Proof of principle study replicating microbial clusters in connection to birth mode and diet in the early life intestine.

The human gut ecosystem starts developing at birth and is influenced by many factors during early life. In this study we make use of a Belgian cohort of 64 children, followed until the age of 6 years, to analyze different phases of microbiota development. We analyzed fecal samples taken before weaning (age 1 month), shortly after weaning (age 6 months), when milk feeding has been discontinued completely (age 1 year), and at the age of 6 years. We performed 16S rRNA gene amplicon sequencing on the collected fecal samples and analyzed the compositional data in relation to dietary metadata and birth mode. Human and formula milk feeding promotes a microbiota dominated by either Bacteroides or Bifidobacterium, respectively. Into later life stages, the microbiota composition follows distinct microbiota clusters, related to abundance dynamics of certain bacterial groups. Furthermore, it becomes apparent that a formula diet leads to early maturation of the infant gut microbiota. Despite other clinical variables within the infant cohort, they did not significantly contribute to the microbiota patterns we observed. Our data provide a proof of principle study of the importance of diet to the development of the microbiota in early life that replicates earlier findings in other cohorts.

Two Years of Genomic Surveillance in Belgium during the SARS-CoV-2 Pandemic to Attain Country-Wide Coverage and Monitor the Introduction and Spread of Emerging Variants.

An adequate SARS-CoV-2 genomic surveillance strategy has proven to be essential for countries to obtain a thorough understanding of the variants and lineages being imported and successfully established within their borders. During 2020, genomic surveillance in Belgium was not structurally implemented but performed by individual research laboratories that had to acquire the necessary funds themselves to perform this important task. At the start of 2021, a nationwide genomic surveillance consortium was established in Belgium to markedly increase the country's genomic sequencing efforts (both in terms of intensity and representativeness), to perform quality control among participating laboratories, and to enable coordination and collaboration of research projects and publications. We here discuss the genomic surveillance efforts in Belgium before and after the establishment of its genomic sequencing consortium, provide an overview of the specifics of the consortium, and explore more details regarding the scientific studies that have been published as a result of the increased number of Belgian SARS-CoV-2 genomes that have become available.

Denaturing gradient gel electrophoresis of neonatal intestinal microbiota in relation to the development of asthma.

BACKGROUND: The extended 'hygiene hypothesis' suggests that the initial composition of the infant gut microbiota is a key determinant in the development of atopic disease. Several studies have demonstrated that the microbiota of allergic and non-allergic infants are different even before the development of symptoms, with a critical time window during the first 6 months of life. The aim of the study was to investigate the association between early intestinal colonisation and the development of asthma in the first 3 years of life using DGGE (denaturing gradient gel electrophoresis). METHODS: In a prospective birth cohort, 110 children were classified according to the API (Asthma Predictive Index). A positive index included wheezing during the first three years of life combined with eczema in the child in the first years of life or with a parental history of asthma. A fecal sample was taken at the age of 3 weeks and analysed with DGGE using universal and genus specific primers. RESULTS: The Asthma Predictive Index was positive in 24/110 (22%) of the children. Using universal V3 primers a band corresponding to a Clostridum coccoides XIVa species was significantly associated with a positive API. A Bacteroides fragilis subgroup band was also significantly associated with a positive API. A final DGGE model, including both bands, allowed correct classification of 73% (80/110) of the cases. CONCLUSION: Fecal colonisation at age 3 weeks with either a Bacteroides fragilis subgroup or a Clostridium coccoides subcluster XIVa species is an early indicator of possible asthma later in life. These findings need to be confirmed in a new longitudinal follow-up study.

Intrauterine exposure to environmental pollutants and body mass index during the first 3 years of life.

OBJECTIVE: We investigated the association between body mass index (BMI) standard deviation score (SDS) and prenatal exposure to hexachlorobenzene, dichlorodiphenyldichloroethylene (DDE), dioxin-like compounds, and polychlorinated biphenyls (PCBs). METHODS: In this prospective birth cohort study, we assessed a random sample of mother-infant pairs (n = 138) living in Flanders, Belgium, with follow-up until the children were 3 years of age. We measured body mass index as standard deviation scores (BMI SDS) of children 1-3 years of age as well as pollutants measured in cord blood. RESULTS: DDE correlated with BMI SDS, with effect modification by maternal smoking and the child's age. At 1 year, children of smoking mothers had higher BMI SDS than did children of nonsmoking mothers. At 3 years, this difference was reduced because of the faster rate of decline in BMI SDS in the former group. This relationship held except for children with high levels of DDE. DDE had a small effect on BMI SDS at 3 years of age in children of nonsmoking mothers (difference in BMI SDS for DDE concentrations between the 90th and 10th percentiles = 0.13). On the other hand, smoking enhanced the relation between DDE and BMI SDS at 3 years (difference in BMI SDS for DDE concentrations between the 90th and 10th percentiles = 0.76). Increasing concentrations of PCBs were associated with higher BMI SDS values at all ages (parameter estimate = 0.003 +/- 0.001; p = 0.03). CONCLUSION: In this study we demonstrated that intrauterine exposure to DDE and PCBs is associated with BMI during early childhood. Future studies are warranted to confirm our findings and to assess possible mechanisms by which these pollutants could alter energy metabolism.

