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Background: Microbiota profiles are strongly influenced by many technical aspects that impact the ability of researchers to compare results. To investigate and identify potential biases introduced by technical variations, we compared several approaches throughout the entire workflow of a microbiome study, from sample collection to sequencing, using commercially availablemock communities (from bacterial strains as well as from DNA) and multiple human fecal samples, including a large set of positive controls created as a random mix of several participant samples. Methods: Human fecal material was sampled, and aliquots were used to test two commercially available stabilization solutions (OMNIgene·GUT and Zymo Research) in comparison to samples frozen immediately upon collection. In addition, the methodology for DNA extraction, input of DNA, or the number of PCR cycles were analyzed. Furthermore, to investigate the potential batch effects in DNA extraction, sequencing, and barcoding, we included 139 positive controls. Results: Samples preserved in both the stabilization buffers limited the overgrowth of Enterobacteriaceae when compared to unpreserved samples stored at room temperature (RT). These stabilized samples stored at RT were different from immediately frozen samples, where the relative abundance of Bacteroidota was higher and Actinobacteriota and Firmicutes were lower. As reported previously, the method used for cell disruption was a major contributor to variation in microbiota composition. In addition, a high number of cycles during PCR lead to an increase in contaminants detected in the negative controls. The DNA extraction had a significant impact on the microbial composition, also observed with the use of different Illumina barcodes during library preparation and sequencing, while no batch effect was observed in replicate runs. Conclusion: Our study reaffrms the importance of themechanical cell disruption method and immediate frozen storage as critical aspects in fecal microbiota studies. A comparison of storage conditions revealed that the bias was limited in RT samples preserved in stabilization systems, and these may be a suitable compromise when logistics are challenging due to the size or location of a study. Moreover, to reduce the effect of contaminants in fecalmicrobiota profiling studies, we suggest the use of ~125 pg input DNA and 25 PCR cycles as optimal parameters during library preparation. [ABSTRACT FROM AUTHOR]

We have identified apuA in Streptococcus suis, which encodes a bifunctional amylopullulanase with conserved [alpha]-amylase and pullulanase substrate-binding domains and catalytic motifs. ApuA exhibited properties typical of a Gram-positive surface protein, with a putative signal sequence and LPKTGE cell-wall-anchoring motif. A recombinant protein containing the predicted N-terminal [alpha]-amylase domain of ApuA was shown to have [alpha]-(1,4) glycosidic activity. Additionally, an apuA mutant of S. suis lacked the pullulanase [alpha]-(1,6) glycosidic activity detected in a cell-surface protein extract of wild-type S. suis. ApuA was required for normal growth in complex medium containing pullulan as the major carbon source, suggesting that this enzyme plays a role in nutrient acquisition in vivo via the degradation of glycogen and food-derived starch in the nasopharyngeal and oral cavities. ApuA was shown to promote adhesion to porcine epithelium and mucus in vitro, highlighting a link between carbohydrate utilization and the ability of S. suis to colonize and infect the host. DOI 10.1099/mic.0.037960-0

Aims: To explore the perceptions of nurses on the factors that influence their readiness to manage intimate partner violence (IPV) in Spain. Design: Qualitative content analysis based on data from six different regions in Spain (Murcia, Region of Valencia, Castile and Leon, Cantabria, Catalonia, Aragon) collected between 2014 and 2016. Methods: 37 personal interviews were carried out with nurses from 27 primary health care (PHC) centres and 10 hospitals. We followed the consolidated criteria for reporting qualitative research guidelines. Qualitative content analysis was supported by Atlas.ti and OpenCode. Results: The results are organised into four categories corresponding to (1) acknowledging IPV as a health issue. An ongoing process; (2) the Spanish healthcare system and PHC service: a favourable space to address IPV although with some limitations; (3) nurses as a key figure for IPV in coordinated care and (4) factors involved in nurses' autonomy in their response to IPV, with their respective subcategories. Conclusion: In practice, nurses perceive responding to IPV as a personal choice, despite the institutional mandate to address IPV as a health issue. There is a need to increase continuous training and ensure IPV is included in the curriculum in university nursing undergraduate degrees, by disseminating the existing IPV protocols. Furthermore, coordination between healthcare professionals needs to be improved in terms of all levels of care and with other institutions. [ABSTRACT FROM AUTHOR]

This note complements and clarifies part of the work of Hawinkel et al. recently published in the journal and suggests some more or less standard tools and methods for carrying out association studies of the microbiome. [ABSTRACT FROM AUTHOR]

