Disruption of the intestinal microbiota is considered an etiological factor in pediatric functional constipation. Scientifically based selection of potential beneficial probiotic strains in ...functional constipation therapy is not feasible due to insufficient knowledge of microbiota composition in affected subjects. The aim of this study was to describe microbial composition and diversity in children with functional constipation, compared to healthy controls.
Fecal samples from 76 children diagnosed with functional constipation according to the Rome III criteria (median age 8.0 years; range 4.2-17.8) were analyzed by IS-pro, a PCR-based microbiota profiling method. Outcome was compared with intestinal microbiota profiles of 61 healthy children (median 8.6 years; range 4.1-17.9). Microbiota dissimilarity was depicted by principal coordinate analysis (PCoA), diversity was calculated by Shannon diversity index. To determine the most discriminative species, cross validated logistic ridge regression was performed.
Applying total microbiota profiles (all phyla together) or per phylum analysis, no disease-specific separation was observed by PCoA and by calculation of diversity indices. By ridge regression, however, functional constipation and controls could be discriminated with 82% accuracy. Most discriminative species were Bacteroides fragilis, Bacteroides ovatus, Bifidobacterium longum, Parabacteroides species (increased in functional constipation) and Alistipes finegoldii (decreased in functional constipation).
None of the commonly used unsupervised statistical methods allowed for microbiota-based discrimination of children with functional constipation and controls. By ridge regression, however, both groups could be discriminated with 82% accuracy. Optimization of microbiota-based interventions in constipated children warrants further characterization of microbial signatures linked to clinical subgroups of functional constipation.
The composition of the gut microbiota is associated with various disease states, most notably inflammatory bowel disease, obesity and malnutrition. This underlines that analysis of intestinal ...microbiota is potentially an interesting target for clinical diagnostics. Currently, the most commonly used sample types are feces and mucosal biopsy specimens. Because sampling method, storage and processing of samples impact microbiota analysis, each sample type has its own limitations. An ideal sample type for use in routine diagnostics should be easy to obtain in a standardized fashion without perturbation of the microbiota. Rectal swabs may satisfy these criteria, but little is known about microbiota analysis on these sample types. In this study we investigated the characteristics and applicability of rectal swabs for gut microbiota profiling in a clinical routine setting in patients presenting with various gastro-intestinal disorders. We found that rectal swabs appeared to be a convenient means of sampling the human gut microbiota. Swabs can be performed on demand, whenever a patient presents; swab-derived microbiota profiles are reproducible, whether they are gathered at home by patients or by medical professionals in an outpatient setting and may be ideally suited for clinical diagnostics and large-scale studies.
The emergence and spread of antibiotic resistance among pathogenic bacteria has been a rising problem for public health in recent decades. It is becoming increasingly recognized that not only ...antibiotic resistance genes (ARGs) encountered in clinical pathogens are of relevance, but rather, all pathogenic, commensal as well as environmental bacteria-and also mobile genetic elements and bacteriophages-form a reservoir of ARGs (the resistome) from which pathogenic bacteria can acquire resistance via horizontal gene transfer (HGT). HGT has caused antibiotic resistance to spread from commensal and environmental species to pathogenic ones, as has been shown for some clinically important ARGs. Of the three canonical mechanisms of HGT, conjugation is thought to have the greatest influence on the dissemination of ARGs. While transformation and transduction are deemed less important, recent discoveries suggest their role may be larger than previously thought. Understanding the extent of the resistome and how its mobilization to pathogenic bacteria takes place is essential for efforts to control the dissemination of these genes. Here, we will discuss the concept of the resistome, provide examples of HGT of clinically relevant ARGs and present an overview of the current knowledge of the contributions the various HGT mechanisms make to the spread of antibiotic resistance.
The aim of this study was to screen how rapidly the human gut microbiota responds to diet in an in vitro model of the proximal colon (TIM-2 system). Two experimental diets were provided to the gut ...bacteria: a high carbohydrate and a high protein diet. The metabolic response and the composition of the microbiota were compared to a control diet simulating an average western meal. Short-chain and branched-chain fatty acids (SCFA and BCFA, respectively) production, in addition to changes in the community composition (profiling), were measured. The activity of the microbiota reflected differences between diets, exhibiting a trade-off between saccharolytic and proteolytic fermentation when compared to the control. Diversity analysis revealed a phylum-specific response depending on the diet tested. Most changes in the microbiome composition occurred during the first 24 h of the experiment. The outcome of this study elucidates the fact that human gut bacteria quickly respond to changes in diet. In addition, it confirms that variations in the concentration of carbohydrates and proteins modify the activity and composition of the microbiota, and these changes can potentially have an impact on the health of the host.