The importance of the development of the intestinal microbiota in infancy.

PURPOSE OF REVIEW: The development of the intestinal microbiota occurs primarily during infancy, and a distortion could potentially contribute to a wide range of diseases. This review summarizes the current understanding of the intestinal microbiota in infants. The potential consequences of different colonization patterns on child health and possible preventive interventions are discussed. RECENT FINDINGS: Recent studies and the use of culture-independent techniques have shown that Bifidobacterium is only a minor component of the infant gut microbiota. These techniques have also introduced the concept of a core microbiome in which metabolic function is more important than the presence of a particular bacterial species. A less diverse gut microbiota with high counts of Bacteroides, Clostridium, Enterobacteriaceae and Staphylococcus early in life has been associated with an increased risk for atopic disease. Changes in infant gut colonization were also found in relation to childhood obesity. Probiotics have no proven preventive effect on the development of asthma and an unconfirmed effect on atopic dermatitis. A prebiotic trial could show a preventive effect on the development of both atopic diseases. SUMMARY: Molecular techniques have improved our understanding of the infant gut ecosystem. The available probiotics for prevention of atopic disease are disappointing, and the results with prebiotics need further confirmation. New studies on the relation between gut microbiota and disease should consider asthma and atopic dermatitis separately. Future trials should focus on high-risk groups, determine their long-term effect and also investigate the effect on Bacteroides and Clostridium.

Prospective evaluation of virulence factors of enterococci isolated from patients with peritonitis: impact on outcome.

The objective of this study was to evaluate the prevalence of 4 virulence factors (VFs) of enterococci (cytolysin [cyl], gelatinase [gel], aggregation substance [agg], and enterococcal surface protein [esp]) and their relationship to outcome in patients with generalized peritonitis in a prospective cohort study. VF expression in each strain was assessed by polymerase chain reaction assay with specific primers. Outcome of the patients was recorded. Ninety-nine strains of Enterococcus were obtained from the peritoneal fluid of 81 patients. Fifty-eight patients had at least 1 strain bearing [cyl] (13.1% of the strains), [gel] (50.5% of the strains), [agg] (40.4% of the strains), and [esp] (34.3% of the strains). The presence of VF of Enterococcus was independently associated with mortality: odds ratio, 5.5; 95% confidence interval, 1.3-28.1. In conclusion, VF accounted for 72% of the patients with enterococci isolated from the peritoneal fluid and was independently associated with mortality in severe peritonitis.

A longitudinal analysis on the association between antibiotic use, intestinal microflora, and wheezing during the first year of life.

OBJECTIVE: To examine the association between the intestinal flora at the age of three weeks and wheezing during the first year of life in a prospective birth cohort study. METHODS: The Asthma and Allergy study is a prospective birth cohort study. A total of 154 children were recruited through maternity clinics. Selection criteria were vaginal delivery at term and uncomplicated perinatal period. Questionnaires were collected with data on the parents, including demography, smoking, and asthma. Data of the child on demographic factors, respiratory symptoms, and risk factors for asthma were collected at the ages of 3 weeks and 6 and 12 months. A fecal sample was collected at 3 weeks of age. RESULTS: The frequency of wheezing averaged on 11.8%, 18.4%, and 23.5% at the three time points. In univariate analyses, increasing total concentration of anerobic bacteria were associated with increased odds of wheezing. Furthermore, several trends were observed between wheezing and Bifidobacterium and Clostridium. A final model showed a significant association between wheezing during the first year of life and antibiotic use, total concentration of anerobic bacteria, while increasing concentrations of Clostridium were protective of wheezing. CONCLUSION: This study demonstrated an association between antibiotics, anerobic bacteria, and wheezing during the first year of life. The effect of antibiotics was probably due to reverse causation. Since Clostridium was protective of wheezing, other anerobic bacteria are probably responsible for the increased risk of wheezing, which remains to be demonstrated.

Early intestinal Bacteroides fragilis colonisation and development of asthma.

