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.

Association of Long-Term Habitual Dietary Fiber Intake since Infancy with Gut Microbiota Composition in Young Adulthood.

BACKGROUND: Dietary fiber is an important health-promoting component of the diet, which is fermented by the gut microbes that produce metabolites beneficial for the host's health. OBJECTIVES: We studied the associations of habitual long-term fiber intake from infancy with gut microbiota composition in young adulthood by leveraging data from the Special Turku Coronary Risk Factor Intervention Project, an infancy-onset 20-y dietary counseling study. METHODS: Fiber intake was assessed annually using food diaries from infancy ≤ age 20 y. At age 26 y, the first postintervention follow-up study was conducted including food diaries and fecal sample collection (N = 357). Cumulative dietary fiber intake was assessed as the area under the curve for energy-adjusted fiber intake throughout the study (age 0-26 y). Gut microbiota was profiled using 16S ribosomal ribonucleic acid amplicon sequencing. The primary outcomes were 1) α diversity expressed as the observed richness and Shannon index, 2) ß diversity using Bray-Curtis dissimilarity scores, and 3) differential abundance of each microbial taxa with respect to the cumulative energy-adjusted dietary fiber intake. RESULTS: Higher cumulative dietary fiber intake was associated with decreased Shannon index (ß = -0.019 per unit change in cumulative fiber intake, P = 0.008). Overall microbial community composition was related to the amount of fiber consumed (permutational analysis of variation R2 = 0.005, P = 0.024). The only genus that was increased with higher cumulative fiber intake was butyrate-producing Butyrivibrio (log2 fold-change per unit change in cumulative fiber intake 0.40, adjusted P = 0.023), whereas some other known butyrate producers such as Faecalibacterium and Subdoligranulum were decreased with higher cumulative fiber intake. CONCLUSIONS: As early-life nutritional exposures may affect the lifetime microbiota composition and disease risk, this study adds novel information on the associations of long-term dietary fiber intake with the gut microbiota. This trial was registered at clinicaltrials.gov as NCT00223600.

An Infancy-Onset 20-Year Dietary Counselling Intervention and Gut Microbiota Composition in Adulthood.

The randomized controlled Special Turku Coronary Risk Factor Intervention Project (STRIP) has completed a 20-year infancy-onset dietary counselling intervention to reduce exposure to atherosclerotic cardiovascular disease risk factors via promotion of a heart-healthy diet. The counselling on, e.g., low intake of saturated fat and cholesterol and promotion of fruit, vegetable, and whole-grain consumption has affected the dietary characteristics of the intervention participants. By leveraging this unique cohort, we further investigated whether this long-term dietary intervention affected the gut microbiota bacterial profile six years after the intervention ceased. Our sub-study comprised 357 individuals aged 26 years (intervention n = 174, control n = 183), whose gut microbiota were profiled using 16S rRNA amplicon sequencing. We observed no differences in microbiota profiles between the intervention and control groups. However, out of the 77 detected microbial genera, the Veillonella genus was more abundant in the intervention group compared to the controls (log2 fold-change 1.58, p < 0.001) after adjusting for multiple comparison. In addition, an association between the study group and overall gut microbiota profile was found only in males. The subtle differences in gut microbiota abundances observed in this unique intervention setting suggest that long-term dietary counselling reflecting dietary guidelines may be associated with alterations in gut microbiota.

Time series analysis of the incidence of acute upper respiratory tract infections, COVID-19 and the use of antibiotics in Finland during the COVID-19 epidemic: a cohort study of 833 444 patients.

OBJECTIVE: To evaluate the trajectories of acute upper respiratory tract infections (URTIs), COVID-19, and the use of antibiotics in Finland during the COVID-19 epidemic. DESIGN: Population-based cohort study. SETTING: Electronic medical records from a nationwide healthcare chain in Finland. PARTICIPANTS: 833 444 patients from a cohort of 1 970 013 Finns who had used medical services between 2017 and 2020. MAIN OUTCOME MEASURES: Number of weekly patients of acute URTIs, COVID-19, and the prescribed number of antibiotics in Finland between 6 January 2020 and 21 June 2020. We estimated the respective expected numbers from 1 March 2020 onward using autoregressive integrated moving average model from 1 January 2017 to 1 March 2020. We assessed the public interest in COVID-19 by collecting Google search trend frequencies. RESULTS: There was a rapid increase in COVID-related internet searches between weeks 10 and 12. At the same time, there was a 106% increase in diagnoses of acute URTIs, from 410 per 100 000 inhabitants to 845 per 100 000. The first COVID-19 cases were diagnosed on week 11. Prescriptions for URTI-related antibiotics declined by 71% (403 per 100 000 to 117 per 100 000) between weeks 11 and 15 while no relevant change took place in prescriptions of antibiotics for urinary tract infections. CONCLUSIONS: At the beginning of the epidemic, many people contacted healthcare professionals with relatively mild symptoms, as indicated by the reduced rate of URTI-antibiotics prescriptions. Our findings indicate that health service providers should be prepared for rapid variations in service demand. Securing access of true COVID-19 patients to proper diagnostics, care and isolation measures may help in preventing the spread of the disease.

