High-throughput DNA extraction strategy for fecal microbiome studies.

Microbiome studies are becoming larger in size to detect the potentially small effect that environmental factors have on our gut microbiomes, or that the microbiome has on our health. Therefore, fast and reproducible DNA isolation methods are needed to handle thousands of fecal samples. We used the Chemagic 360 chemistry and Magnetic Separation Module I (MSMI) instrument to compare two sample preservatives and four different pre-treatment protocols to find an optimal method for DNA isolation from thousands of fecal samples. The pre-treatments included bead beating, sample handling in tube and plate format, and proteinase K incubation. The optimal method offers a sufficient yield of high-quality DNA without contamination. Three human fecal samples (adult, senior, and infant) with technical replicates were extracted. The extraction included negative controls (OMNIgeneGUT, DNA/RNA shield fluid, and Chemagic Lysis Buffer 1) to detect cross-contamination and ZymoBIOMICS Gut Microbiome Standard as a positive control to mimic the human gut microbiome and assess sensitivity of the extraction method. All samples were extracted using Chemagic DNA Stool 200 H96 kit (PerkinElmer, Finland). The samples were collected in two preservatives, OMNIgeneGUT and DNA/RNA shield fluid. DNA quantity was measured using Qubit-fluorometer, DNA purity and quality using gel electrophoresis, and taxonomic signatures with 16S rRNA gene-based sequencing with V3V4 and V4 regions. Bead beating increased bacterial diversity. The largest increase was detected in gram-positive genera Blautia, Bifidobacterium, and Ruminococcus. Preservatives showed minor differences in bacterial abundances. The profiles between the V3V4 and V4 regions differed considerably with lower diversity samples. Negative controls showed signs from genera abundant in fecal samples. Technical replicates of the Gut Standard and stool samples showed low variation. The selected isolation protocol included recommended steps from manufacturer as well as bead beating. Bead beating was found to be necessary to detect hard-to-lyse bacteria. The protocol was reproducible in terms of DNA yield among different stool replicates and the ZymoBIOMICS Gut Microbiome Standard. The MSM1 instrument and pre-treatment in a 96-format offered the possibility of automation and handling of large sample collections. Both preservatives were feasible in terms of sample handling and had low variation in taxonomic signatures. The 16S rRNA target region had a high impact on the composition of the bacterial profile. IMPORTANCE: Next-generation sequencing (NGS) is a widely used method for determining the composition of the gut microbiota. Due to the differences in the gut microbiota composition between individuals, microbiome studies have expanded into large population studies to maximize detection of small effects on microbe-host interactions. Thus, the demand for a rapid and reliable microbial profiling is continuously increasing, making the optimization of high-throughput 96-format DNA extraction integral for NGS-based downstream applications. However, experimental protocols are prone to bias and errors from sample collection and storage, to DNA extraction, primer selection and sequencing, and bioinformatics analyses. Methodological bias can contribute to differences in microbiome profiles, causing variability across studies and laboratories using different protocols. To improve consistency and confidence of the measurements, the standardization of microbiome analysis methods has been recognized in many fields.

Differences in Gut Microbiota Profiles and Microbiota Steroid Hormone Biosynthesis in Men with and Without Prostate Cancer.

