Lactococcus cell envelope proteases enable lactococcal growth in minimal growth media supplemented with high molecular weight proteins of plant and animal origin.

Lactic acid bacteria (LAB) have evolved into fastidious microorganisms that require amino acids from environmental sources. Some LAB have cell envelope proteases (CEPs) that drive the proteolysis of high molecular weight proteins like casein in milk. CEP activity is typically studied using casein as the predominant substrate, even though CEPs can hydrolyze other protein sources. Plant protein hydrolysis by LAB has rarely been connected to the activity of specific CEPs. This study aims to show the activity of individual CEPs using LAB growth in a minimal growth medium supplemented with high molecular weight casein or potato proteins. Using Lactococcus cremoris MG1363 as isogenic background to express CEPs, we demonstrate that CEP activity is directly related to growth in the protein-supplemented minimal growth media. Proteolysis is analyzed based on the amino acid release, allowing a comparison of CEP activities and analysis of amino acid utilization by L. cremoris MG1363. This approach provides a basis to analyze CEP activity on plant-based protein substrates as casein alternatives and to compare activity of CEP homologs.

Delivery of E. coli Nissle to the mouse gut by mucoadhesive microcontainers does not improve its competitive ability against strains linked to ulcerative colitis.

For patients with ulcerative colitis (UC), administration of the probiotic E. coli Nissle (EcN) holds promise for alleviation of disease symptoms. The mechanisms are unclear, but it has been hypothesised that a capacity of the probiotic to outcompete potentially detrimental UC-associated E. coli strains plays an important role. However, this could previously not be confirmed in a mouse model of competition between EcN and two UC-associated strains, as reported by Petersen et al. 2011. In the present study, we re-evaluated the idea, hypothesising that delivery of EcN by a micro device dosing system (microcontainers), designed for delivery into the intestinal mucus, could support colonisation and confer a competition advantage compared to classical oral dosing. Six groups of mice were pre-colonised with one of two UC-associated E. coli strains followed by oral delivery of EcN, either in capsules containing microcontainers with freeze-dried EcN powder, capsules containing freeze-dried EcN powder, or as a fresh sucrose suspension. Co-colonisation between the probiotic and the disease-associated strains was observed regardless of dosing method, and no competition advantages linked to microcontainer delivery were identified within this setup. Other approaches are thus needed if the competitive capacity of EcN in the gut should be improved.

Antibiotic induced restructuring of the gut microbiota does not affect oral uptake and accumulation of perfluorooctane sulfonic acid (PFOS) in rats.

Perfluorooctane sulfonic acid (PFOS) is a manmade legacy compound belonging to the group of persistent per- and polyfluorinated substances (PFAS). While many adverse health effects of PFOS have been identified, knowledge about its effect on the intestinal microbiota is scarce. The microbial community inhabiting the gut of mammals plays an important role in health, for instance by affecting the uptake, excretion, and bioavailability of some xenobiotic toxicants. Here, we investigated (i) the effect of vancomycin-mediated microbiota modulation on the uptake of PFOS in adult Sprague-Dawley rats, and (ii) the effects of PFOS exposure on the rat microbiota composition. Four groups of twelve rats were exposed daily for 7 days with either 3 mg/kg PFOS plus 8 mg/kg vancomycin, only PFOS, only vancomycin, or a corn oil control. Vancomycin-induced modulation of the gut microbiota composition did not affect uptake of branched and linear PFOS over a period of 7 days, measured in serum samples. 16S rRNA amplicon sequencing of faecal and intestinal samples revealed that vancomycin treatment lowered microbial alpha-diversity, while PFOS increased the microbial diversity in vancomycin-treated as well as in non-antibiotic treated animals, possibly because an observed decrease in the Enterobacteriaceae abundance allows other microbial species to propagate. Colonic short-chain fatty acids were significantly lower in vancomycin-treated animals but remained unaffected by PFOS. Our results suggest that PFOS exposure may disturb the intestinal microbiota, but that antibiotic-induced modulation of the intestinal ecosystem does not affect systemic uptake of PFOS in rats.

Oropharyngeal Microbiota Clusters in Children with Asthma or Wheeze Associate with Allergy, Blood Transcriptomic Immune Pathways, and Exacerbation Risk.

