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Semaglutide, a glucagon-like peptide-1 receptor agonist, is an antidiabetic medication that has recently been approved for the treatment of obesity as well. Semaglutide is postulated to be a promising candidate for the treatment of non-alcoholic steatohepatitis (NASH). Here, Ldlr-/-.Leiden mice received a fast-food diet (FFD) for 25 weeks, followed by another 12 weeks on FFD with daily subcutaneous injections of semaglutide or vehicle (control). Plasma parameters were evaluated, livers and hearts were examined, and hepatic transcriptome analysis was performed. In the liver, semaglutide significantly reduced macrovesicular steatosis (−74%, p < 0.001) and inflammation (−73%, p < 0.001) and completely abolished microvesicular steatosis (−100%, p < 0.001). Histological and biochemical assessment of hepatic fibrosis showed no significant effects of semaglutide. However, digital pathology revealed significant improvements in the degree of collagen fiber reticulation (−12%, p < 0.001). Semaglutide did not affect atherosclerosis relative to controls. Additionally, we compared the transcriptome profile of FFD-fed Ldlr-/-.Leiden mice with a human gene set that differentiates human NASH patients with severe fibrosis from those with mild fibrosis. In FFD-fed Ldlr-/-.Leiden control mice, this gene set was upregulated as well, while semaglutide predominantly reversed this gene expression. Using a translational model with advanced NASH, we demonstrated that semaglutide is a promising candidate with particular potential for the treatment of hepatic steatosis and inflammation, while for the reversal of advanced fibrosis, combinations with other NASH agents may be necessary.

Oral health and dentistry are essential components of systems medicine, which has received lesser attention in comparison to other medical fields, such as cancer biology. In this context, oral polymorphonuclear neutrophils (oPMNs) play an important role in the maintenance of oral health. To the best of our knowledge, this is the first study to report original observations on the transcriptional responses of oPMNs during experimentally induced gingivitis, by temporarily refraining from regular oral care. Oral rinses were prospectively collected at four different time points for oPMNs isolation from healthy volunteers: day 1 (start of the experimental gingivitis challenge), day 9 (during challenge), day 14 (end of the challenge), and day 21 (postchallenge). Transcriptome of oPMNs was determined by RNA sequencing. Differentially expressed genes (DEGs) were selected at p < 0.01 level, and evaluated for pathway regulation using Ingenuity Pathway Analysis suite. We found four major clusters of DEGs, consisting of 256 initial response DEGs (day 9 only), 221 late response DEGs (day 14 only), 53 persistent responsive DEGs (consistent at day 9 and 14), and 524 DEGs showing responses only in the postchallenge phase (day 21 only). Pathway analysis of the initial and late response DEGs showed involvement in many immune regulatory pathways and PMN function, whereas DEGs at day 21 were associated with epithelial adherence signaling and other miscellaneous related signaling pathways. The results from this pilot study showed that oPMNs mediate oral inflammatory processes, suggesting their immunomodulatory role in oral equilibrium.

Patients with metabolic syndrome are often prescribed statins to prevent the development of cardiovascular disease. Conversely, data on their effects on non-alcoholic steatohepatitis (NASH) are lacking. We evaluated these effects by feeding APOE*3-Leiden mice a Western-type diet (WTD) with or without atorvastatin to induce NASH and hepatic fibrosis. Besides the well-known plasma cholesterol lowering (−30%) and anti-atherogenic effects (severe lesion size −48%), atorvastatin significantly reduced hepatic steatosis (−22%), the number of aggregated inflammatory cells in the liver (−80%) and hepatic fibrosis (−92%) compared to WTD-fed mice. Furthermore, atorvastatin-treated mice showed less immunohistochemically stained areas of inflammation markers. Atorvastatin prevented accumulation of free cholesterol in the form of cholesterol crystals (−78%). Cholesterol crystals are potent inducers of the NLRP3 inflammasome pathway and atorvastatin prevented its activation, which resulted in reduced expression of the pro-inflammatory cytokines interleukin (IL)-1β (−61%) and IL-18 (−26%). Transcriptome analysis confirmed strong reducing effects of atorvastatin on inflammatory mediators, including NLRP3, NFκB and TLR4. The present study demonstrates that atorvastatin reduces hepatic steatosis, inflammation and fibrosis and prevents cholesterol crystal formation, thereby precluding NLRP3 inflammasome activation. This may render atorvastatin treatment as an attractive approach to reduce NAFLD and prevent progression into NASH in dyslipidemic patients.

Metabolic disorders, such as obesity and type 2 diabetes have a large impact on global health, especially in industrialized countries. Tissue-specific chronic low-grade inflammation is a key contributor to complications in metabolic disorders. To support therapeutic approaches to these complications, it is crucial to gain a deeper understanding of the inflammatory dynamics and to monitor them on the individual level. To this end, blood-based biomarkers reflecting the tissue-specific inflammatory dynamics would be of great value. Here, we describe an in silico approach to select candidate biomarkers for tissue-specific inflammation by using a priori mechanistic knowledge from pathways and tissue-derived molecules. The workflow resulted in a list of candidate markers, in part consisting of literature confirmed biomarkers as well as a set of novel, more innovative biomarkers that reflect inflammation in the liver and adipose tissue. The first step of biomarker verification was on murine tissue gene-level by inducing hepatic inflammation and adipose tissue inflammation through a high-fat diet. Our data showed that in silico predicted hepatic markers had a strong correlation to hepatic inflammation in the absence of a relation to adipose tissue inflammation, while others had a strong correlation to adipose tissue inflammation in the absence of a relation to liver inflammation. Secondly, we evaluated the human translational value by performing a curation step in the literature using studies that describe the regulation of the markers in human, which identified 9 hepatic (such as Serum Amyloid A, Haptoglobin, and Interleukin 18 Binding Protein) and 2 adipose (Resistin and MMP-9) inflammatory biomarkers at the highest level of confirmation. Here, we identified and pre-clinically verified a set of in silico predicted biomarkers for liver and adipose tissue inflammation which can be of great value to study future development of therapeutic/lifestyle interventions to combat metabolic inflammatory complications.