Background: Although prostate cancer (PCa) is the most common cancer in men in Western countries, there is significant variability in geographical incidence. This might result from genetic factors, discrepancies in screening policies, or differences in lifestyle. Gut microbiota has recently been associated with cancer progression, but its role in PCa is unclear. Objective: Characterization of the gut microbiota and its functions associated with PCa. Design setting and participants: In a prospective multicenter clinical trial (NCT02241122), the gut microbiota profiles of 181 men with a clinical suspicion of PCa were assessed utilizing 16S rRNA sequencing. Outcome measurements and statistical analysis: Sequences were assigned to operational taxonomic units, differential abundance analysis, and α- and ß-diversities, and predictive functional analyses were performed. Plasma steroid hormone levels corresponding to the predicted microbiota steroid hormone biosynthesis profiles were investigated. Results and limitations: Of 364 patients, 181 were analyzed, 60% of whom were diagnosed with PCa. Microbiota composition and diversity were significantly different in PCa, partially affected by Prevotella 9, the most abundant genus of the cohort, and significantly higher in PCa patients. Predictive functional analyses revealed higher 5-α-reductase, copper absorption, and retinol metabolism in the PCa-associated microbiome. Plasma testosterone was associated negatively with the predicted microbial 5-α-reductase level. Conclusions: Gut microbiota of the PCa patients differed significantly compared with benign individuals. Microbial 5-α-reductase, copper absorption, and retinol metabolism are potential mechanisms of action. These findings support the observed association of lifestyle, geography, and PCa incidence. Patient summary: In this report, we found that several microbes and potential functions of the gut microbiota are altered in prostate cancer compared with benign cases. These findings suggest that gut microbiota could be the link between environmental factors and prostate cancer.

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

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

Infant gut microbiota and negative and fear reactivity.

BACKGROUND: Studies indicate that gut microbiota is related to neurodevelopmental and behavioral outcomes. Accordingly, early gut microbiota composition (GMC) has been linked to child temperament, but research is still scarce. The aim of this study was to examine how early GMC at 2.5 months is associated with child negative and fear reactivity at 8 and 12 months since they are potentially important intermediate phenotypes of later child psychiatric disorders. METHODS: Our study population was 330 infants enrolled in the longitudinal FinnBrain Birth Cohort Study. Gut microbiota composition was analyzed using stool sample 16s rRNA sequencing. Negative and fear reactivity were assessed using the Laboratory Temperament Assessment Battery (Lab-TAB) at child's age of 8 months (n =150) and the Infant Behavior Questionnaire-Revised Short Form (IBQ-R SF) at child's age of 12 months (n = 276). CONCLUSIONS: We found a positive association between alpha diversity and reported fear reactivity and differing microbial community composition based on negative reactivity for boys. Isobutyric acid correlated with observed negative reactivity, however, this association attenuated in the linear model. Several genera were associated with the selected infant temperament traits. This study adds to the growing literature on links between infant gut microbiota and temperament informing future mechanistic studies.

Hair cortisol, cortisone and DHEA concentrations and the composition of microbiota in toddlers.

Animal research suggests that the gut microbiota and the HPA axis communicate in a bidirectional manner. However, human data, especially on early childhood, remain limited. In this exploratory design, we investigated the connections between long-term HPA axis functioning, measured as cortisol, cortisone or dehydroepiandrosterone concentrations and their ratios from hair segments of three centimeters, and gut microbiota profiles, (measured as diversity and bacterial composition by 16 S rRNA sequencing) in healthy 2.5-year-old toddlers (n = 135) recruited from the FinnBrain Birth Cohort Study. The alpha diversity of the microbiota was studied by linear regression. Beta diversity analyses with weighted UniFrac or Bray-Curtis distances were performed using PERMANOVA. The bacterial core genus level analyses were conducted using DESeq2 and ALDEx2. These analyses suggested that hair sample concentrations of separate hormones, cortisol/cortisone and cortisol/dehydroepiandrosterone ratios were associated with various gut bacterial genera such as the Veillonella, the [Ruminococcus] torques group and [Eubacterium] hallii group, although multiple testing correction attenuated the p-values. Alpha or beta diversity was not linked with either steroid concentrations or ratios. These findings in toddlers suggest that long-term HPA axis activity may be related to genera abundancies but not to ecosystem-level measures in gut microbiota. The influence of these observed interrelations on later child health and development warrants further research.

Optimal sampling and analysis methods for clinical diagnostics of vaginal microbiome.