Introduction: Prevention of infectious diseases in the elderly is essential to establish healthy aging. Yet, immunological aging impairs successful vaccination of the elderly. Predictive biomarkers for vaccine responsiveness in middle-aged adults may help to identify responders and non-responders before reaching old age. Therefore, we aimed to determine biomarkers associated with low and high responsiveness toward a primary vaccination in middle-aged adults, for which a tetravalent meningococcal vaccine was used as a model. Methods: Middle-aged adults (50-65 years of age, N = 100), receiving a tetravalent meningococcal vaccination, were divided into low and high responders using the functional antibody titers at 28 days postvaccination. A total of 48 parameters, including absolute numbers of immune cells and serum levels of cytokines and biochemical markers, were determined prevaccination in all participants. Heat maps and multivariate redundancy analysis (RDA) were used to reveal immune phenotype characteristics and associations of the low and high responders. Results: Several significant differences in prevaccination immune markers were observed between the low and high vaccine responders. Moreover, RDA analysis revealed a significant association between the prevaccination immune phenotype and vaccine responsiveness. In particular, our analysis pointed at high numbers of CD4 T cells, especially naïve CD4 and regulatory T subsets, to be associated with low vaccine responsiveness. In addition, low responders showed lower prevaccination IL-1Ra levels than high responders. Conclusion: This explorative biomarker study shows associations between the prevaccination immune phenotype and vaccine responsiveness after a primary meningococcal vaccination in middle-aged adults. Consequently, these results provide a basis for predictive biomarker discovery for vaccine responsiveness that will require validation in larger cohort studies. [ABSTRACT FROM AUTHOR]

An abnormal colonization pattern of the preterm gut may affect immune maturation and exert a long-term influence on the intestinal bacterial composition and host health. However, follow-up studies assessing the evolution of the fecal microbiota of infants that were born preterm are very scarce. In this work, the bacterial compositions of fecal samples, obtained from sixteen 2-year-old infants were evaluated using a phylogenetic microarray; subsequently, the results were compared with those obtained in a previous study from samples of meconium and feces collected from the same infants while they stayed in the neonatal intensive care unit (NICU). In parallel, the concentration of a wide range of cytokines, chemokines, growth factors and immunoglobulins were determined in meconium and fecal samples. Globally, a higher bacterial diversity and a lower interindividual variability were observed in 2-year-olds' feces, when compared to the samples obtained during their first days of life. Hospital-associated fecal bacteria, that were dominant during the NICU stay, seemed to be replaced, two years later, by genera, which are usually predominant in the healthy adult microbiome. The immune profile of the meconium and fecal samples differed, depending on the sampling time, showing different immune maturation statuses of the gut. [ABSTRACT FROM AUTHOR]

The colonization and development of gut microbiota immediately after birth is highly variable and depends on several factors, such as delivery mode and modality of feeding during the first months of life. A cohort of 31 mother and neonate pairs, including 25 at-term caesarean (CS) and 6 vaginally (V) delivered neonates (DNs), were included in this study and 121 meconium/faecal samples were collected at days 1 through 30 following birth. Operational taxonomic units (OTUs) were assessed in 69 stool samples by phylogenetic microarray HITChip and inter- and intra-individual distributions were established by inter-OTUs correlation matrices and OTUs co-occurrence or co-exclusion networks. 1H-NMR metabolites were determined in 70 stool samples, PCA analysis was performed on 55 CS DNs samples, and metabolome/OTUs co-correlations were assessed in 45 CS samples, providing an integrated map of the early microbiota OTUs-metabolome. A microbiota “core” of OTUs was identified that was independent of delivery mode and lactation stage, suggesting highly specialized communities that act as seminal colonizers of microbial networks. Correlations among OTUs, metabolites, and OTUs-metabolites revealed metabolic profiles associated with early microbial ecological dynamics, maturation of milk components, and host physiology. [ABSTRACT FROM AUTHOR]

Background: Primary sclerosing cholangitis (PSC) is a cholestatic liver disease that is strongly associated with a particular phenotype of inflammatory bowel disease (IBD) with right-sided colonic involvement. In IBD, several studies demonstrated significant aberrancies in the intestinal microbiota in comparison with healthy controls. We aimed to explore the link between IBD and PSC by studying the intestinal mucosa-adherent microbiota in PSC and ulcerative colitis (UC) patients and noninflammatory controls. Methods: We included 12 PSC patients, 11 UC patients, and nine noninflammatory controls. The microbiota composition was determined in ileocecal biopsies from each patient by 16S rRNA based analyses using the human intestinal tract chip. Results: Profiling of the mucosa-adherent microbiota of PSC patients, UC patients, and noninflammatory controls revealed that these groups did not cluster separately based on microbiota composition. At the genus-like level, the relative abundance of uncultured Clostridiales II was significantly lower (almost 2-fold) in PSC (0.26 ± 0.10%) compared with UC (0.41 ± 0.29%) and controls (0.49 ± 0.25%) (p = 0.02). Diversity and richness in the microbiota composition differed across the groups and were significantly lower in PSC patients compared with noninflammatory controls (p = 0.04 and p = 0.02, respectively). No significant differences were found in evenness. Conclusions: Reduced amounts of uncultured Clostridiales II in PSC biopsies in comparison with UC and healthy controls can be considered a signature of a compromised gut, as we have recently observed that this group of as yet uncultured Firmicutes correlates significantly with health. [ABSTRACT FROM AUTHOR]