BACKGROUND: The 'hygiene hypothesis' suggests that early exposure to microbes can be protective against atopic disease. The intestinal microbial flora could operate as an important postnatal regulator of the Th1/Th2 balance. The aim of the study was to investigate the association between early intestinal colonisation and the development of asthma in the first 3 years of life. METHODS: In a prospective birth cohort, 117 children were classified according to the Asthma Predictive Index. A positive index included wheezing during the first three years of life combined with eczema in the child in the first years of life or with a parental history of asthma. A faecal sample was taken at the age of 3 weeks and cultured on selective media. RESULTS: Asthma Predictive Index was positive in 26/117 (22%) of the children. The prevalence of colonisation with Bacteroides fragilis was higher at 3 weeks in index+ compared to index- children (64% vs. 34% p < 0,05). Bacteroides fragilis and Total Anaerobes counts at 3 weeks were significantly higher in children with a positive index as compared with those without. After adjusting for confounders a positive association was found between Bacteroides fragilis colonisation and Asthma Predictive Index (odds ratio: 4,4; confidence interval: 1,7 - 11,8). CONCLUSION: Bacteroides fragilis colonisation at age 3 weeks is an early indicator of possible asthma later in life. This study could provide the means for more accurate targeting of treatment and prevention and thus more effective and better controlled modulation of the microbial milieu.

Distribution of HCV genotypes in Belgium from 2008 to 2015.

BACKGROUND: The knowledge of circulating HCV genotypes and subtypes in a country is crucial to guide antiviral therapy and to understand local epidemiology. Studies investigating circulating HCV genotypes and their trends have been conducted in Belgium. However they are outdated, lack nationwide representativeness or were not conducted in the general population. METHODS: In order to determine the distribution of different circulating HCV genotypes in Belgium, we conducted a multicentre study with all the 19 Belgian laboratories performing reimbursed HCV genotyping assays. Available genotype and subtype data were collected for the period from 2008 till 2015. Furthermore, a limited number of other variables were collected: some demographic characteristics from the patients and the laboratory technique used for the determination of the HCV genotype. RESULTS: For the study period, 11,033 unique records collected by the participating laboratories were used for further investigation. HCV genotype 1 was the most prevalent (53.6%) genotype in Belgium, with G1a and G1b representing 19.7% and 31.6%, respectively. Genotype 3 was the next most prevalent (22.0%). Further, genotype 4, 2, and 5 were responsible for respectively 16.1%, 6.2%, and 1.9% of HCV infections. Genotype 6 and 7 comprise the remaining <1%. Throughout the years, a stable distribution was observed for most genotypes. Only for genotype 5, a decrease as a function of the year of analysis was observed, with respectively 3.6% for 2008, 2.3% for 2009 and 1.6% for the remaining years. The overall M:F ratio was 1.59 and was mainly driven by the high M:F ratio of 3.03 for patients infected with genotype 3. Patients infected with genotype 3 are also younger (mean age 41.7 years) than patients infected with other genotypes (mean age above 50 years for all genotypes). The patients for whom a genotyping assay was performed in 2008 were younger than those from 2015. Geographical distribution demonstrates that an important number of genotyped HCV patients live outside the Belgian metropolitan cities. CONCLUSION: This national monitoring study allowed a clear and objective view of the circulating HCV genotypes in Belgium and will help health authorities in the establishment of cost effectiveness determinations before implementation of new treatment strategies. This baseline characterization of the circulating genotypes is indispensable for a continuous surveillance, especially for the investigation of the possible impact of migration from endemic regions and prior to the increasing use of highly potent direct-acting antiviral (DAA) agents.

Differences in gut microbiota composition between obese and lean children: a cross-sectional study.

BACKGROUND: An altered gut microbiota composition has recently been linked to obesity. The principal aim of this study is to investigate and compare the gut microbiota composition in obese and lean children. Secondly, associations between analysed gut bacterial species, dietary compounds, energy intake and biochemical blood parameters are evaluated. METHODS: In this prospective cross-sectional study, 26 overweight/obese (mean BMI: 28.7 ± 6.5) and 27 lean (mean BMI: 16.5 ± 2.1) children aged 6 to 16 were included. Faecal samples were collected and subjected to selective plating and quantitative real-time PCR (qPCR) in order to determine the concentrations of bacterial species belonging to the genera: Bacteroides, Bifidobacterium, Clostridium, Staphylococcus and Lactobacillus. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was applied for an in-depth identification of species of Bacteroides fragilis group. Differences in the concentrations of gut bacterial species between obese and lean children were statistically analysed using Mann Whitney U test. Subsequently, random forest analysis and multiple linear regression analysis were performed in order to test associations between gut bacterial species, dietary compounds and blood parameters. RESULTS: Obese children showed an elevated Firmicutes-to-Bacteroidetes ratio compared with lean children. Furthermore, low relative proportions of B. vulgatus and high concentrations of Lactobacillus spp. were observed in the obese microbiota. In all children, Staphylococcus spp. were positively associated with energy intake. Additionally, in obese children, Lactobacillus spp. were positively associated with plasma hs-CRP. CONCLUSIONS: Our findings corroborate a significant difference in the gut microbiota composition of important bacterial species between obese and lean children. In future, non-invasive manipulation of gut microbiota composition in early infancy could offer a new approach to manage childhood obesity and associated disorders.