Next-generation sequencing-based microbiological analysis is a complex way to profile vaginal microbiome samples since each step affects the results gained. Methodologies for sample collection lack golden standards. We compared Puritan DNA/RNA swab (PS) and Copan FLOQ swab (CS) and provided consistent and reliable microbiome profiles analyzed by 16S rRNA gene sequencing. We collected two consecutive vaginal samples utilizing PS with room temperature storing and CS with instant freezing from 26 women. Variable region 4 of bacterial 16S rRNA gene was amplified with single PCR by custom-designed dual-indexed primers and sequenced with Illumina MiSeq system. Read quality control, operational taxonomic unit tables, and alpha and beta diversities analysis were performed, and community richness, diversity, and evenness were evaluated and compared between the two samplings and tests. Nineteen sample pairs produced detectable, intact DNA during the extraction protocol and/or further microbial profiles. Alpha bacterial diversity indices were independent on the collection protocol. No significant statistical differences were found in the measured beta diversity metrics between the collection methods. Of the women, 43% had Lactobacillus-dominated vaginal microbiome profile despite of collection method. Previously reported important vaginal microbiome phyla Actinobacteria, Bacteroidetes, Firmicutes, Fusobacteria, and Proteobacteria were present in the sample set although their relative abundances varied among individuals. PS and CS enable constant vaginal microbiota sampling. The PS method with no need for instant freezing is suitable for on-site collections at clinics. Furthermore, it seems to be possible to take two samples instead of one with constant microbiological results.

Appendiceal microbiome in uncomplicated and complicated acute appendicitis: A prospective cohort study.

BACKGROUND: Uncomplicated and complicated acute appendicitis seem to be two different forms of this common abdominal emergency. The contribution of appendiceal microbiota to appendicitis pathogenesis has been suggested, but differences between uncomplicated and complicated appendicitis are largely unknown. We compared the appendiceal microbiota in uncomplicated and complicated acute appendicitis. METHODS: This prospective single-center clinical cohort study was conducted as part of larger multicenter MAPPAC trial enrolling adult patients with computed tomography or clinically confirmed uncomplicated or complicated acute appendicitis. The microbial composition of the appendiceal lumen was determined using 16S rRNA gene amplicon sequencing. RESULTS: Between April 11, 2017, and March 29, 2019, 118 samples (41 uncomplicated and 77 complicated appendicitis) were available. After adjusting for age, sex, and BMI, alpha diversity in complicated appendicitis was higher (Shannon p = 0.011, Chao1 p = 0.006) compared to uncomplicated appendicitis. Microbial compositions were different between uncomplicated and complicated appendicitis (Bray-Curtis distance, P = 0.002). Species poor appendiceal microbiota composition with specific predominant bacteria was present in some patients regardless of appendicitis severity. CONCLUSION: Uncomplicated and complicated acute appendicitis have different appendiceal microbiome profiles further supporting the disconnection between these two different forms of acute appendicitis. STUDY REGISTRATION: ClinicalTrials.gov NCT03257423.

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

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

Gut Microbiota and Serum Metabolome in Elite Cross-Country Skiers: A Controlled Study.

Exercise has been shown to affect gut the microbiome and metabolic health, with athletes typically displaying a higher microbial diversity. However, research on the gut microbiota and systemic metabolism in elite athletes remains scarce. In this study, we compared the gut microbiota profiles and serum metabolome of national team cross-country skiers at the end of an exhausting training and competitive season to those of normally physically-active controls. The gut microbiota were analyzed using 16S rRNA amplicon sequencing. Serum metabolites were analyzed using nuclear magnetic resonance. Phylogenetic diversity and the abundance of several mucin-degrading gut microbial taxa, including Akkermansia, were lower in the athletes. The athletes had a healthier serum lipid profile than the controls, which was only partly explained by body mass index. Butyricicoccus associated positively with HDL cholesterol, HDL2 cholesterol and HDL particle size. The Ruminococcus torques group was less abundant in the athlete group and positively associated with total cholesterol and VLDL and LDL particles. We found the healthier lipid profile of elite athletes to co-occur with known health-beneficial gut microbes. Further studies should elucidate these links and whether athletes are prone to mucin depletion related microbial changes during the competitive season.