Maternal prenatal psychological distress and hair cortisol levels associate with infant fecal microbiota composition at 2.5 months of age.

BACKGROUND: Maternal prenatal stress associates with infant developmental outcomes, but the mechanisms underlying this association are not fully understood. Alterations in the composition and function of infant intestinal microbiota may mediate some of the observed health effects, a viewpoint that is supported by animal studies along with a small human study showing that exposure to prenatal stress modifies the offspring's intestinal microbiota. In the current study, we aim to investigate the associations between maternal prenatal psychological distress (PPD) and hair cortisol concentration (HCC) with infant fecal microbiota composition in a large prospective human cohort. METHODS: The study population was drawn from FinnBrain Birth Cohort Study. Maternal PPD was measured with standardized questionnaires (EPDS, SCL, PRAQ-R2, Daily Hassles) three times during pregnancy (n = 398). A measure addressing the chronicity of PPD was composed separately for each questionnaire. HCC was measured from a five cm segment at gestational week 24 (n = 115), thus covering the early and mid-pregnancy. Infant fecal samples were collected at the age of 2.5 months and analyzed with 16S rRNA amplicon sequencing. RESULTS: Maternal chronic PPD (all symptom measures) showed positive associations (FDR < 0.01) with bacterial genera from phylum Proteobacteria, with potential pathogens, in infants. Further, chronic PPD (SCL, PRAQ-R2, and Daily Hassles negative scale) associated negatively with Akkermansia. HCC associated negatively with Lactobacillus. Neither maternal chronic PPD nor HCC associated with infant fecal microbiota diversity. CONCLUSION: Chronic maternal PPD symptoms and elevated HCC associate with alterations in infant intestinal microbiota composition. In keeping with the earlier literature, maternal PPD symptoms were associated with increases in genera fromProteobacteria phylum. Further research is needed to understand how these microbiota changes are linked with later child health outcomes.

Six-Week Endurance Exercise Alters Gut Metagenome That Is not Reflected in Systemic Metabolism in Over-weight Women.

Recent studies suggest that exercise alters the gut microbiome. We determined whether six-weeks endurance exercise, without changing diet, affected the gut metagenome and systemic metabolites of overweight women. Previously sedentary overweight women (n = 19) underwent a six-weeks endurance exercise intervention, but two were excluded due to antibiotic therapy. The gut microbiota composition and functions were analyzed by 16S rRNA gene amplicon sequencing and metagenomics. Body composition was analyzed with DXA X-ray densitometer and serum metabolomics with NMR metabolomics. Total energy and energy-yielding nutrient intakes were analyzed from food records using Micro-Nutrica software. Serum clinical variables were determined with KONELAB instrument. Soluble Vascular Adhesion Protein 1 (VAP-1) was measured with ELISA and its' enzymatic activity as produced hydrogen peroxide. The exercise intervention was effective, as maximal power and maximum rate of oxygen consumption increased while android fat mass decreased. No changes in diet were observed. Metagenomic analysis revealed taxonomic shifts including an increase in Akkermansia and a decrease in Proteobacteria. These changes were independent of age, weight, fat % as well as energy and fiber intake. Training slightly increased Jaccard distance of genus level ß-diversity. Training did not alter the enriched metagenomic pathways, which, according to Bray Curtis dissimilarity analysis, may have been due to that only half of the subjects' microbiomes responded considerably to exercise. Nevertheless, tranining decreased the abundance of several genes including those related to fructose and amino acid metabolism. These metagenomic changes, however, were not translated into major systemic metabolic changes as only two metabolites, phospholipids and cholesterol in large VLDL particles, decreased after exercise. Training also decreased the amine oxidase activity of pro-inflammatory VAP-1, whereas no changes in CRP were detected. All clinical blood variables were within normal range, yet exercise slightly increased glucose and decreased LDL and HDL. In conclusion, exercise training modified the gut microbiome without greatly affecting systemic metabolites or body composition. Based on our data and existing literature, we propose that especially Akkermansia and Proteobacteria are exercise-responsive taxa. Our results warrant the need for further studies in larger cohorts to determine whether exercise types other than endurance exercise also modify the gut metagenome.