The development of obesity is characterized by the metabolic overload of tissues and subsequent organ inflammation. The health effects of krill oil (KrO) on obesity-associated inflammation remain largely elusive, because long-term treatments with KrO have not been performed to date. Therefore, we examined the putative health effects of 28 weeks of 3% (w/w) KrO supplementation to an obesogenic diet (HFD) with fat derived mostly from lard. The HFD with KrO was compared to an HFD control group to evaluate the effects on fatty acid composition and associated inflammation in epididymal white adipose tissue (eWAT) and the liver during obesity development. KrO treatment increased the concentrations of EPA and DHA and associated oxylipins, including 18-HEPE, RvE2 and 14-HDHA in eWAT and the liver. Simultaneously, KrO decreased arachidonic acid concentrations and arachidonic-acid-derived oxylipins (e.g., HETEs, PGD2, PGE2, PGF2α, TXB2). In eWAT, KrO activated regulators of adipogenesis (e.g., PPARγ, CEBPα, KLF15, STAT5A), induced a shift towards smaller adipocytes and increased the total adipocyte numbers indicative for hyperplasia. KrO reduced crown-like structures in eWAT, and suppressed HFD-stimulated inflammatory pathways including TNFα and CCL2/MCP-1 signaling. The observed eWAT changes were accompanied by reduced plasma leptin and increased plasma adiponectin levels over time, and improved insulin resistance (HOMA-IR). In the liver, KrO suppressed inflammatory signaling pathways, including those controlled by IL-1β and M-CSF, without affecting liver histology. Furthermore, KrO deactivated hepatic REL-A/p65-NF-κB signaling, consistent with increased PPARα protein expression and a trend towards an increase in IkBα. In conclusion, long-term KrO treatment increased several anti-inflammatory PUFAs and oxylipins in WAT and the liver. These changes were accompanied by beneficial effects on general metabolism and inflammatory tone at the tissue level. The stimulation of adipogenesis by KrO allows for safe fat storage and may, together with more direct PPAR-mediated anti-inflammatory mechanisms, attenuate inflammation.

Graphical abstract, Highlights • Type 2 diabetes (T2DM) increases the risk of recurrence in myocardial infarction patients. • A gut microbiota profile is associated to the further T2DM development. • Microbiome data improved the prediction of T2DM development when added to clinical parameters. • A risk score including the most predictive genera was associated with the probability of T2DM. • A high risk score was associated with a higher hepatic insulin resistance and β-cell dysfunction., Introduction A distinctive gut microbiome have been linked to type 2 diabetes mellitus (T2DM). Objectives We aimed to evaluate whether gut microbiota composition, in addition to clinical biomarkers, could improve the prediction of new incident cases of diabetes in patients with coronary heart disease. Methods All the patients from the CORDIOPREV (Clinical Trials.gov.Identifier: NCT00924937) study without T2DM at baseline were included (n = 462). Overall, 107 patients developed it after a median of 60 months. The gut microbiota composition was determined by 16S rRNA gene sequencing and predictive models were created using hold-out method. Results A gut microbiota profile associated with T2DM development was determined through a microbiome-based predictive model. The addition of microbiome data to clinical parameters (variables included in FINDRISC risk score and the diabetes risk score of the American Diabetes Association, HDL, triglycerides and HbA1c) improved the prediction increasing the area under the curve from 0.632 to 0.946. Furthermore, a microbiome-based risk score including the ten most discriminant genera, was associated with the probability of develop T2DM. Conclusion These results suggest that a microbiota profile is associated to the T2DM development. An integrate predictive model of microbiome and clinical data that can improve the prediction of T2DM is also proposed, if is validated in independent populations to prevent this disease.

BackgroundNon-alcoholic fatty liver disease (NAFLD) is a complex multifactorial disorder that is characterised by dysfunctional lipid metabolism and cholesterol homeostasis, and a related chronic inflammatory response. NAFLD has become the most common cause of chronic liver disease in many countries, and its prevalence continues to rise in parallel with increasing rates of obesity. Here, we evaluated the putative NAFLD-attenuating effects of a multicomponent medicine consisting of 24 natural ingredients: Hepar compositum (HC-24).MethodsLdlr-/-.Leiden mice were fed a high-fat diet (HFD) with a macronutrient composition and cholesterol content comparable to human diets for 24 weeks to induce obesity-associated metabolic dysfunction, including hepatic steatosis and inflammation. HC-24 or vehicle control was administered intraperitoneally 3 times/week (1.5 ml/kg) for the last 18 weeks of the study. Histological analyses of liver and adipose tissue were combined with extensive hepatic transcriptomics analysis. Transcriptomics results were further substantiated with ELISA, immunohistochemical and liver lipid analyses.ResultsHFD feeding induced obesity and metabolic dysfunction including adipose tissue inflammation and increased gut permeability. In the liver, HFD-feeding resulted in a disturbance of cholesterol homeostasis and an associated inflammatory response. HC-24 did not affect body weight, metabolic risk factors, adipose tissue inflammation or gut permeability. While HC-24 did not alter total liver steatosis, there was a pronounced reduction in lobular inflammation in HC-24-treated animals, which was associated with modulation of genes and proteins involved in inflammation (e.g., neutrophil chemokine Cxcl1) and cholesterol homeostasis (i.e., predicted effect on ‘cholesterol’ as an upstream regulator, based on gene expression changes associated with cholesterol handling). These effects were confirmed by CXCL1 ELISA, immunohistochemical staining of neutrophils and biochemical analysis of hepatic free cholesterol content. Intrahepatic free cholesterol levels were found to correlate significantly with the number of inflammatory aggregates in the liver, thereby providing a potential rationale for the observed anti-inflammatory effects of HC-24.ConclusionsFree cholesterol accumulates in the liver of Ldlr-/-.Leiden mice under physiologically translational dietary conditions, and this is associated with the development of hepatic inflammation. The multicomponent medicine HC-24 reduces accumulation of free cholesterol and has molecular and cellular anti-inflammatory effects in the liver.

The prevalence of non-alcoholic steatohepatitis (NASH) is rapidly increasing and associated with cardiovascular disease (CVD), the major cause of mortality in NASH patients. Although sharing common risk factors, the mechanisms by which NASH may directly contribute to the development to CVD remain poorly understood. The aim of this study is to gain insight into key molecular processes of NASH that drive atherosclerosis development. Thereto, a time-course study was performed in Ldlr−/−.Leiden mice fed a high-fat diet to induce NASH and atherosclerosis. The effects on NASH and atherosclerosis were assessed and transcriptome analysis was performed. Ldlr−/−.Leiden mice developed obesity, hyperlipidemia and insulin resistance, with steatosis and hepatic inflammation preceding atherosclerosis development. Transcriptome analysis revealed a time-dependent increase in pathways related to NASH and fibrosis followed by an increase in pro-atherogenic processes in the aorta. Gene regulatory network analysis identified specific liver regulators related to lipid metabolism (SC5D, LCAT and HMGCR), inflammation (IL1A) and fibrosis (PDGF, COL3A1), linked to a set of aorta target genes related to vascular inflammation (TNFA) and atherosclerosis signaling (CCL2 and FDFT1). The present study reveals pathogenic liver processes that precede atherosclerosis development and identifies hepatic key regulators driving the atherogenic pathways and regulators in the aorta.