Rationale: Children with preschool wheezing or school-age asthma are reported to have airway microbial imbalances. Objectives: To identify clusters in children with asthma or wheezing using oropharyngeal microbiota profiles. Methods: Oropharyngeal swabs from the U-BIOPRED (Unbiased Biomarkers for the Prediction of Respiratory Disease Outcomes) pediatric asthma or wheezing cohort were characterized using 16S ribosomal RNA gene sequencing, and unsupervised hierarchical clustering was performed on the Bray-Curtis ß-diversity. Enrichment scores of the Molecular Signatures Database hallmark gene sets were computed from the blood transcriptome using gene set variation analysis. Children with severe asthma or severe wheezing were followed up for 12-18 months, with assessment of the frequency of exacerbations. Measurements and Main Results: Oropharyngeal samples from 241 children (age range, 1-17 years; 40% female) revealed four taxa-driven clusters dominated by Streptococcus, Veillonella, Rothia, and Haemophilus. The clusters showed significant differences in atopic dermatitis, grass pollen sensitization, FEV1% predicted after salbutamol, and annual asthma exacerbation frequency during follow-up. The Veillonella cluster was the most allergic and included the highest percentage of children with two or more exacerbations per year during follow-up. The oropharyngeal clusters were different in the enrichment scores of TGF-ß (transforming growth factor-ß) (highest in the Veillonella cluster) and Wnt/ß-catenin signaling (highest in the Haemophilus cluster) transcriptomic pathways in blood (all q values <0.05). Conclusions: Analysis of the oropharyngeal microbiota of children with asthma or wheezing identified four clusters with distinct clinical characteristics (phenotypes) that associate with risk for exacerbation and transcriptomic pathways involved in airway remodeling. This suggests that further exploration of the oropharyngeal microbiota may lead to novel pathophysiologic insights and potentially new treatment approaches.

Warmer winters result in reshaping of the European beech forest soil microbiome (bacteria, archaea and fungi)-With potential implications for ecosystem functioning.

In temperate regions, climate warming alters temperature and precipitation regimes. During winter, a decline in insulating snow cover changes the soil environment, where especially frost exposure can have severe implications for soil microorganisms and subsequently for soil nutrient dynamics. Here, we investigated winter climate change responses in European beech forests soil microbiome. Nine study sites with each three treatments (snow exclusion, insolation, and ambient) were investigated. Long-term adaptation to average climate was explored by comparing across sites. Triplicated treatment plots were used to evaluate short-term (one single winter) responses. Community profiles of bacteria, archaea and fungi were created using amplicon sequencing. Correlations between the microbiome, vegetation and soil physicochemical properties were found. We identify core members of the forest-microbiome and link them to key processes, for example, mycorrhizal symbiont and specialized beech wood degraders (fungi) and nitrogen cycling (bacteria, archaea). For bacteria, the shift of the microbiome composition due to short-term soil temperature manipulations in winter was similar to the community differences observed between long-term relatively cold to warm conditions. The results suggest a strong link between the changes in the microbiomes and changes in environmental processes, for example, nitrogen dynamics, driven by variations in winter climate.

Impact of intensive lifestyle intervention on gut microbiota composition in type 2 diabetes: a post-hoc analysis of a randomized clinical trial.

Type 2 diabetes (T2D) management is based on combined pharmacological and lifestyle intervention approaches. While their clinical benefits are well studied, less is known about their effects on the gut microbiota. We aimed to investigate if an intensive lifestyle intervention combined with conventional standard care leads to a different gut microbiota composition compared to standard care alone treatment in individuals with T2D, and if gut microbiota is associated with the clinical benefits of the treatments. Ninety-eight individuals with T2D were randomized to either an intensive lifestyle intervention combined with standard care group (N = 64), or standard care alone group (N = 34) for 12 months. All individuals received standardized, blinded, target-driven medical therapy, and individual counseling. The lifestyle intervention group moreover received intensified physical training and dietary plans. Clinical characteristics and fecal samples were collected at baseline, 3-, 6-, 9-, and 12-month follow-up. The gut microbiota was profiled with 16S rRNA gene amplicon sequencing. There were no statistical differences in the change of gut microbiota composition between treatments after 12 months, except minor and transient differences at month 3. The shift in gut microbiota alpha diversity at all time windows did not correlate with the change in clinical characteristics, and the gut microbiota did not mediate the treatment effect on clinical characteristics. The clinical benefits of intensive lifestyle and/or pharmacological interventions in T2D are unlikely to be explained by, or causally related to, changes in the gut microbiota composition.

The effect of early probiotic exposure on the preterm infant gut microbiome development.