Distinct Diet-Microbiota-Metabolism Interactions in Overweight and Obese Pregnant Women: a Metagenomics Approach.

Diet and gut microbiota are known to modulate metabolic health. Our aim was to apply a metagenomics approach to investigate whether the diet-gut microbiota-metabolism and inflammation relationships differ in pregnant overweight and obese women. This cross-sectional study was conducted in overweight (n = 234) and obese (n = 152) women during early pregnancy. Dietary quality was measured by a validated index of diet quality (IDQ). Gut microbiota taxonomic composition and species diversity were assessed by metagenomic profiling (Illumina HiSeq platform). Markers for glucose metabolism (glucose, insulin) and low-grade inflammation (high sensitivity C-reactive protein [hsCRP], glycoprotein acetylation [GlycA]) were analyzed from blood samples. Higher IDQ scores were positively associated with a higher gut microbiota species diversity (r = 0.273, P = 0.007) in obese women, but not in overweight women. Community composition (beta diversity) was associated with the GlycA level in the overweight women (P = 0.04) but not in the obese. Further analysis at the species level revealed a positive association between the abundance of species Alistipes finegoldii and the GlycA level in overweight women (logfold change = 4.74, P = 0.04). This study has been registered at ClinicalTrials.gov under registration no. NCT01922791 (https://clinicaltrials.gov/ct2/show/NCT01922791). IMPORTANCE We observed partially distinct diet-gut microbiota-metabolism and inflammation responses in overweight and obese pregnant women. In overweight women, gut microbiota community composition and the relative abundance of A. finegoldii were associated with an inflammatory status. In obese women, a higher dietary quality was related to a higher gut microbiota diversity and a healthy inflammatory status.

Skin microbiota diversity among genetically unrelated individuals of Indian origin.

Background: Human skin harbors complex transient and resident microbial communities that show intra- & inter-individual variation due to various environmental and host-associated factors such as skin site, diet, age, gender, genetics, or the type and use of cosmetics. This variation remains largely uncharacterized in the Indian population; hence, the present study aims to characterize the variation in skin microbiota among individuals of Indian origin and quantify associations with age, diet, and geography. Methods: Axillary sweat samples from genetically unrelated individuals (N = 58) residing in the three geographical locations of Maharashtra, India, were collected using a sterile cotton swab. Bacterial DNA was extracted using a standard protocol and checked for quality. Variable regions (V3-V4) of the 16S rRNA gene were sequenced using the Illumina platform. We used standard methods from microbiota bioinformatics, including alpha and beta diversity, community typing, and differential abundance, to quantify the association of skin microbiota with age, diet, and geographical location. Results: Our study indicated the prevalence of phyla- Firmicutes, Proteobacteria, and Actinobacteria, consistent with previous reports on skin microbiota composition of the world population level. The alpha diversity (Shannon index) was significantly associated with the age group (Kruskal-Wallis test, p = 0.02), but not with geography (p = 0.62) or diet (p = 0.74). The overall skin microbiota community composition was significantly associated with geographical location based on Community State Types (CST) analysis and PERMANOVA (R2 = 0.07, p = 0.01). Differential abundance analysis at the genus level indicated a distinctively high abundance of Staphylococcus and Corynebacterium among individuals of the Pune district. Pseudomonas and Anaerococcus were abundant in individuals from Ahmednagar whereas, Paenibacillus, Geobacillus, Virgibacillus, Jeotgalicoccus, Pullulanibacillus, Delsulfosporomusa, Citinovibrio, and Calditerricola were abundant in individuals from Nashik district. Conclusion: Our work provides one of the first characterizations of skin microbiota variation in different sub-populations in India. The analysis quantifies the level of individuality, as contrasted to the other factors of age, geography, and diet, thus helping to evaluate the applicability of skin microbiota profiles as a potential biomarker to stratify individuals.