Non-alcoholic steatohepatitis (NASH) is the most rapidly growing liver disease that is nevertheless without approved pharmacological treatment. Despite great effort in developing novel NASH therapeutics, many have failed in clinical trials. This has raised questions on the adequacy of preclinical models. Elafibranor is one of the drugs currently in late stage development which had mixed results for phase 2/interim phase 3 trials. In the current study we investigated the response of elafibranor in APOE*3Leiden.CETP mice, a translational animal model that displays histopathological characteristics of NASH in the context of obesity, insulin resistance and hyperlipidemia. To induce NASH, mice were fed a high fat and cholesterol (HFC) diet for 15 weeks (HFC reference group) or 25 weeks (HFC control group) or the HFC diet supplemented with elafibranor (15 mg/kg/d) from week 15–25 (elafibranor group). The effects on plasma parameters and NASH histopathology were assessed and hepatic transcriptome analysis was used to investigate the underlying pathways affected by elafibranor. Elafibranor treatment significantly reduced steatosis and hepatic inflammation and precluded the progression of fibrosis. The underlying disease pathways of the model were compared with those of NASH patients and illustrated substantial similarity with molecular pathways involved, with 87% recapitulation of human pathways in mice. We compared the response of elafibranor in the mice to the response in human patients and discuss potential pitfalls when translating preclinical results of novel NASH therapeutics to human patients. When taking into account that due to species differences the response to some targets, like PPAR-α, may be overrepresented in animal models, we conclude that elafibranor may be particularly useful to reduce hepatic inflammation and could be a pharmacologically useful agent for human NASH, but probably in combination with other agents.

Epidemiological studies have reported positive associations between serum perfluorooctanoic acid (PFOA) and total and non-high-density lipoprotein cholesterol (non-HDL-C) although the magnitude of effect of PFOA on cholesterol lacks consistency. The objectives of this study were to evaluate the effect of PFOA on plasma cholesterol and triglyceride metabolism at various plasma PFOA concentrations relevant to humans, and to elucidate the mechanisms using APOE*3-Leiden.CETP mice, a model with a human-like lipoprotein metabolism. APOE*3-Leiden.CETP mice were fed a Western-type diet with PFOA (10, 300, 30 000 ng/g/d) for 4–6 weeks. PFOA exposure did not alter plasma lipids in the 10 and 300 ng/g/d dietary PFOA dose groups. At 30 000 ng/g/d, PFOA decreased plasma triglycerides (TG), total cholesterol (TC), and non-HDL-C, whereas HDL-C was increased. The plasma lipid alterations could be explained by decreased very low-density lipoprotein (VLDL) production and increased VLDL clearance by the liver through increased lipoprotein lipase activity. The concomitant increase in HDL-C was mediated by decreased cholesteryl ester transfer activity and changes in gene expression of proteins involved in HDL metabolism. Hepatic gene expression and pathway analysis confirmed the changes in lipoprotein metabolism that were mediated for a major part through activation of the peroxisome proliferator-activated receptor (PPAR)α. Our data confirmed the findings from a phase 1 clinical trial in humans that demonstrated high serum or plasma PFOA levels resulted in lower cholesterol levels. The study findings do not show an increase in cholesterol at environmental or occupational levels of PFOA exposure, thereby indicating these findings are associative rather than causal.

Thermophilic bacilli such as Anoxybacillus and Geobacillus are important contaminants in dairy powder products. Remarkably, one of the common contaminants, Geobacillus thermoglucosidans, showed poor growth in skim milk, whereas significant growth of G. thermoglucosidans was observed in the presence of an Anoxybacillus flavithermus dairy isolate. In the present study, we investigated the underlying reason for this growth dependence of G. thermoglucosidans. Whole-genome sequences of 4 A. flavithermus strains and 4 G. thermoglucosidans strains were acquired, with special attention given to carbohydrate utilization clusters and proteolytic enzymes. Focusing on traits relevant for dairy environments, comparative genomic analysis revealed that all G. thermoglucosidans strains lacked the genes necessary for lactose transport and metabolism, showed poor growth in skim milk, and produced white colonies on X-gal plates, indicating the lack of β-galactosidase activity. The A. flavithermus isolates scored positive in these tests, consistent with the presence of a putative lactose utilization gene cluster. All tested isolates from both species showed proteolytic activity on milk plate count agar plates. Adding glucose or galactose to liquid skim milk supported growth of G. thermoglucosidans isolates, in line with the presence of the respective monosaccharide utilization gene clusters in the genomes. Analysis by HPLC of A. flavithermus TNO-09.006 culture filtrate indicated that the previously described growth dependence of G. thermoglucosidans in skim milk was based on the supply of glucose and galactose by A. flavithermus TNO-09.006.

Background & Aims The incidence of nonalcoholic steatohepatitis (NASH) is increasing. The pathophysiological mechanisms of NASH and the sequence of events leading to hepatic fibrosis are incompletely understood. The aim of this study was to gain insight into the dynamics of key molecular processes involved in NASH and to rank early markers for hepatic fibrosis. Methods A time-course study in low-density lipoprotein–receptor knockout. Leiden mice on a high-fat diet was performed to identify the temporal dynamics of key processes contributing to NASH and fibrosis. An integrative systems biology approach was used to elucidate candidate markers linked to the active fibrosis process by combining transcriptomics, dynamic proteomics, and histopathology. The translational value of these findings were confirmed using human NASH data sets. Results High-fat-diet feeding resulted in obesity, hyperlipidemia, insulin resistance, and NASH with fibrosis in a time-dependent manner. Temporal dynamics of key molecular processes involved in the development of NASH were identified, including lipid metabolism, inflammation, oxidative stress, and fibrosis. A data-integrative approach enabled identification of the active fibrotic process preceding histopathologic detection using a novel molecular fibrosis signature. Human studies were used to identify overlap of genes and processes and to perform a network biology-based prioritization to rank top candidate markers representing the early manifestation of fibrosis. Conclusions An early predictive molecular signature was identified that marked the active profibrotic process before histopathologic fibrosis becomes manifest. Early detection of the onset of NASH and fibrosis enables identification of novel blood-based biomarkers to stratify patients at risk, development of new therapeutics, and help shorten (pre)clinical experimental time frames., Graphical abstract

Non-alcoholic steatohepatitis (NASH) is a fast-growing liver disorder that is associated with an increased incidence of cardiovascular disease and type 2 diabetes. Animal models adequately mimicking this condition are scarce. We herein investigate whether Ldlr&minus, /&minus, Leiden mice on different high-fat diets represent a suitable NASH model. Ldlr&minus, Leiden mice were fed a healthy chow diet or fed a high-fat diet (HFD) containing lard or a fast food diet (FFD) containing milk fat. Additionally, the response to treatment with obeticholic acid (OCA) was evaluated. Both high-fat diets induced obesity, hyperlipidemia, hyperinsulinemia, and increased alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels. Mice on both diets developed progressive macro- and microvesicular steatosis, hepatic inflammation, and fibrosis, along with atherosclerosis. HFD induced more severe hyperinsulinemia, while FFD induced more severe hepatic inflammation with advanced (F3) bridging fibrosis, as well as more severe atherosclerosis. OCA treatment significantly reduced hepatic inflammation and fibrosis, and it did not affect atherosclerosis. Hepatic transcriptome analysis was compared with human NASH and illustrated similarity. The present study defines a translational model of NASH with progressive liver fibrosis and simultaneous atherosclerosis development. By adaptation of the fat content of the diet, either insulin resistance (HFD) or hepatic inflammation and fibrosis (FFD) can be aggravated.