Premature birth, especially if born before week 32 of gestation, is associated with increased risk of neonatal morbidity and mortality. Prophylactic use of probiotics has been suggested to protect preterm infants via supporting a healthy gut microbiota (GM) development, but the suggested strains and doses vary between studies. In this study, we profiled the GM of 5, 10 and 30-day fecal samples from two cohorts of preterm neonates (born <30 weeks of gestation) recruited in the same neonatal intensive care unit. One cohort (n = 165) was recruited from September 2006 to January 2009 before probiotics were introduced in the clinic. The second cohort (n = 87) was recruited from May 2010 to October 2011 after introducing Lacticaseibacillus rhamnosus GG and Bifidobacterium animalis ssp. lactis BB-12 supplementation policy. Through V3-V4 region 16S rRNA gene amplicon sequencing, a distinct increase of L. rhamnosus and B. animalis was found in the fecal samples of neonates supplemented with probiotics. During the first 30 days of life, the preterm GM went through similarly patterned progression of bacterial populations. Staphylococcus and Weissella dominated in early samples, but was gradually overtaken by Veillonella, Enterococcus and Enterobacteriaceae. Probiotic supplementation was associated with pronounced reduction of Weissella, Veillonella spp. and the opportunistic pathogen Klebsiella. Potential nosocomial pathogens Citrobacter and Chryseobacterium species also gradually phased out. In conclusion, probiotic supplementation to preterm neonates affected gut colonization by certain bacteria, but did not change the overall longitudinal bacterial progression in the neonatal period.Abbreviations: GM: Gut microbiota; ASV: Amplicon sequence variant; NEC: Necrotizing enterocolitis; DOL: Days of life; NICU: Neonatal intensive care unit; ESPGHAN: European Society for Pediatric Gastroenterology, Hepatology and Nutrition; Db-RDA: Distance-based redundancy analysis; PERMANOVA: Permutational multivariate analysis of variance; ANCOM: Analysis of compositions of microbiomes; LGG: Lacticaseibacillus (former Lactobacillus) rhamnosus GG; BB-12: Bifidobacterium animalis ssp. lactis BB-12; DGGE: Denaturing Gradient Gel Electrophoresis.

The Airway Microbiota Modulates Effect of Azithromycin Treatment for Episodes of Recurrent Asthma-like Symptoms in Preschool Children: A Randomized Clinical Trial.

Rationale: Childhood asthma is often preceded by recurrent episodes of asthma-like symptoms, which can be triggered by both viral and bacterial agents. Recent randomized controlled trials have shown that azithromycin treatment reduces episode duration and severity through yet undefined mechanisms. Objectives: To study the influence of the airway microbiota on the effect of azithromycin treatment during acute episodes of asthma-like symptoms. Methods: Children from the COPSAC2010 (Copenhagen Prospective Studies on Asthma in Childhood 2010) cohort with recurrent asthma-like symptoms aged 12-36 months were randomized during acute episodes to azithromycin or placebo as previously reported. Before randomization, hypopharyngeal aspirates were collected and examined by 16S ribosomal RNA gene amplicon sequencing. Measurements and Main Results: In 139 airway samples from 68 children, episode duration after randomization was associated with microbiota richness (7.5% increased duration per 10 additional operational taxonomic units [OTUs]; 95% confidence interval, 1-14%; P = 0.025), with 15 individual OTUs (including several Neisseria and Veillonella), and with microbial pneumotypes defined from weighted UniFrac distances (longest durations in a Neisseria-dominated pneumotype). Microbiota richness before treatment increased the effect of azithromycin by 10% per 10 additional OTUs, and more OTUs were positively versus negatively associated with an increased azithromycin effect (82 vs. 58; P = 0.0032). Furthermore, effect modification of azithromycin was found for five individual OTUs (three OTUs increased and two OTUs decreased the effect; q < 0.05). Conclusions: The airway microbiota in acute episodes of asthma-like symptoms is associated with episode duration and modifies the effect of azithromycin treatment of the episodes in preschool children with recurrent asthma-like symptoms. Clinical trial registered with www.clinicaltrials.gov (NCT01233297).

Modeling transfer of vaginal microbiota from mother to infant in early life.