Towards standardized and reproducible research in skin microbiomes.

Skin is a complex organ serving a critical role as a barrier and mediator of interactions between the human body and its environment. Recent studies have uncovered how resident microbial communities play a significant role in maintaining the normal healthy function of the skin and the immune system. In turn, numerous host-associated and environmental factors influence these communities' composition and diversity across the cutaneous surface. In addition, specific compositional changes in skin microbiota have also been connected to the development of several chronic diseases. The current era of microbiome research is characterized by its reliance on large data sets of nucleotide sequences produced with high-throughput sequencing of sample-extracted DNA. These approaches have yielded new insights into many previously uncharacterized microbial communities. Application of standardized practices in the study of skin microbial communities could help us understand their complex structures, functional capacities, and health associations and increase the reproducibility of the research. Here, we overview the current research in human skin microbiomes and outline challenges specific to their study. Furthermore, we provide perspectives on recent advances in methods, analytical tools and applications of skin microbiomes in medicine and forensics.

Infant fecal microbiota composition and attention to emotional faces.

The gut microbiota has been suggested to influence neurodevelopment in rodents. Preliminary human studies have associated fecal microbiota composition with features of emotional and cognitive development as well as differences in thalamus-amygdala connectivity. Currently, microbiota-gut-brain axis studies cover heterogenous set of infant and child brain developmental phenotypes, while microbiota associations with more fine-grained aspects of brain development remain largely unknown. Here (N = 122, 53% boys), we investigated the associations between infant fecal microbiota composition and infant attention to emotional faces, as bias for faces is strong in infancy and deviations in early processing of emotional facial expressions may influence the trajectories of social-emotional development. The fecal microbiota composition was assessed at 2.5 months of age and analyzed with 16S rRNA gene sequencing. Attention to emotional faces was assessed with an age-appropriate face-distractor paradigm, using neutral, happy, fearful, and scrambled faces and salient distractors, at 8 months of age. We observed an association between a lower abundance of Bifidobacterium and a higher abundance of Clostridium with an increased "fear bias," that is, attention toward fearful versus happy/neutral faces. This data suggests an association between early microbiota and later fear bias, a well-established infant phenotype of emotionally directed attention. However, the clinical significance or causality of our findings remains to be assessed. (PsycInfo Database Record (c) 2022 APA, all rights reserved).

Gut microbiota diversity but not composition is related to saliva cortisol stress response at the age of 2.5 months.

Human brain and intestinal microbes reportedly maintain a constant bidirectional connection through diverse neural, endocrine, immune, and metabolic pathways. Increasing evidence indicates that this communication system, referred to as microbiota-gut-brain axis, enables the gut microbes to influence several aspects of brain function and behavior, including hypothalamic-pituitary-adrenal (HPA) axis stress responses, and on the other hand, stress can affect gut microbiota. However, the role of gut microbiota in the HPA axis functioning in humans remains to be specified especially in early life. This study aimed at identifying the potential link between the cortisol stress response and the gut microbiota at the age of 2.5 months. Fecal microbiota profiles were acquired by 16S rRNA gene sequencing, while salivary cortisol responses after an exposure to a mild acute stressor represented the HPA axis reactivity. We observed that a blunted cortisol stress response was weakly associated with a diverse gut microbiota diversity at the age of 2.5 months. Gut microbiota composition was not associated with cortisol stress responsiveness, but rather with covariates, i.e. factors that influence gut microbiota composition and colonization.LAY SUMMARYThis exploratory study aimed at identifying possible links between cortisol stress responses and fecal microbiota composition in early infancy. In a well-characterized study population of 2.5-month-old infants, we observed that an attenuated cortisol stress responsiveness after a mild stressor was weakly associated with a diverse fecal microbiota. Our results suggest that the gut microbiota composition is associated with environmental factors, such as delivery mode and number of siblings, rather than with cortisol stress responsiveness, in this age group.