Obesity characterized by adiposity and ectopic fat accumulation is associated with the development of non-alcoholic fatty liver disease (NAFLD). Treatments that stimulate lipid utilization may prevent the development of obesity and comorbidities. This study evaluated the potential anti-obesogenic hepatoprotective effects of combined treatment with L-carnitine and nicotinamide riboside, i.e., components that can enhance fatty acid transfer across the inner mitochondrial membrane and increase nicotinamide adenine nucleotide (NAD+) levels, which are necessary for &beta, oxidation and the TCA cycle, respectively. Ldlr &minus, /&minus, Leiden mice were treated with high-fat diet (HFD) supplemented with L-carnitine (LC, 0.4% w/w), nicotinamide riboside (NR, 0.3% w/w) or both (COMBI) for 21 weeks. L-carnitine plasma levels were reduced by HFD and normalized by LC. NR supplementation raised its plasma metabolite levels demonstrating effective delivery. Although food intake and ambulatory activity were comparable in all groups, COMBI treatment significantly attenuated HFD-induced body weight gain, fat mass gain (&minus, 17%) and hepatic steatosis (&minus, 22%). Also, NR and COMBI reduced hepatic 4-hydroxynonenal adducts. Upstream-regulator gene analysis demonstrated that COMBI reversed detrimental effects of HFD on liver metabolism pathways and associated regulators, e.g., ACOX, SCAP, SREBF, PPARGC1B, and INSR. Combination treatment with LC and NR exerts protective effects on metabolic pathways and constitutes a new approach to attenuate HFD-induced obesity and NAFLD.

OBJECTIVES: A key to a successful personalized nutrition (PN) strategy is tailoring the behavior intervention to achieve sustained behavior change consistent with desired health outcomes. The objective of this study was to evaluate the impact of a digitally-delivered, systems biology-based PN program (Habit, LLC, Oakland, CA), that included nutrition and behavior advice, and meals. METHODS: In this single-arm, multi-phase, open-label study (clinicaltrials.gov: NCT03424395), participants (n = 107; 67% female), 30–59 years of age, were provided a Fitbit® Activity Tracker and instructions to complete an at-home kit that included collection of DNA and capillary blood before and after (30 and 120 min) consuming a mixed macronutrient challenge beverage. Lifestyle behavior data and activity levels were collected at baseline. Participants completed a 10-week control phase and a 10-week PN program intervention phase that included tailored nutrition and behavior advice, and meals (2 meals/day, 5 days/week). Validated quality of life, perceived stress, diet self-efficacy, and eating behavior questionnaires were administered to assess behavior before and after the intervention. Data from 73 compliant participants (66% female) were included in the analysis. Anthropometric and biological results were used to categorize participants into subgroups including Protein Seekers (PS) and Range Seekers (RS). RESULTS: Participants reported feeling fuller faster after the intervention, relative to before (P = 0.002), which could be linked to the dietary recommendations and behavior advice. Analysis by gender showed men reported more control over their eating patterns during negative events at baseline, compared to women (P = 0.005). This difference was not evident at the end of the intervention with women reporting slightly more, and men slightly less control. PS entered the study with a higher weight and BMI (P

Personalized nutrition may be more effective in changing lifestyle behaviors compared to population-based guidelines. This single-arm exploratory study evaluated the impact of a 10-week personalized systems nutrition (PSN) program on lifestyle behavior and health outcomes. Healthy men and women (n = 82) completed the trial. Individuals were grouped into seven diet types, for which phenotypic, genotypic and behavioral data were used to generate personalized recommendations. Behavior change guidance was also provided. The intervention reduced the intake of calories (−256.2 kcal, p &lt, 0.0001), carbohydrates (−22.1 g, 0.0039), sugar (−13.0 g, 0.0001), total fat (−17.3 g, 0.0001), saturated fat (−5.9 g, p = 0.0003) and PUFA (−2.5 g, p = 0.0065). Additionally, BMI (−0.6 kg/m2, 0.0001), body fat (−1.2%, p = 0.0192) and hip circumference (−5.8 cm, 0.0001) were decreased after the intervention. In the subgroup with the lowest phenotypic flexibility, a measure of the body’s ability to adapt to environmental stressors, LDL (−0.44 mmol/L, p = 0.002) and total cholesterol (−0.49 mmol/L, 0.0001) were reduced after the intervention. This study shows that a PSN program in a workforce improves lifestyle habits and reduces body weight, BMI and other health-related outcomes. Health improvement was most pronounced in the compromised phenotypic flexibility subgroup, which indicates that a PSN program may be effective in targeting behavior change in health-compromised target groups.

Background Antibiotic therapy is commonly used in animal agriculture. Antibiotics excreted by the animals can contaminate farming environments, resulting in long term exposure of animals to sub-inhibitory levels of antibiotics. Little is known on the effect of this exposure on antibiotic resistance. In this study, we aimed to investigate the long term effects of sub-inhibitory levels of antibiotics on the gut microbiota composition and resistome of veal calves in vivo. Forty-two veal calves were randomly assigned to three groups. The first group (OTC-high) received therapeutic oral dosages of 1 g oxytetracycline (OTC), twice per day, during 5 days. The second group (OTC-low) received an oral dose of OTC of 100–200 μg per day during 7 weeks, mimicking animal exposure to environmental contamination. The third group (CTR) did not receive OTC, serving as unexposed control. Antibiotic residue levels were determined over time. The temporal effects on the gut microbiota and antibiotic resistance gene abundance was analysed by metagenomic sequencing. Results In the therapeutic group, OTC levels exceeded MIC values. The low group remained at sub-inhibitory levels. The control group did not reach any significant OTC levels. 16S rRNA gene-based analysis revealed significant changes in the calf gut microbiota. Time-related changes accounted for most of the variation in the sequence data. Therapeutic application of OTC had transient effect, significantly impacting gut microbiota composition between day 0 and day 2. By metagenomic sequence analysis we identified six antibiotic resistance genes representing three gene classes (tetM, floR and mel) that differed in relative abundance between any of the intervention groups and the control. qPCR was used to validate observations made by metagenomic sequencing, revealing a peak of tetM abundance at day 28–35 in the OTC-high group. No increase in resistance genes abundance was seen in the OTC-low group. Conclusions Under the conditions tested, sub-therapeutic administration of OTC did not result in increased tetM resistance levels as observed in the therapeutic group. Electronic supplementary material The online version of this article (10.1186/s12864-018-5419-x) contains supplementary material, which is available to authorized users.

