatic cancer mice subjected to an ERS diet displayed significant retardation in tumor growth. In in vitro growth experiments, the fecal microbial cultures from mice fed with an ERS diet showed enhanced growth on residual substrates, higher production of formate and lactate, and decreased amounts of propionate, compared to fecal microbiota from mice fed with the control diet. CONCLUSION: A positive effect of the ERS diet on composition and metabolism of mouse fecal microbiota shown in vitro is associated with the decrease of tumor progression in the in vivo PC xenograft mouse model. These results suggest that engineered dietary interventions could be supportive as a synergistic approach to enhance the efficacy of existing cancer treatments in pancreatic cancer patients.

Rat NEURL1 3'UTR is alternatively spliced and targets mRNA to dendrites.

Neuralized, an E3 ubiquitin ligase, interacts with and positively modulates the Notch pathway by promoting ubiquitination and subsequent endocytosis of its ligands. NEURL1 mRNA is dendritically localised in the dentate gyrus of an adult rat brain, implying that it may be locally translated, but its transport mechanisms remain unstudied. Here, we report the presence of a previously unknown, shorter splice-variant of rat NEURL1 3'UTR (1477bp in length), and identify it as a potential target of nonsense-mediated decay. We show that endogenous NEURL1 mRNAs with both longer and shorter 3'UTRs are enriched in the neurites of cultured rat primary hippocampal neurons. Both NEURL1 3'UTRs can mediate transport of reporter mRNAs into dendrites in primary hippocampal neurons. By analysing the dendritic trafficking capacity of reporter mRNAs linked to various regions of longer or shorter NEURL1 3'UTR, we localise the dendritic targeting element (DTE) of spliced version of NEURL1 3'UTR to its first half, corresponding to the nucleotides 1-148 and 416-914 of the full-length 3'UTR. In contrast, the dendritic targeting capacity of the full-length NEURL1 3'UTR is abolished by splitting its 3'UTR in two halves (nt 1-914 and nt 915-1744), suggesting that slightly different DTE might mediate dendritic transport of the two transcripts.