Recurrent Clostridium difficile infection (CDI) can be effectively treated by infusion of a healthy donor faeces suspension. However, it is unclear what factors determine treatment efficacy. By using a phylogenetic microarray platform, we assessed composition, diversity and dynamics of faecal microbiota before, after and during follow-up of the transplantation from a healthy donor to different patients, to elucidate the mechanism of action of faecal infusion. Global composition and network analysis of the microbiota was performed in faecal samples from nine patients with recurrent CDI. Analyses were performed before and after duodenal donor faeces infusion, and during a follow-up of 10 weeks. The microbiota data were compared with that of the healthy donors. All patients successfully recovered. Their intestinal microbiota changed from a low-diversity diseased state, dominated by Proteobacteria and Bacilli, to a more diverse ecosystem resembling that of healthy donors, dominated by Bacteroidetes and Clostridium groups, including butyrate-producing bacteria. We identified specific multi-species networks and signature microbial groups that were either depleted or restored as a result of the treatment. The changes persisted over time. Comprehensive and deep analyses of the microbiota of patients before and after treatment exposed a therapeutic reset from a diseased state towards a healthy profile. The identification of microbial groups that constitute a niche for C. difficile overgrowth, as well as those driving the reinstallation of a healthy intestinal microbiota, could contribute to the development of biomarkers predicting recurrence and treatment outcome, identifying an optimal microbiota composition that could lead to targeted treatment strategies. [ABSTRACT FROM AUTHOR]

Aims/hypothesis: Recent studies indicate that an aberrant gut microbiota is associated with the development of type 1 diabetes, yet little is known about the microbiota in children who have diabetes at an early age. To this end, the microbiota of children aged 1-5 years with new-onset type 1 diabetes was compared with the microbiota of age-matched healthy controls. Methods: A deep global analysis of the gut microbiota composition was established by phylogenetic microarray analysis using a Human Intestinal Tract Chip (HITChip). Results: Principal component analyses highlighted the importance of age when comparing age-matched pairs. In pairs younger than 2.9 years, the combined abundance of the class Bacilli (notably streptococci) and the phylum Bacteroidetes was higher in diabetic children, whereas the combined abundance of members of Clostridium clusters IV and XIVa was higher in the healthy controls. Controls older than 2.9 years were characterised by a higher fraction of butyrate-producing species within Clostridium clusters IV and XIVa than was seen in the corresponding diabetic children or in children from the younger age groups, while the diabetic children older than 2.9 years could be differentiated by having an increased microbial diversity. Conclusions/interpretation: The results from both age groups suggest that non-diabetic children have a more balanced microbiota in which butyrate-producing species appear to hold a pivotal position. [ABSTRACT FROM AUTHOR]

Objective Intestinal microbiota have been suggested to contribute to the development of obesity, but the mechanism remains elusive. The relationship between microbiota composition, intestinal permeability, and inflammation in nonobese and obese subjects was investigated. Design and Methods Fecal microbiota composition of 28 subjects (BMI 18.6-60.3 kg m−2) was analyzed by a phylogenetic profiling microarray. Fecal calprotectin and plasma C-reactive protein levels were determined to evaluate intestinal and systemic inflammation. Furthermore, HbA1c, and plasma levels of transaminases and lipids were analyzed. Gastroduodenal, small intestinal, and colonic permeability were assessed by a multisaccharide test. Results Based on microbiota composition, the study population segregated into two clusters with predominantly obese (15/19) or exclusively nonobese (9/9) subjects. Whereas intestinal permeability did not differ between clusters, the obese cluster showed reduced bacterial diversity, a decreased Bacteroidetes/Firmicutes ratio, and an increased abundance of potential proinflammatory Proteobacteria. Interestingly, fecal calprotectin was only detectable in subjects within the obese microbiota cluster ( n = 8/19, P = 0.02). Plasma C-reactive protein was also increased in these subjects ( P = 0.0005), and correlated with the Bacteroidetes/Firmicutes ratio ( rs = −0.41, P = 0.03). Conclusions Intestinal microbiota alterations in obese subjects are associated with local and systemic inflammation, suggesting that the obesity-related microbiota composition has a proinflammatory effect. [ABSTRACT FROM AUTHOR]