Treatment with the second and third generation BCR-ABL1 tyrosine kinase inhibitors (TKIs) increases cardiovascular risk in chronic myeloid leukemia (CML) patients. We investigated the vascular adverse effects of three generations of TKIs in a translational model for atherosclerosis, the APOE*3Leiden.CETP mouse. Mice were treated for sixteen weeks with imatinib (150 mg/kg BID), nilotinib (10 and 30 mg/kg QD) or ponatinib (3 and 10 mg/kg QD), giving similar drug exposures as in CML-patients. Cardiovascular risk factors were analyzed longitudinally, and histopathological analysis of atherosclerosis and transcriptome analysis of the liver was performed. Imatinib and ponatinib decreased plasma cholesterol (imatinib, -69%, p < 0.001; ponatinib 3 mg/kg, -37%, p < 0.001; ponatinib 10 mg/kg-44%, p < 0.001) and atherosclerotic lesion area (imatinib, -78%, p < 0.001; ponatinib 3 mg/kg, -52%, p = 0.002; ponatinib 10 mg/kg, -48%, p = 0.006), which were not affected by nilotinib. In addition, imatinib increased plaque stability. Gene expression and pathway analysis demonstrated that ponatinib enhanced the mRNA expression of coagulation factors of both the contact activation (intrinsic) and tissue factor (extrinsic) pathways. In line with this, ponatinib enhanced plasma levels of FVII, whereas nilotinib increased plasma FVIIa activity. While imatinib showed a beneficial cardiovascular risk profile, nilotinib and ponatinib increased the cardiovascular risk through induction of a pro-thrombotic state.KEYWORDS: animal model cardiovascular disease; atherosclerosis; cardiovascular side effects; cholesterol; chronic myeloid leukemia; coagulation/thrombosis; lipids and lipoprotein metabolism

Concerns have been raised about whether preclinical models sufficiently mimic molecular disease processes observed in nonalcoholic steatohepatitis (NASH) patients, bringing into question their translational value in studies of therapeutic interventions in the process of NASH/fibrosis. We investigated the representation of molecular disease patterns characteristic for human NASH in high‐fat diet (HFD)‐fed Ldlr‐/‐.Leiden mice and studied the effects of obeticholic acid (OCA) on these disease profiles. Multiplatform serum metabolomic profiles and genome‐wide liver transcriptome from HFD‐fed Ldlr‐/‐.Leiden mice were compared with those of NASH patients. Mice were profiled at the stage of mild (24 weeks HFD) and severe (34 weeks HFD) fibrosis, and after OCA intervention (24‐34 weeks; 10 mg/kg/day). Effects of OCA were analyzed histologically, biochemically, by immunohistochemistry, using deuterated water technology (de novo collagen formation), and by its effect on the human‐based transcriptomics and metabolomics signatures. The transcriptomics and metabolomics profile of Ldlr‐/‐.Leiden mice largely reflected the molecular signature of NASH patients. OCA modulated the expression of these molecular profiles and quenched specific proinflammatory‐profibrotic pathways. OCA attenuated specific facets of cellular inflammation in liver (F4/80‐positive cells) and reduced crown‐like structures in adipose tissue. OCA reduced de novo collagen formation and attenuated further progression of liver fibrosis, but did not reduce fibrosis below the level before intervention. Conclusion: HFD‐fed Ldlr‐/‐.Leiden mice recapitulate molecular transcriptomic and metabolomic profiles of NASH patients, and these signatures are modulated by OCA. Intervention with OCA in developing fibrosis reduces collagen deposition and de novo synthesis but does not resolve already manifest fibrosis in the period studied. These data show that human molecular signatures can be used to evaluate the translational character of preclinical models for NASH.

OBJECTIVES: This study evaluates the impact of a digitally-delivered systems biology-based personalized nutrition (PN) program (Habit LLC, Oakland, CA), that included nutrition and behavior advice, and meals. METHODS: In this single-arm, multi-phase, open-label study (clinicaltrials.gov: NCT03424395), participants (n = 107; 67% female) were provided a Fitbit® Activity Tracker and completed an at-home kit that included collection of DNA and capillary blood before and after (30 and 120 min) consuming a mixed macronutrient challenge beverage. Participants completed a 10-week run-in and 10-week PN program intervention phase. The intervention included personalized nutrition and behavior advice and meals (2 meals/day; 5 days/week) tailored to dietary subgroups. Here we report on changes in diet, anthropometrics, and biomarkers of two subgroups (Range Seekers [RS] and Protein Seekers [PS]) that differed in anthropometrics and biological measures at baseline and were provided different dietary recommendations. RESULTS: At baseline, there were no differences between groups in nutrients assessed, however, compared to RS (n = 48), PS (n = 22) had higher BMI, weight, body fat %, waist and hip circumference (all P

A novel generic approach for stress profiling was applied to Listeria monocytogenes strain F2365. This food-borne pathogen was exposed to gradients of five different stresses of increasing intensity, typically ranging from moderate to lethal conditions. The stress factors included heat, acidic pH, a detergent disinfectant, an oxidant, and hyperosmotic conditions. In addition to CFU counts and lag time, five different molecular viability parameters were measured by fluorescence-based assays, including membrane integrity, membrane potential, esterase activity, redox activity, and intracellular pH stability. The last was measured by our recently invented real-time viability assay. Exposure to all stresses resulted in clear dose-response relationships for all viability parameters with the exception of hyperosmotic conditions. A statistical analysis showed strong correlations for (i) the growth parameters plate counts and lag times, (ii) the enzyme-associated functions redox and esterase activity, and (iii) the membrane-associated pH stability and membrane integrity. Results indicated a pronounced difference in the susceptibilities of the measured parameters depending on the stress factor applied. However, at relatively high stress intensities, all of the viability parameters became affected independent of the stress factor. Applications of the approach presented here include studies on the mechanism of action of unknown compounds with biocidal activity and a comparative analysis of the severities of the impact of stress conditions of interest. It appears that a meaningful evaluation of the impact of mild stress conditions can be obtained only through measurement of multiple viability parameters.

Failure of food preservation is frequently caused by thermostable spores of members of the Bacillaceae family, which show a wide spectrum of resistance to cleaning and preservation treatments. We constructed and validated a mixed-species genotyping array for 6 Bacillus species, including Bacillus subtilis, Bacillus licheniformis, Bacillus pumilus, Bacillus sporothermodurans, Bacillus cereus and Bacillus coagulans, and 4 Geobacillus species, including Geobacillus stearothermophilus, Geobacillus thermocatenulatus, Geobacillus toebii and Geobacillus sp., in order to track food spoilage isolates from ingredient to product. The discriminating power of the array was evaluated with sets of 42 reference and 20 test strains. Bacterial isolates contain a within-species-conserved core genome comprising 68–88% of the entire genome and a non-conserved accessory genome comprising 7–22%. The majority of the core genome markers do not hybridise between species, thus they allow for efficient discrimination at the species level. The accessory genome array markers provide high-resolution discrimination at the level of individual isolates from a single species. In conclusion, the reported mixed-species microarray contains discriminating markers that allow rapid and cost-effective typing of Bacillus food spoilage bacteria in a wide variety of food products. Keywords: Bacillus; Geobacillus; Genotyping; Comparative genome hybridisation; Microarray; Tracking and tracing; Food spoilage; Bacterial spores

Spores of thermophilic spore-forming bacteria are a common cause of contamination in dairy products. Here, we report draft genome sequences of four thermophilic strains from a milk-processing plant or standard milk, namely, a Geobacillus thermoglucosidans isolate (TNO-09.023), Geobacillus stearothermophilus TNO-09.027, and two Anoxybacillus flavithermus isolates (TNO-09.014 and TNO-09.016).