Prebiotic Xylo-Oligosaccharides Ameliorate High-Fat-Diet-Induced Hepatic Steatosis in Rats.

Understanding the importance of the gut microbiota (GM) in non-alcoholic fatty liver disease (NAFLD) has raised the hope for therapeutic microbes. We have shown that high hepatic fat content associated with low abundance of Faecalibacterium prausnitzii in humans and, further, the administration of F. prausnitzii prevented NAFLD in mice. Here, we aimed at targeting F. prausnitzii by prebiotic xylo-oligosaccharides (XOS) to treat NAFLD. First, the effect of XOS on F. prausnitzii growth was assessed in vitro. Then, XOS was supplemented or not with high (HFD, 60% of energy from fat) or low (LFD) fat diet for 12 weeks in Wistar rats (n = 10/group). XOS increased F. prausnitzii growth, having only a minor impact on the GM composition. When supplemented with HFD, XOS ameliorated hepatic steatosis. The underlying mechanisms involved enhanced hepatic ß-oxidation and mitochondrial respiration. Nuclear magnetic resonance (1H-NMR) analysis of cecal metabolites showed that, compared to the HFD, the LFD group had a healthier cecal short-chain fatty acid profile and on the HFD, XOS reduced cecal isovalerate and tyrosine, metabolites previously linked to NAFLD. Cecal branched-chain fatty acids associated positively and butyrate negatively with hepatic triglycerides. In conclusion, XOS supplementation can ameliorate NAFLD by improving hepatic oxidative metabolism and affecting GM.

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

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

Prospective multicentre cohort trial on acute appendicitis and microbiota, aetiology and effects of antimicrobial treatment: study protocol for the MAPPAC (Microbiology APPendicitis ACuta) trial.

INTRODUCTION: Based on the epidemiological and clinical data, acute appendicitis can present either as uncomplicated or complicated. The aetiology of these different appendicitis forms remains unknown. Antibiotic therapy has been shown to be safe, efficient and cost-effective for CT-confirmed uncomplicated acute appendicitis. Despite appendicitis being one of the most common surgical emergencies, there are very few reports on appendicitis aetiology and pathophysiology focusing on the differences between uncomplicated and complicated appendicitis. Microbiology APPendicitis ACuta (MAPPAC) trial aims to evaluate these microbiological and immunological aspects including immune response in the aetiology of these different forms also assessing both antibiotics non-responders and appendicitis recurrence. In addition, MAPPAC aims to determine antibiotic and placebo effects on gut microbiota composition and antimicrobial resistance. METHODS AND ANALYSIS: MAPPAC is a prospective clinical trial with both single-centre and multicentre arm conducted in close synergy with concurrent trials APPendicitis ACuta II (APPAC II) (per oral (p.o.) vs intravenous+p.o. antibiotics, NCT03236961) and APPAC III (double-blind trial placebo vs antibiotics, NCT03234296) randomised clinical trials. Based on the enrolment for these trials, patients with CT-confirmed uncomplicated acute appendicitis are recruited also to the MAPPAC study. In addition to these conservatively treated randomised patients with uncomplicated acute appendicitis, MAPPAC will recruit patients with uncomplicated and complicated appendicitis undergoing appendectomy. Rectal and appendiceal swabs, appendicolith, faecal and serum samples, appendiceal biopsies and clinical data are collected during the hospital stay for microbiological and immunological analyses in both study arms with the longitudinal study arm collecting faecal samples also during follow-up up to 12 months after appendicitis treatment. ETHICS AND DISSEMINATION: This study has been approved by the Ethics Committee of the Hospital District of Southwest Finland (Turku University Hospital, approval number ATMK:142/1800/2016) and the Finnish Medicines Agency. Results of the trial will be published in peer-reviewed journals. TRIAL REGISTRATION NUMBER: NCT03257423.