Early-life microbiota has been linked to the development of chronic inflammatory diseases. It has been hypothesized that maternal vaginal microbiota is an important initial seeding source and therefore might have lifelong effects on disease risk. To understand maternal vaginal microbiota's role in seeding the child's microbiota and the extent of delivery mode-dependent transmission, we studied 665 mother-child dyads from the COPSAC2010 cohort. The maternal vaginal microbiota was evaluated twice in the third trimester and compared with the children's fecal (at 1 week, 1 month, and 1 year of age) and airway microbiota (at 1 week, 1 month, and 3 months). Based on the concept of weighted transfer ratios (WTRs), we have identified bacterial orders for which the WTR displays patterns indicate persistent or transient transfer from the maternal vaginal microbiome, as well as orders that are shared at later time points independent of delivery mode, indicating a common reservoir.

The T-shirt microbiome is distinct between individuals and shaped by washing and fabric type.

Activity of the microbial population in clothing causes unpleasant odor and textile deterioration. However, little is known about how the textile microbial community is shaped. In this study, we developed a method for extracting DNA from small amounts of detergent-washed clothing, and applied it to both worn and unworn, washed and unwashed cotton and polyester samples of the axillary region of T-shirts from 10 male subjects. The combined application of 16S rRNA gene amplicon sequencing and quantitative PCR allowed us to estimate the absolute abundances of bacteria in the samples. We found that the T-shirt microbiome was highly individual, both in composition, diversity and microbial biomass. Fabric type was influential where Acinetobacter was more abundant in cotton. Intriguingly, unworn cotton T-shirts had a native microbiome dominated by Acinetobacter, whereas unworn polyester had no detectable bacterial microbiome. The native textile microbiome did not seem to have any effect on the microbial composition emerging from wearing the garment. Surprisingly, washing in mild detergent had only minor effects on the composition and biomass of the microbial community, and only few Amplicon Sequence Variants (ASV)s were found to decrease in abundance after washing. Individual variations between test subjects shaped the microbial community more than the type of fabric or wash with detergent. The individuality of T-shirt microbiomes and specificity of the washing procedure suggests that personalized laundry regimes could be applied to increase efficient removal of undesired bacteria.

The microbiome of captive hamadryas baboons.

BACKGROUND: The hamadryas baboon (Papio hamadryas) is a highly social primate that lives in complex multilevel societies exhibiting a wide range of group behaviors akin to humans. In contrast to the widely studied human microbiome, there is a paucity of information on the host-associated microbiomes of nonhuman primates (NHPs). Here, our goal was to understand the microbial composition throughout different body sites of cohabiting baboons. RESULTS: We analyzed 170 oral, oropharyngeal, cervical, uterine, vaginal, nasal and rectal samples from 16 hamadryas baboons via 16S rRNA gene sequencing. Additionally, raw Miseq sequencing data from 1041 comparable publicly available samples from the human oral cavity, gut and vagina were reanalyzed using the same pipeline. We compared the baboon and human microbiome of the oral cavity, gut and vagina, showing that the baboon microbiome is distinct from the human. Baboon cohabitants share similar microbial profiles in their cervix, uterus, vagina, and gut. The oral cavity, gut and vagina shared more bacterial amplicon sequence variants (ASVs) in group living baboons than in humans. The shared ASVs had significantly positive correlations between most body sites, suggesting a potential bacterial exchange throughout the body. No significant differences in gut microbiome composition were detected within the maternity line and between maternity lines, suggesting that the offspring gut microbiota is shaped primarily through bacterial exchange among cohabitants. Finally, Lactobacillus was not so predominant in baboon vagina as in the human vagina but was the most abundant genus in the baboon gut. CONCLUSIONS: This study is the first to provide comprehensive analyses of the baboon microbiota across different body sites. We contrast this to human body sites and find substantially different microbiomes. This group of cohabitating baboons generally showed higher microbial diversity and remarkable similarities between body sites than were observed in humans. These data and findings from one group of baboons can form the basis of future microbiome studies in baboons and be used as a reference in research where the microbiome is expected to impact human modeling with baboons.

Stability and resilience of the intestinal microbiota in children in daycare - a 12 month cohort study.