Background To date, women are most often diagnosed with bacterial vaginosis (BV) using microscopy based Nugent scoring or Amsel criteria. However, the accuracy is less than optimal. The aim of the present study was to confirm the identity of known BV-associated composition profiles and evaluate indicators for BV using three molecular methods. Methods Evaluation of indicators for BV was carried out by 16S rRNA amplicon sequencing of the V5-V7 region, a tailor-made 16S rRNA oligonucleotide-based microarray, and a PCR-based profiling technique termed IS-profiling, which is based on fragment variability of the 16S-23S rRNA intergenic spacer region. An inventory of vaginal bacterial species was obtained from 40 females attending a Dutch sexually transmitted infection outpatient clinic, of which 20 diagnosed with BV (Nugent score 7–10), and 20 BV negative (Nugent score 0–3). Results Analysis of the bacterial communities by 16S rRNA amplicon sequencing revealed two clusters in the BV negative women, dominated by either Lactobacillus iners or Lactobacillus crispatus and three distinct clusters in the BV positive women. In the former, there was a virtually complete, negative correlation between L. crispatus and L. iners. BV positive subjects showed cluster profiles that were relatively high in bacterial species diversity and dominated by anaerobic species, including Gardnerella vaginalis, and those belonging to the Families of Lachnospiraceae and Leptotrichiaceae. Accordingly, the Gini-Simpson index of species diversity, and the relative abundance Lactobacillus species appeared consistent indicators for BV. Under the conditions used, only the 16S rRNA amplicon sequencing method was suitable to assess species diversity, while all three molecular composition profiling methods were able to indicate Lactobacillus abundance in the vaginal microbiota. Conclusion An affordable and simple molecular test showing a depletion of the genus Lactobacillus in combination with an increased species diversity of vaginal microbiota could serve as an alternative and practical diagnostic method for the assessment of BV. Electronic supplementary material The online version of this article (doi:10.1186/s12879-016-1513-3) contains supplementary material, which is available to authorized users.

Invasive group A streptococcal (GAS) disease reemerged in The Netherlands in the late 1980s. To seek an explanation for this resurgence, the genetic compositions of 22 M1 and 19 M28 GAS strains isolated in The Netherlands between 1960s and the mid-1990s were analyzed by using a mixed-genome DNA microarray. During this four-decade period, M1 and especially M28 strains acquired prophages on at least eight occasions. All prophages carried a superantigen (s peA2 , spe C, spe K) or a streptodornase ( sdaD2 , sdn ), both associated with invasive GAS disease. Invasive and noninvasive GAS strains did not differ in prophage acquisition, suggesting that there was an overall increase in the pathogenicity of M1 and M28 strains over the last four decades rather than emergence of hypervirulent subclones. The increased overall pathogenic potential may have contributed to the reemergence of invasive GAS disease in The Netherlands.

Group A streptococci (GAS), or Streptococcus pyogenes , are associated with a remarkable variety of diseases, ranging from superficial infections to life-threatening diseases such as toxic-shock-like syndrome (TSS). GAS strains belonging to M types M1 and M3 are associated with TSS. This study aims to obtain insight into the gene profiles underlying different M types and disease manifestations. Genomic differences between 76 clinically well characterized GAS strains collected in The Netherlands were examined using a mixed-genome microarray. Inter-M-type genomic differences clearly outweighed intra-M-type genome variation. Phages were major contributors to observed genome diversification. We identified four novel genes, including two genes encoding fibronectin-binding-like proteins, which are highly specific to a subset of M types and thus may contribute to M-type-associated disease manifestations. All M12 strains were characterized by the unique absence of the citrate lyase complex and reduced growth under hypoxic, nutrient-deprived conditions. Furthermore, six virulence factors, including genes encoding a complement-inhibiting protein ( sic ), an exotoxin ( speA ), iron(III) binding factor, collagen binding factor ( cpa ), and fibrinogen binding factor ( prt2-like ), were unique to M1 and/or M3 strains. These virulence factors may contribute to the potential of these strains to cause TSS. Finally, in contrast to M-type-specific virulence profiles, we did not identify a common virulence profile among strains associated with TSS irrespective of their M type.

Due to the spread of resistance, antibiotic exposure receives increasing attention. Ecological consequences for the different niches of individual microbiomes are, however, largely ignored. Here, we report the effects of widely used antibiotics (clindamycin, ciprofloxacin, amoxicillin, and minocycline) with different modes of action on the ecology of both the gut and the oral microbiomes in 66 healthy adults from the United Kingdom and Sweden in a two-center randomized placebo-controlled clinical trial. Feces and saliva were collected at baseline, immediately after exposure, and 1, 2, 4, and 12 months after administration of antibiotics or placebo. Sequences of 16S rRNA gene amplicons from all samples and metagenomic shotgun sequences from selected baseline and post-antibiotic-treatment sample pairs were analyzed. Additionally, metagenomic predictions based on 16S rRNA gene amplicon data were performed using PICRUSt. The salivary microbiome was found to be significantly more robust, whereas the antibiotics negatively affected the fecal microbiome: in particular, health-associated butyrate-producing species became strongly underrepresented. Additionally, exposure to different antibiotics enriched genes associated with antibiotic resistance. In conclusion, healthy individuals, exposed to a single antibiotic treatment, undergo considerable microbial shifts and enrichment in antibiotic resistance in their feces, while their salivary microbiome composition remains unexpectedly stable. The health-related consequences for the gut microbiome should increase the awareness of the individual risks involved with antibiotic use, especially in a (diseased) population with an already dysregulated microbiome. On the other hand, understanding the mechanisms behind the resilience of the oral microbiome toward ecological collapse might prove useful in combating microbial dysbiosis elsewhere in the body., IMPORTANCE Many health care professionals use antibiotic prophylaxis strategies to prevent infection after surgery. This practice is under debate since it enhances the spread of antibiotic resistance. Another important reason to avoid nonessential use of antibiotics, the impact on our microbiome, has hardly received attention. In this study, we assessed the impact of antibiotics on the human microbial ecology at two niches. We followed the oral and gut microbiomes in 66 individuals from before, immediately after, and up to 12 months after exposure to different antibiotic classes. The salivary microbiome recovered quickly and was surprisingly robust toward antibiotic-induced disturbance. The fecal microbiome was severely affected by most antibiotics: for months, health-associated butyrate-producing species became strongly underrepresented. Additionally, there was an enrichment of genes associated with antibiotic resistance. Clearly, even a single antibiotic treatment in healthy individuals contributes to the risk of resistance development and leads to long-lasting detrimental shifts in the gut microbiome.