Gut microbiota composition is associated with temperament traits in infants.

BACKGROUND: One of the key behavioral phenotypes in infancy are different temperament traits, and certain early life temperament traits have been shown to precede later mental health problems. Differences in the gut microbiota composition (GMC) have been suggested to link with neurodevelopment. For example, toddler temperament traits have been found to associate with differences in GMC; however, studies in infants are lacking although infancy is a rapid period of neurodevelopment as well as GM development. Thus, we aimed to investigate association between infant GMC and temperament. METHODS: The study population (n = 301, 53% boys) was drawn from the FinnBrain Birth Cohort Study. Stool samples were collected from the 2.5-month-old infants and sequenced with 16S Illumina MiSeq platform. GMC taxonomic composition (at Genus and OTU level), observed sample clusters, diversity and richness were investigated in relation to the maternal reports of Infant Behavior Questionnaire -Revised (IBQ-R) at the age of 6 months. RESULTS: Three sample clusters (Bifidobacterium/Enterobacteriaceae, Bacteroides, V. Dispar) based on GMC were identified, of which Bifidobacterium/Enterobacteriaceae-cluster presented with higher scores on the IBQ-R main dimension regulation and its subscale duration of orienting compared to Bacteroides-cluster. The clusters associated with temperament in a sex-dependent manner. The IBQ-R main dimension surgency (positive emotionality) was associated positively both with genus Bifidobacterium and Streptococcus. Alpha diversity had a negative association with negative emotionality and fear reactivity. CONCLUSION: This is the first study demonstrating associations, but not causal connections, between GMC and temperament in young infants in a prospective design.

Prevalence of Complications Leading to a Health Care Contact After Transrectal Prostate Biopsies: A Prospective, Controlled, Multicenter Study Based on a Selected Study Cohort.

BACKGROUND: Transrectal ultrasound-guided prostate biopsy (TRUS-Bx) is typically considered a safe procedure. However, infectious complications have been increasing. OBJECTIVE: To determine the contemporary rate of biopsy-related infectious and noninfectious complications after TRUS-Bx, and identify potential risk factors associated with the complications. DESIGN, SETTING, AND PARTICIPANTS: This was a prospective multicenter study and a substudy of a trial investigating the role of magnetic resonance imaging (MRI) in prostate cancer diagnosis (multi-IMPROD, NCT02241122). INTERVENTION: TRUS-Bx was performed for all patients included in the study. Ciprofloxacin, levofloxacin, or fosfomycin was administered for antibiotic prophylaxis. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: On inclusion, patients completed a detailed questionnaire and underwent MRI scanning. Antibiotic prophylaxis was prospectively recorded. After collection of a rectal swab, TRUS-Bx (total of 14-18 biopsy cores) was performed and. The rectal swabs were cultured and the antimicrobial susceptibility profile of Escherichia coli strains was analyzed. Biopsy complications leading to a visit to a health care unit were recorded and potential risk factors for complications were analyzed. RESULTS AND LIMITATIONS: Twelve of the 294 patients (4.1%) had a biopsy-related complication, of which two (0.7%) were infectious and managed in the outpatient setting. Some 11% of the patients had an E. coli strain resistant to the prophylactic antibiotic administered. CONCLUSIONS: The risk of an infectious or noninfectious complication after TRUS-Bx is very low, although the FQ resistance rate in the study population was significant. Accordingly, the present TRUS-Bx procedure and antibiotic prophylaxis are efficient in guarding against biopsy complications, but regional resistance rates may affect the generalizability of the results. PATIENT SUMMARY: We examined the rate of complications after prostate biopsies in 294 patients. The risk of having a biopsy-related complication was low (4.1%). The rate of infectious complications was reasonably low (0.7%) although antibiotic resistance to the prophylactic antibiotic regimen was significant (11%).