Green tea catechins may play a role in body weight regulation through interactions with the gut microbiota.
We examined whether green tea supplementation for 12 weeks induces changes in composition ...of the human gut microbiota.
58 Caucasian men and women were included in a randomized, placebo-controlled design. For 12 weeks, subjects consumed either green tea (>0.56 g/d epigallocatechin-gallate + 0.28 ∼ 0.45 g/d caffeine) or placebo capsules. Fecal samples were collected twice (baseline, vs. week 12) for analyses of total bacterial profiles by means of IS-profiling, a 16S-23S interspacer region-based profiling method.
No significant changes between baseline and week 12 in subjects receiving green tea or placebo capsules, and no significant interactions between treatment (green tea or placebo) and time (baseline and week 12) were observed for body composition. Analysis of the fecal samples in subjects receiving green tea and placebo showed similar bacterial diversity and community structures, indicating there were no significant changes in bacterial diversity between baseline and week 12 in subjects receiving green tea capsules or in subjects receiving placebo capsules. No significant interactions were observed between treatment (green tea or placebo) and time (baseline and week 12) for the gut microbial diversity. Although, there were no significant differences between normal weight and overweight subjects in response to green tea, we did observe a reduced bacterial alpha diversity in overweight as compared to normal weight subjects (p = 0.002).
Green tea supplementation for 12 weeks did not have a significant effect on composition of the gut microbiota.
ClinicalTrials.gov NCT01556321.
The neonatal period, during which the initial gut microbiota is acquired, is a critical phase. The healthy development of the infant's microbiome can be interrupted by external perturbations, like ...antibiotics, which are associated with profound effects on the gut microbiome and various disorders later in life. The aim of this study was to investigate the development of intestinal microbiota and the effect of antibiotic exposure during the first three months of life in term infants. Fecal samples were collected from healthy infants and infants who received antibiotics in the first week of life at one week, one month, and three months after birth. Microbial composition was analyzed using IS-pro and compared between antibiotics-treated and untreated infants. In total, 98 infants, divided into four groups based on feeding type and delivery mode, were analyzed. At one week of age, samples clustered into two distinct groups, which were termed "settler types", based on their Bacteroidetes abundance. Caesarean-born infants belonged to the low-Bacteroidetes settler type, but vaginally-born infants were divided between the two groups. The antibiotics effect was assessed within a subgroup of 45 infants, vaginally-born and exclusively breastfed, to minimize the effect of other confounders. Antibiotics administration resulted in lower Bacteroidetes diversity and/or a delay in Bacteroidetes colonization, which persisted for three months, and in a differential development of the microbiota. Antibiotics resulted in pronounced effects on the Bacteroidetes composition and dynamics. Finally, we hypothesize that stratification of children's cohorts based on settler types may reveal group effects that might otherwise be masked.
Large-scale cohort studies are currently being designed to investigate the human microbiome in health and disease. Adequate sampling strategies are required to limit bias due to shifts in microbial ...communities during sampling and storage. Therefore, we examined the impact of different sampling and storage conditions on the stability of fecal microbial communities in healthy and diseased subjects. Fecal samples from 10 healthy controls, 10 irritable bowel syndrome and 8 inflammatory bowel disease patients were collected on site, aliquoted immediately after defecation and stored at -80 °C, -20 °C for 1 week, at +4°C or room temperature for 24 hours. Fecal transport swabs (FecalSwab, Copan) were collected and stored for 48-72 hours at room temperature. We used pyrosequencing of the 16S gene to investigate the stability of microbial communities. Alpha diversity did not differ between all storage methods and -80 °C, except for the fecal swabs. UPGMA clustering and principal coordinate analysis showed significant clustering by test subject (p < 0.001) but not by storage method. Bray-Curtis dissimilarity and (un)weighted UniFrac showed a significant higher distance between fecal swabs and -80 °C versus the other methods and -80 °C samples (p < 0.009). The relative abundance of Ruminococcus and Enterobacteriaceae did not differ between the storage methods versus -80 °C, but was higher in fecal swabs (p < 0.05). Storage up to 24 hours (at +4 °C or room temperature) or freezing at -20 °C did not significantly alter the fecal microbial community structure compared to direct freezing of samples from healthy subjects and patients with gastrointestinal disorders.
Determination of pathogen-specific bacterial DNA load (BDL) in blood has been shown to be directly correlated with severity of infection in patients with bacteremia. In the diagnostic work-up of ...patients with Staphylococcus aureus bacteremia (SAB), determination of the primary focus is imperative, because of implications for treatment duration, and ultimately prognosis. Here we investigate whether measurement of BDL in patients with SAB can distinguish between intravascular and extravascular foci of infection.