Impact of Antimicrobial Treatment for Acute Otitis Media on Carriage Dynamics of Penicillin-Susceptible and Penicillin-Nonsusceptible Streptococcus pneumoniae.

Background: Despite concerns that antimicrobial treatment of prevalent infections may select for drug-resistant bacteria, the effects of antimicrobial treatment on colonization dynamics have not been well quantified. Methods: We measured impacts of antimicrobial treatment on nasopharyngeal carriage of penicillin-susceptible Streptococcus pneumoniae (PSSP) and penicillin-nonsusceptible (PNSP) lineages at the end of treatment and 15, 30, and 60 days after treatment in a previously conducted randomized, double-blinded, placebo-controlled trial of amoxicillin-clavulanate for stringently defined acute otitis media. Results: In intention-to-treat analyses, immediate treatment with amoxicillin-clavulanate reduced PSSP carriage prevalence by 88% (95% confidence interval [CI], 76%-96%) at the end of treatment and by 27% (-3%-49%) after 60 days but did not alter PNSP carriage prevalence. By the end of treatment, 7% of children who carried PSSP at enrollment remained colonized in the amoxicillin-clavulanate arm, compared with 61% of PSSP carriers who received placebo; impacts of amoxicillin-clavulanate persisted at least 60 days after treatment among children who carried PSSP at enrollment. Amoxicillin-clavulanate therapy reduced PSSP acquisition by >80% over 15 days. Among children who carried PNSP at enrollment, no impacts on carriage prevalence of S. pneumoniae, PSSP, or PNSP were evident at follow-up visits. Conclusions: Although the absolute risk of carrying PNSP was unaffected by treatment, antimicrobial therapy conferred a selective impact on colonizing pneumococci by accelerating clearance and delaying acquisition of PSSP.

Enterobacter cloacae administration induces hepatic damage and subcutaneous fat accumulation in high-fat diet fed mice.

Accumulating evidence indicates that gut microbiota plays a significant role in obesity, insulin resistance and associated liver disorders. Family Enterobacteriaceae and especially Enterobacter cloacae strain B29 have been previously linked to obesity and hepatic damage. The underlying mechanisms, however, remain unclear. Therefore, we comprehensively examined the effects of E. cloacae subsp. cloacae (ATCC® 13047™) administration on host metabolism of mice fed with high-fat diet (HFD). C57BL/6N mice were randomly divided into HFD control, chow control, and E. cloacae treatment groups. The E. cloacae treatment group received live bacterial cells in PBS intragastrically twice a week, every other week for 13 weeks. Both control groups received PBS intragastrically. After the 13-week treatment period, the mice were sacrificed for gene and protein expression and functional analyses. Our results show that E. cloacae administration increased subcutaneous fat mass and the relative proportion of hypertrophic adipocytes. Both subcutaneous and visceral fat had signs of decreased insulin signaling and elevated lipolysis that was reflected in higher serum glycerol levels. In addition, E. cloacae -treated mice had significantly higher hepatic AST and AST/ALT ratio, and their liver histology indicated fibrosis, demonstrating that E. cloacae subsp. cloacae administration promotes hepatic damage in HFD fed mice.

Antimicrobial characterization of silver nanoparticle-coated surfaces by "touch test" method.