Background Spoilage of food products is frequently caused by bacterial spores and lactic acid bacteria. Identification of these organisms by classic cultivation methods is limited by their ability to form colonies on nutrient agar plates. In this study, we adapted and optimized 16S rRNA amplicon sequencing for quantification of bacterial spores in a canned food matrix and for monitoring the outgrowth of spoilage microbiota in a ready-to-eat food matrix. Results The detection limit of bar-coded 16S rRNA amplicon sequencing was determined for the number of bacterial spores in a canned food matrix. Analysis of samples from a canned food matrix spiked with a mixture of equinumerous spores from the thermophiles, Geobacillus stearothermophilus and Geobacillus thermoglucosidans, and the mesophiles, Bacillus sporothermodurans, Bacillus cereus, and Bacillus subtilis, led to the detection of these spores with an average limit of 2 × 102 spores ml−1. The data were normalized by setting the number of sequences resulting from DNA of an inactivated bacterial species, present in the matrix at the same concentration in all samples, to a fixed value for quantitative sample-to-sample comparisons. The 16S rRNA amplicon sequencing method was also employed to monitor population dynamics in a ready-to-eat rice meal, incubated over a period of 12 days at 7 °C. The most predominant outgrowth was observed by the genera Leuconostoc, Bacillus, and Paenibacillus. Analysis of meals pre-treated with weak acids showed inhibition of outgrowth of these three genera. The specificity of the amplicon synthesis was improved by the design of oligonucleotides that minimize the amplification of 16S rRNA genes from chloroplasts originating from plant-based material present in the food. Conclusion This study shows that the composition of complex spoilage populations, including bacterial spores, can be monitored in complex food matrices by bar-coded amplicon sequencing in a quantitative manner. In order to allow sample-to-sample comparisons, normalizations based on background DNA are described. This method offers a solution for the identification and quantification of spoilage microbiota, which cannot be cultivated under standard laboratory conditions. The study indicates variable detection limits among species of bacterial spores resulting from differences in DNA extraction efficiencies. Electronic supplementary material The online version of this article (doi:10.1186/s40168-015-0096-3) contains supplementary material, which is available to authorized users.

One of the major concerns in the production of dairy concentrates is the risk of contamination by heat-resistant spores from thermophilic bacteria. In order to acquire more insight in the composition of microbial communities occurring in the dairy concentrate industry, a bar-coded 16S amplicon sequencing analysis was carried out on milk, final products, and fouling samples taken from dairy concentrate production lines. The analysis of these samples revealed the presence of DNA from a broad range of bacterial taxa, including a majority of mesophiles and a minority of (thermophilic) spore-forming bacteria. Enrichments of fouling samples at 55°C showed the accumulation of predominantly Brevibacillus and Bacillus , whereas enrichments at 65°C led to the accumulation of Anoxybacillus and Geobacillus species. Bacterial population analysis of biofilms grown using fouling samples as an inoculum indicated that both Anoxybacillus and Geobacillus preferentially form biofilms on surfaces at air-liquid interfaces rather than on submerged surfaces. Three of the most potent biofilm-forming strains isolated from the dairy factory industrial samples, including Geobacillus thermoglucosidans , Geobacillus stearothermophilus , and Anoxybacillus flavithermus , have been characterized in detail with respect to their growth conditions and spore resistance. Strikingly, Geobacillus thermoglucosidans , which forms the most thermostable spores of these three species, is not able to grow in dairy intermediates as a pure culture but appears to be dependent for growth on other spoilage organisms present, probably as a result of their proteolytic activity. These results underscore the importance of abiotic and microbiotic factors in niche colonization in dairy factories, where the presence of thermophilic sporeformers can affect the quality of end products.

Spores of thermophilic spore-forming bacteria are a common cause of contamination in dairy products. We isolated the thermophilic strain Anoxybacillus flavithermus TNO-09.006 from a milk-processing plant, and we report the complete genome of this isolate consisting of a single chromosome of 2.65 Mb.

Bacterial spore formers are prime organisms of concern in the food industry. Spores from the genus Bacillus are extremely stress resistant, most notably exemplified by high thermotolerance. This sometimes allows surviving spores to germinate and grow out to vegetative cells causing food spoilage and possible intoxication. Similar issues though more pending toward spore toxigenicity are observed for the anaerobic Clostridia. The paper indicates the nature of stress resistance and highlights contemporary molecular approaches to analyze the mechanistic basis of it in Bacilli. A molecular comparison between a laboratory strain and a food borne isolate, very similar at the genomic level to the laboratory strain but generating extremely heat resistant spores, is discussed. The approaches cover genome-wide genotyping, proteomics and genome-wide expression analyses studies. The analyses aim at gathering sufficient molecular information to be able to put together an initial framework for dynamic modelling of spore germination and outgrowth behaviour. Such emerging models should be developed both at the population and at the single spore level. Tools and challenges in achieving the latter are succinctly discussed. © 2010 Elsevier Ltd.

Bacterial strains differ in their ability to cause hospital outbreaks. Using comparative genomic hybridization, Enterobacter cloacae complex isolates were studied to identify genetic markers specific for Enterobacter cloacae complex outbreak strains. No outbreak-specific genes were found that were common in all investigated outbreak strains. Therefore, the aim of our study was to identify specific genetic markers for an Enterobacter hormaechei outbreak strain (EHOS) that caused a nationwide outbreak in The Netherlands. Most EHOS isolates carried a large conjugative plasmid (pQC) containing genes encoding heavy-metal resistance, mobile elements, pili-associated proteins and exported proteins as well as multiple-resistance genes. Furthermore, the chromosomally encoded high-pathogenicity island (HPI) was highly associated with the EHOS strain. In addition, other DNA fragments were identified that were associated with virulence: three DNA fragments known to be located on a virulence plasmid (pLVPK), as well as phage- and plasmid-related sequences. Also, four DNA fragments encoding putative pili with the most homology to pili of Salmonella enterica were associated with the EHOS. Finally, four DNA fragments encoding putative outer-membrane proteins were negatively associated with the EHOS. In conclusion, resistance and putative virulence genes were identified in the EHOS that may have contributed to increased epidemicity. The high number of genes detected in the EHOS that were related to transferable elements reflects the genomic plasticity of the E. cloacae complex and may explain the emergence of the EHOS in the hospital environment.