In a consecutive cohort of 43 patients with positive blood cultures with Staphylococcus aureus, we performed a quantitative PCR on whole blood to detect the bacterial DNA load. Infections were classified into 3 categories: i) soft tissue infections and phlebitis, ii) deep-seated infections and iii) endocarditis and other intravascular infections. Bacterial DNA loads and inflammatory parameters in the three categories were analyzed and compared.
Median BDL in patients with endocarditis and other intravascular infections was 1015 cfu/ml, significantly higher than BDL in the other two categories (28 and 31 cfu/ml respectively). In contrast, CRP and leukocytes were not significantly different between the three patient categories. BDL could be detected in all patients with intravascular causes and levels were generally 10-30 times higher than in the other infection categories. Median BDL in non-survivors was 85 cfu/ml, which was higher than in survivors with a median BDL of 29 cfu/ml, although not significant.
In Staphylococcus aureus bacteremia pathogen-specific BDL is distinctly higher in patients with intravascular infections compared to extravascular origins. As measurement of BDL by PCR can easily be implemented in routine diagnostics, it can improve the diagnostic work-up of SAB by rapidly identifying the subset of patients who need higher dosages of antibiotics and additional measures to improve outcome.
To achieve an optimal effect in patients with sepsis at the emergency department (ED), the gentamicin peak-concentration should be sufficiently high (i.e. peak-concentration/MIC ≥8-10). ICU patients ...with sepsis often need higher gentamicin doses to achieve sufficiently high peak-concentrations. The aim of this study is to investigate which dose is needed to reach adequate peak-concentrations in patients presenting with sepsis at the ED.
Patients with sepsis at the ED were included from August 2015 until February 2017. Peak-concentrations were measured in blood 30 minutes after the first gentamicin dose. The study consisted of three phases. In the first phase, peak-concentrations were measured after a standard dose of 5mg/kg. In the second phase, a simulation ((peak-concentration/actual dose) × simulated dose) was performed to determine which dose was needed to reach adequate gentamicin peak-concentrations of ≥16mg/L. In the third phase, peak-concentrations were measured for the best simulated dose.
In phase one, of 86 patients who received a dose of 5mg/kg, 34 (39.5%) patients did not reach the target peak-concentration of ≥16mg/L, and 73 (84.9%) did not reach ≥20mg/L. In phase two, the simulation showed that with a dose of 7mg/kg 83 (96.5%) patients would reach peak-concentrations ≥16mg/L, and 67 (77.9%) of ≥20mg/L. In phase three, 53 patients received a dose of 7mg/kg, of whom 45 (84.9%) reached peak-concentrations of ≥16mg/L, and 31 (58.5%) of ≥20mg/L.
Patients with sepsis at the ED need higher doses of gentamicin. A dose of 7mg/kg is needed to achieve adequate peak-concentrations in the majority of patients.
The availability of valid Severe Acute Respiratory Syndrome Coronvirus-2 (SARS-CoV-2) serological tests overcome the problem of underestimated cumulative Coronavirus Disease 2019 (COVID-19) cases ...during the first months of the pandemic in The Netherlands. The possibility to reliably determine the number of truly infected persons, enabled us to study initial drivers for exposure risk in the absence of routine testing. Numerous activities or circumstances can accelerate virus spread, here defined as exposure factors. Hence, we aimed to evaluate a wide variety of demographic, behavioural and social exposure factors associated with seropositivity during the first eight months of the pandemic in Limburg, The Netherlands.
SARS-CoV-2 point-seroprevalence was determined cross-sectionally to indicate previous infection in a convenience sample of minimal 10,000 inhabitants of the study province. All adult (18+ years) inhabitants of the study province were eligible to register themselves for participation. Once the initial 10,000 registrations were reached, a reserve list was kept to ensure sufficient participants. Possible exposure factors were mapped by means of an extensive questionnaire. Associated exposure factors were determined using univariable and multivariable logistic regression models.
Seropositivity was established in 19.5% (n = 1,948) of the 10,001 participants (on average 49 years old (SD = 15; range 18-90 years), majority women (n = 5,829; 58.3%). Exposure factors associated with seropositivity included current education, working in healthcare and not working from home, and being a member of three or four associations or clubs. Specifically for February-March 2020, visiting an après-ski bar during winter sports in Austria, travelling to Spain, celebrating carnival, and participating in a singing activity or ball sport were associated with seropositivity.
Our results confirm that relevant COVID-19 exposure factors generally reflected circumstances where social distancing was impossible, and the number and duration of contacts was high, in particular for indoor activities.
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