Bacterial infections, especially by antimicrobial resistant (AMR) bacteria, are an increasing problem worldwide. AMR is especially a problem with health care-associated infections due to bacteria in hospital environments being easily transferred from patient to patient and from patient to environment, and thus, solutions to prevent bacterial transmission are needed. Hand washing is an effective tool for preventing bacterial infections, but other approaches such as nanoparticle-coated surfaces are also needed. In the current study, direct and indirect liquid flame spray (LFS) method was used to produce silver nanoparticle-coated surfaces. The antimicrobial properties of these nanoparticle surfaces were evaluated with the "touch test" method against Escherichia coli and Staphylococcus aureus. It was shown in this study that in glass samples one silver nanoparticle-coating cycle can inhibit E. coli growth, whereas at least two coating cycles were needed to inhibit S. aureus growth. Silver nanoparticle-coated polyethylene (PE) and PE terephthalate samples did not inhibit bacterial growth as effectively as glass samples: three nanoparticle-coating cycles were needed to inhibit E. coli growth, and more than 30 coating cycles were needed until S. aureus growth was inhibited. To conclude, with the LFS method, it is possible to produce nanostructured large-area antibacterial surfaces which show antibacterial effect against clinically relevant pathogens. Results indicate that the use of silver nanoparticle surfaces in hospital environments could prevent health care-associated infections in vivo.

Faecalibacterium prausnitzii treatment improves hepatic health and reduces adipose tissue inflammation in high-fat fed mice.

Faecalibacterium prausnitzii is considered as one of the most important bacterial indicators of a healthy gut. We studied the effects of oral F. prausnitzii treatment on high-fat fed mice. Compared to the high-fat control mice, F. prausnitzii-treated mice had lower hepatic fat content, aspartate aminotransferase and alanine aminotransferase, and increased fatty acid oxidation and adiponectin signaling in liver. Hepatic lipidomic analyses revealed decreases in several species of triacylglycerols, phospholipids and cholesteryl esters. Adiponectin expression was increased in the visceral adipose tissue, and the subcutaneous and visceral adipose tissues were more insulin sensitive and less inflamed in F. prausnitzii-treated mice. Further, F. prausnitzii treatment increased muscle mass that may be linked to enhanced mitochondrial respiration, modified gut microbiota composition and improved intestinal integrity. Our findings show that F. prausnitzii treatment improves hepatic health, and decreases adipose tissue inflammation in mice and warrant the need for further studies to discover its therapeutic potential.

Gut Microbiota Analysis Results Are Highly Dependent on the 16S rRNA Gene Target Region, Whereas the Impact of DNA Extraction Is Minor.

Next-generation sequencing (NGS) is currently the method of choice for analyzing gut microbiota composition. As gut microbiota composition is a potential future target for clinical diagnostics, it is of utmost importance to enhance and optimize the NGS analysis procedures. Here, we have analyzed the impact of DNA extraction and selected 16S rDNA primers on the gut microbiota NGS results. Bacterial DNA from frozen stool specimens was extracted with 5 commercially available DNA extraction kits. Special attention was paid to the semiautomated DNA extraction methods that could expedite the analysis procedure, thus being especially suitable for clinical settings. The microbial composition was analyzed with 2 distinct protocols: 1 targeting the V3-V4 and the other targeting the V4-V5 area of the bacterial 16S rRNA gene. The overall effect of DNA extraction on the gut microbiota 16S rDNA profile was relatively small, whereas the 16S rRNA gene target region had an immense impact on the results. Furthermore, semiautomated DNA extraction methods clearly appeared suitable for NGS procedures, proposing that application of these methods could importantly reduce hands-on time and human errors without compromising the validity of results.

Correction: Adipocytes as a Link Between Gut Microbiota-Derived Flagellin and Hepatocyte Fat Accumulation.

[This corrects the article DOI: 10.1371/journal.pone.0152786.].

Adipocytes as a Link Between Gut Microbiota-Derived Flagellin and Hepatocyte Fat Accumulation.

While the role of both elevated levels of circulating bacterial cell wall components and adipose tissue in hepatic fat accumulation has been recognized, it has not been considered that the bacterial components-recognizing adipose tissue receptors contribute to the hepatic fat content. In this study we found that the expression of adipose tissue bacterial flagellin (FLG)-recognizing Toll-like receptor (TLR) 5 associated with liver fat content (r = 0.699, p = 0.003) and insulin sensitivity (r = -0.529, p = 0.016) in humans (n = 23). No such associations were found for lipopolysaccharides (LPS)-recognizing TLR4. To study the underlying molecular mechanisms of these associations, human HepG2 hepatoma cells were exposed in vitro to the conditioned culture media derived from FLG or LPS-challenged human adipocytes. The adipocyte-mediated effects were also compared to the effects of direct HepG2 exposure to FLG and LPS. We found that the media derived from FLG-treated adipocytes stimulated fat accumulation in HepG2 cells, whereas either media derived from LPS-treated adipocytes or direct FLG or LPS exposure did not. This is likely due to that FLG-treatment of adipocytes increased lipolysis and secretion of glycerol, which is known to serve a substrate for triglyceride synthesis in hepatocytes. Similarly, only FLG-media significantly decreased insulin signaling-related Akt phosphorylation, IRS1 expression and mitochondrial respiratory chain ATP5A. In conclusion, our results suggest that the FLG-induced TLR5 activation in adipocytes increases glycerol secretion from adipocytes and decreases insulin signaling and mitochondrial functions, and increases fat accumulation in hepatocytes. These mechanisms could, at least partly, explain the adipose tissue TLR5 expression associated with liver fat content in humans.