Enterococcus faecium, an ubiquous colonizer of humans and animals, has evolved in the last 15 years from an avirulent commensal to the third most frequently isolated nosocomial pathogen among intensive care unit patients in the United States. E. faecium combines multidrug resistance with the potential of horizontal resistance gene transfer to even more pathogenic bacteria. Little is known about the evolution and virulence of E. faecium, and genomic studies are hampered by the absence of a completely annotated genome sequence. To further unravel its evolution, we used a mixed whole-genome microarray and hybridized 97 E. faecium isolates from different backgrounds (hospital outbreaks (n = 18), documented infections (n = 34) and asymptomatic carriage of hospitalized patients (n = 15), and healthy persons (n = 15) and animals (n = 21)). Supported by Bayesian posterior probabilities (PP = 1.0), a specific clade containing all outbreak-associated strains and 63% of clinical isolates was identified. Sequencing of 146 of 437 clade-specific inserts revealed mobile elements (n = 74), including insertion sequence (IS) elements (n = 42), phage genes (n = 6) and plasmid sequences (n = 26), hypothetical (n = 58) and membrane proteins (n = 10), and antibiotic resistance (n = 9) and regulatory genes (n = 11), mainly located on two contigs of the unfinished E. faecium DO genome. Split decomposition analysis, varying guanine cytosine content, and aberrant codon adaptation indices all supported acquisition of these genes through horizontal gene transfer with IS16 as the predicted most prominent insert (98% sensitive, 100% specific). These findings suggest that acquisition of IS elements has facilitated niche adaptation of a distinct E. faecium subpopulation by increasing its genome plasticity. Increased genome plasticity was supported by higher diversity indices (ratio of average genetic similarities of pulsed-field gel electrophoresis and multi locus sequence typing) for clade-specific isolates. Interestingly, the previously described multi locus sequence typing–based clonal complex 17 largely overlapped with this clade. The present data imply that the global emergence of E. faecium, as observed since 1990, represents the evolution of a subspecies with a presumably better adaptation than other E. faecium isolates to the constraints of a hospital environment., Author Summary Whole-genome sequencing has become instrumental in investigating the genome contents of bacteria. However, there is enormous diversity within bacterial populations, and annotation of multiple genomes is costly and elaborate. For investigating diversity and phylogeny within bacterial species, comparative genomic hybridization is an attractive alternative that may provide fundamental insights into the factors (genes) distinguishing bacterial subpopulations. Enterococcus faecium, a worldwide emerging nosocomial pathogen usually resistant to multiple antibiotics, causes infections in immunocompromised patients. Using comparative genomic hybridization of 97 E. faecium strains isolated from different epidemiological niches worldwide, a subpopulation of E. faecium strains was identified that was associated with invasive infections and hospital outbreaks. Approximately 13% of the E. faecium pangenome was highly specific for this subpopulation, and, based on phylogenetic clustering, it should be considered a subspecies. We hypothesize that extensive variation within specific functional genes and high prevalence of mobile elements, mostly insertion sequence elements, contributed to the success of this genetic subset in its competition with other enterococci in hospital settings, creating a novel globally dispersed nosocomial subspecies. These findings fully confirmed previous phylogenetic studies based on multi locus sequence typing that had also revealed a genetic subset of E. faecium, clonal complex 17. Identification of genes specific for clonal complex 17 is a first step in elucidating how global spread and adaptation to the hospital environment of this emerging nosocomial pathogen has occurred.

Background: The variation of microbial communities associated with the human body can be the cause of many factors, including the human genetic makeup, diet, age, surroundings, and sexual behavior. In this study, we investigated the effects of intimate kissing on the oral microbiota of 21 couples by self-administered questionnaires about their past kissing behavior and by the evaluation of tongue and salivary microbiota samples in a controlled kissing experiment. In addition, we quantified the number of bacteria exchanged during intimate kissing by the use of marker bacteria introduced through the intake of a probiotic yoghurt drink by one of the partners prior to a second intimate kiss.Results: Similarity indices of microbial communities show that average partners have a more similar oral microbiota composition compared to unrelated individuals, with by far most pronounced similarity for communities associated with the tongue surface. An intimate kiss did not lead to a significant additional increase of the average similarity of the oral microbiota between partners. However, clear correlations were observed between the similarity indices of the salivary microbiota of couples and self-reported kiss frequencies, and the reported time passed after the latest kiss. In control experiments for bacterial transfer, we identified the probiotic Lactobacillus and Bifidobacterium marker bacteria in most kiss receivers, corresponding to an average total bacterial transfer of 80 million bacteria per intimate kiss of 10 s.Conclusions: This study indicates that a shared salivary microbiota requires a frequent and recent bacterial exchange and is therefore most pronounced in couples with relatively high intimate kiss frequencies. The microbiota on the dorsal surface of the tongue is more similar among partners than unrelated individuals, but its similarity does not clearly correlate to kissing behavior, suggesting an important role for specific selection mechanisms resulting from a shared lifestyle, environment, or genetic factors from the host. Furthermore, our findings imply that some of the collective bacteria among partners are only transiently present, while others have found a true niche on the tongue's surface allowing long-term colonization.

Background Discrimination between clinical and environmental strains within many bacterial species is currently underexplored. Genomic analyses have clearly shown the enormous variability in genome composition between different strains of a bacterial species. In this study we have used Legionella pneumophila, the causative agent of Legionnaire's disease, to search for genomic markers related to pathogenicity. During a large surveillance study in The Netherlands well-characterized patient-derived strains and environmental strains were collected. We have used a mixed-genome microarray to perform comparative-genome analysis of 257 strains from this collection. Results Microarray analysis indicated that 480 DNA markers (out of in total 3360 markers) showed clear variation in presence between individual strains and these were therefore selected for further analysis. Unsupervised statistical analysis of these markers showed the enormous genomic variation within the species but did not show any correlation with a pathogenic phenotype. We therefore used supervised statistical analysis to identify discriminating markers. Genetic programming was used both to identify predictive markers and to define their interrelationships. A model consisting of five markers was developed that together correctly predicted 100% of the clinical strains and 69% of the environmental strains. Conclusions A novel approach for identifying predictive markers enabling discrimination between clinical and environmental isolates of L. pneumophila is presented. Out of over 3000 possible markers, five were selected that together enabled correct prediction of all the clinical strains included in this study. This novel approach for identifying predictive markers can be applied to all bacterial species, allowing for better discrimination between strains well equipped to cause human disease and relatively harmless strains.

Background In bacteriology, the ability to grow in selective media and to form colonies on nutrient agar plates is routinely used as a retrospective criterion for the detection of living bacteria. However, the utilization of indicators for bacterial viability-such as the presence of specific transcripts or membrane integrity-would overcome bias introduced by cultivation and reduces the time span of analysis from initiation to read out. Therefore, we investigated the correlation between transcriptional activity, membrane integrity and cultivation-based viability in the Gram-positive model bacterium Bacillus subtilis. Results We present microbiological, cytological and molecular analyses of the physiological response to lethal heat stress under accurately defined conditions through systematic sampling of bacteria from a single culture exposed to gradually increasing temperatures. We identified a coherent transcriptional program including known heat shock responses as well as the rapid expression of a small number of sporulation and competence genes, the latter only known to be active in the stationary growth phase. Conclusion The observed coordinated gene expression continued even after cell death, in other words after all bacteria permanently lost their ability to reproduce. Transcription of a very limited number of genes correlated with cell viability under the applied killing regime. The transcripts of the expressed genes in living bacteria – but silent in dead bacteria-include those of essential genes encoding chaperones of the protein folding machinery and can serve as molecular biomarkers for bacterial cell viability.