Intestinal microflora and body mass index during the first three years of life: an observational study.

BACKGROUND: Recent research on obesity has demonstrated that the intestinal microflora can have an important influence on host energy balance. The aim of the study was to investigate the relationship between the intestinal microflora and the body mass index in the first 3 years of life. RESULTS: In a prospective study, a faecal sample from 138 infants was taken at the age of 3, 26 and 52 weeks and cultured on selective media for 6 bacterial genera. Between the age of 1 and 3 years the Body Mass Index Standard Deviation Score (BMI SDS) of these children was determined. The association between the intestinal flora and BMI SDS was assessed for each bacterial genus. A positive correlation was found between the Bacteroides fragilis concentration and the BMI SDS at the age of 3 and 26 weeks. The Staphylococcus concentration showed a negative correlation with the BMI SDS at the age of 3 and 52 weeks. A low intestinal ratio of Staphylococcus/Bacteroides fragilis at the age of 3 weeks, corresponding to a low Staphylococcus and a high Bacteroides fragilis concentration, was associated with a higher BMI SDS during the first three years of life. CONCLUSION: High intestinal Bacteroides fragilis and low Staphylococcus concentrations in infants between the age of 3 weeks and 1 year were associated with a higher risk of obesity later in life. This study could provide new targets for a better and more effective modulation of the intestinal microflora in infants.

Development of a multiplex PCR for the detection of asa1, gelE, cylA, esp, and hyl genes in enterococci and survey for virulence determinants among European hospital isolates of Enterococcus faecium.

A multiplex PCR for the simultaneous detection of five virulence genes (asa1, gelE, cylA, esp, and hyl) in enterococci was developed. The presence of these genes was investigated in 153 clinical and 118 fecal Enterococcus faecium isolates from inpatients at an increased risk of developing infections (such as patients in intensive care units and hematology wards) from 13 hospitals in eight European countries. Of the 271 E. faecium isolates, 135 were vancomycin resistant E. faecium (VREF) isolates and 136 were vancomycin susceptible E. faecium (VSEF) isolates. Susceptibilities to ampicillin, gentamicin, streptomycin, vancomycin, teicoplanin, ramoplanin, quinupristin-dalfopristin, and linezolid were tested by the microdilution method. Overall, the prevalence of esp was significantly higher (P = 0.03) in clinical VREF isolates (92%) than in fecal VREF isolates (73%). In Italy, the prevalence of esp was significantly higher (P = 0.02) in VREF isolates (91%) than in VSEF isolates (68%), whereas in the United Kingdom, hyl was significantly more prevalent (P = 0.01) in VREF isolates (71%) than in VSEF isolates (29%). No significant differences were found for the other countries. Pulsed-field gel electrophoresis was used to check the clonality among the strains tested and showed the spread of two center-specific (esp-positive) VREF clones in Italy and one center-specific (hyl-positive) clone in the United Kingdom. These clones were resistant to ampicillin, gentamicin, and streptomycin. The multiplex PCR reported in this study is a convenient and rapid method for the simultaneous detection of the virulence genes asa1, gelE, cylA, esp, and hyl in enterococci. Molecular analysis showed the intrahospital spread of esp-positive VREF clones (in Italy) and hyl-positive VREF clones (in the United Kingdom); the role of hyl remains to be elucidated.

A potential virulence gene, hylEfm, predominates in Enterococcus faecium of clinical origin.

An open reading frame (hyl(Efm)) with homologies to previously described hyaluronidase genes has been identified in nonstool isolates of Enterococcus faecium. E. faecium isolates (n=577) from diverse sources were screened for the presence of hyl(Efm) and esp(Efm), a putative virulence gene associated with epidemic E. faecium strains. The presence of esp(Efm) was roughly twice that of hyl(Efm), but both were found primarily in vancomycin-resistant E. faecium isolates in nonstool cultures obtained from patients hospitalized in the United States. These data suggest that specific E. faecium strains may be enriched in determinants that make them more likely to cause clinical infections. Differences in the prevalence of these strains may help explain variations in the clinical importance of multiresistant E. faecium across different continents.