Role of Nasopharyngeal Bacteria and Respiratory Viruses in Acute Symptoms of Young Children.

BACKGROUND: The spectrum of acute symptoms in young outpatient children with respiratory tract infection (RTI) is variable, and it cannot be explained by the diagnosis of acute otitis media (AOM) versus uncomplicated RTI. We studied that the variation of symptoms is explained by the nasopharyngeal bacteria and/or respiratory viruses. METHODS: Children aged 6-35 months with acute symptoms with AOM (n = 201) or without AOM (n = 225) were eligible in this cross-sectional study. We analyzed their nasopharyngeal samples for pathogenic bacteria by culture and for respiratory viruses by polymerase chain reaction. We surveyed 17 symptoms (fever, respiratory, ear related, nonspecific, gastrointestinal) with a structured questionnaire. RESULTS: Fever had a positive association with influenza viruses [odds ratio (OR): 6.61; 95% confidence interval (CI): 1.66-26.27], human metapneumovirus (OR: 3.84; 95% CI: 1.25-11.77), coronaviruses (OR: 3.45; 95% CI: 1.53-7.75) and parainfluenza viruses (OR: 2.18; 95% CI: 1.07-4.47). Rhinitis (OR: 5.07; 95% CI: 1.93-13.36), nasal congestion (OR: 2.03; 95% CI: 1.25-3.31) and cough (OR: 1.91; 95% CI: 1.15-3.17) had positive associations with Moraxella catarrhalis. Furthermore, cough had a positive association with respiratory syncytial virus (OR: 7.20; 95% CI: 1.59-32.71) and parainfluenza viruses (OR: 2.79; 95% CI: 1.02-7.69). CONCLUSIONS: The variation of acute symptoms in young children may be influenced by both nasopharyngeal bacteria and respiratory viruses. Our results showed a strong association between fever and respiratory viruses; rhinitis, nasal congestion and cough were associated with M. catarrhalis in the presence of viruses. Further studies are required to determine the possible synergistic role of M. catarrhalis in symptoms of RTI.

Detection of Group A Streptococcus from Pharyngeal Swab Samples by Bacterial Culture Is Challenged by a Novel mariPOC Point-of-Care Test.

mariPOC is a novel point-of-care test system for rapid detection of respiratory tract infections. We compared the performance of the mariPOC test to that of bacterial culture for detecting group A streptococcus (GAS) in 219 pharyngitis patients (ages 1-64 years) and 109 healthy asymptomatic controls (ages 19-69 years). In addition, 42 patient samples were analyzed by quantitative PCR (qPCR). Of the 219 pharyngeal patient samples, 32 were positive in a GAS bacterial culture (prevalence 15%) and 65 (30%) in the mariPOC test. The amount of GAS in samples reported positive by the mariPOC test and negative by culture was, on average, 10-fold less than that of those positive in both methods. This indicated that the negative results in bacterial cultures were due to lower sensitivity. The qPCR results were positive and in line with the mariPOC results in 43% of the discordant samples studied. Two GAS culture-positive samples were negative by the mariPOC test. The prevalences of GAS in the control subjects were 2% and 6% by culture and mariPOC results, respectively. We conclude that the mariPOC antigen detection test is more sensitive than the conventional bacterial culture for the detection of GAS among symptomatic pharyngitis patients. The higher prevalence of GAS by the mariPOC test among symptomatic patients was probably not due to carriership, since among the control patients, the difference in the prevalence of GAS by the mariPOC test and culture was not nearly as high, 15% versus 4%, respectively. Clinical trials are needed to show the clinical importance of our findings.

Tackling antibiotic resistance: the environmental framework.

Antibiotic resistance is a threat to human and animal health worldwide, and key measures are required to reduce the risks posed by antibiotic resistance genes that occur in the environment. These measures include the identification of critical points of control, the development of reliable surveillance and risk assessment procedures, and the implementation of technological solutions that can prevent environmental contamination with antibiotic resistant bacteria and genes. In this Opinion article, we discuss the main knowledge gaps, the future research needs and the policy and management options that should be prioritized to tackle antibiotic resistance in the environment.

Toll-like receptor 5 in obesity: the role of gut microbiota and adipose tissue inflammation.

OBJECTIVE: This study aimed at establishing bacterial flagellin-recognizing toll-like receptor 5 (TLR5) as a novel link between gut microbiota composition, adipose tissue inflammation, and obesity. METHODS: An adipose tissue microarray database was used to compare women having the highest (n = 4, H-TLR) and lowest (n = 4, L-TLR) expression levels of TLR5-signaling pathway genes. Gut microbiota composition was profiled using flow cytometry and FISH. Standard laboratory techniques were used to determine anthropometric and clinical variables. In vivo results were verified using cultured human adipocytes. RESULTS: The H-TLR group had higher flagellated Clostridium cluster XIV abundance and Firmicutes-to-Bacteroides ratio. H-TLR subjects had obese phenotype characterized by greater waist circumference, fat %, and blood pressure (P < 0.05 for all). They also had higher leptin and lower adiponectin levels (P < 0.05 for both). Six hundred and sixty-eight metabolism- and inflammation-related adipose tissue genes were differentially expressed between the groups. In vitro studies confirmed that flagellin activated TLR5 inflammatory pathways, decreased insulin signaling, and increased glycerol secretion. CONCLUSIONS: The in vivo findings suggest that flagellated Clostridium cluster XIV bacteria contribute to the development of obesity through distorted adipose tissue metabolism and inflammation. The in vitro studies in adipocytes show that the underlying mechanisms of the human findings may be due to flagellin-activated TLR5 signaling.

Fermentable fibres condition colon microbiota and promote diabetogenesis in NOD mice.

AIMS/HYPOTHESIS: Gut microbiota (GM) and diet both appear to be important in the pathogenesis of type 1 diabetes. Fermentable fibres (FFs), of which there is an ample supply in natural, diabetes-promoting diets, are used by GM as a source of energy. Our aim was to determine whether FFs modify GM and diabetes incidence in the NOD mouse. METHODS: Female NOD mice were weaned to a semisynthetic diet and the effects of FF supplementation on diabetes incidence and insulitis were evaluated. Real-time quantitative PCR was employed to determine the effects imposed to gene transcripts in the colon and lymph nodes. Changes to GM were analysed by next-generation sequencing. RESULTS: NOD mice fed semisynthetic diets free from FFs were largely protected from diabetes while semisynthetic diets supplemented with the FFs pectin and xylan (PX) resulted in higher diabetes incidence. Semisynthetic diet free from FFs altered GM composition significantly; addition of PX changed the composition of the GM towards that found in natural-diet-fed mice and increased production of FF-derived short-chain fatty acid metabolites in the colon. The highly diabetogenic natural diet was associated with expression of proinflammatory and stress-related genes in the colon, while the semisynthetic diet free from FFs promoted Il4, Il22, Tgfß and Foxp3 transcripts in the colon and/or pancreatic lymph node. PX in the same diet counteracted these effects and promoted stress-related IL-18 activation in gut epithelial cells. 16S RNA sequencing revealed each diet to give rise to its particular GM composition, with different Firmicutes to Bacteroidetes ratios, and enrichment of mucin-degrading Ruminococcaceae following diabetes-protective FF-free diet. CONCLUSIONS/INTERPRETATION: FFs condition microbiota, affect colon homeostasis and are important components of natural, diabetes-promoting diets in NOD mice.

Cefotaxime-resistant Salmonella enterica in travelers returning from Thailand to Finland.

During 1993-2011, cefotaxime resistance among Salmonella enterica isolates from patients in Finland increased substantially. Most of these infections originated in Thailand; many were qnr positive and belonged to S. enterica serovar Typhimurium and S. enterica monophasic serovar 4,[5],12:i:-. Although cefotaxime-resistant salmonellae mainly originate in discrete geographic areas, they represent a global threat.