BACKGROUND: We performed a 12-month cohort study of the stability and resilience of the intestinal microbiota of healthy children in daycare in Denmark in relation to diarrheal events and exposure to known risk factors for gastrointestinal health such as travelling and antibiotic use. In addition, we analyzed how gut microbiota recover from such exposures. RESULTS: We monitored 32 children in daycare aged 1-6 years. Fecal samples were submitted every second month during a one-year observational period. Information regarding exposures and diarrheal episodes was obtained through questionnaires. Bacterial communities were identified using 16S rRNA gene sequencing. The core microbiota (mean abundance > 95%) dominated the intestinal microbiota, and none of the tested exposures (diarrheal events, travel, antibiotic use) were associated with decreases in the relative abundance of the core microbiota. Samples exhibited lower intra-individual variation than inter-individual variation. Half of all the variation between samples was explained by which child a sample originated from. Age explained 7.6-9.6% of the variation, while traveling, diarrheal events, and antibiotic use explained minor parts of the beta diversity. We found an age-dependent increase of alpha diversity in children aged 1-3 years, and while diarrheal events caused a decrease in alpha diversity, a recovery time of 40-45 days was observed. Among children having had a diarrheal event, we observed a 10x higher relative abundance of Prevotella. After travelling, a higher abundance of two Bacteroides species and 40% less Lachnospiraceae were seen. Antibiotic use did not correlate with changes in the abundance of any bacteria. CONCLUSION: We present data showing that Danish children in daycare have stable intestinal microbiota, resilient to the exposures investigated. An early age-dependent increase in the diversity was demonstrated. Diarrheal episodes decreased alpha diversity with an estimated recovery time of 40-45 days.

Short- and long-term impacts of azithromycin treatment on the gut microbiota in children: A double-blind, randomized, placebo-controlled trial.

BACKGROUND: Macrolides are commonly prescribed for respiratory infections and asthma-like episodes in children. While their clinical benefits have been proved, concerns regarding the side-effects of their therapeutic use have been raised. Here we assess the short- and long-term impacts of azithromycin on the gut microbiota of young children. METHODS: We performed a randomized, double-blind, placebo-controlled trial in a group of children aged 12-36 months, diagnosed with recurrent asthma-like symptoms from the COPSAC2010 cohort. Each acute asthma-like episode was randomized to a 3-day course of azithromycin oral solution of 10 mg/kg per day or placebo. Azithromycin reduced episode duration by half, which was the primary end-point and reported previously. The assessment of gut microbiota after treatment was the secondary end-point and reported in this study. Fecal samples were collected 14 days after randomization (N = 59, short-term) and again at age 4 years (N = 49, long-term, of whom N = 18 were placebo treated) and investigated by 16S rRNA gene amplicon sequencing. FINDINGS: Short-term, azithromycin caused a 23% reduction in observed richness and 13% reduction in Shannon diversity. Microbiota composition was shifted primarily in the Actinobacteria phylum, especially a reduction of abundance in the genus Bifidobacterium. Long-term (13-39 months after treatment), we did not observe any differences between the azithromycin and placebo recipients in their gut microbiota composition. INTERPRETATION: Azithromycin treatment induced a perturbation in the gut microbiota 14 days after randomization but did not have long-lasting effects on the gut microbiota composition. However, it should be noted that our analyses included a limited number of fecal samples for the placebo treated group at age 4 years. FUND: Lundbeck Foundation, Danish Ministry of Health, Danish Council for Strategic Research, Capital Region Research Foundation, China Scholarship Council.

Enrichment and characterization of an environmental microbial consortium displaying efficient keratinolytic activity.

Keratin refers to a group of insoluble and recalcitrant protein materials. Slaughterhouses produce large amount of keratinous byproducts, which are either disposed or poorly valorized through costly thermochemical processes for animal feed formulation. Learning from nature, keratinolytic microbial consortia stand as a cost-efficient and environmental friendly way to valorize this recalcitrant resource. Directed selection was applied to enrich soil-born microbial consortia, using sequential batch cultivations in keratin medium, while measuring enzymes activity and monitoring consortia compositions via 16S rRNA gene amplicon sequencing. A promising microbial consortium KMCG6, featuring mainly members of Bacteroidetes and Proteobacteria, was obtained. It possessed keratinolytic activity with <25% residual substrate remaining, which also displayed a high degradation reproducibility level after long-term cryopreservation. This work represents an advance in the field of α-keratin degradation with potential for practical applications.

The developing hypopharyngeal microbiota in early life.

BACKGROUND: The airways of healthy humans harbor a distinct microbial community. Perturbations in the microbial community have been associated with disease, yet little is known about the formation and development of a healthy airway microbiota in early life. Our goal was to understand the establishment of the airway microbiota within the first 3 months of life. We investigated the hypopharyngeal microbiota in the unselected COPSAC2010 cohort of 700 infants, using 16S rRNA gene sequencing of hypopharyngeal aspirates from 1 week, 1 month, and 3 months of age. RESULTS: Our analysis shows that majority of the hypopharyngeal microbiota of healthy infants belong to each individual's core microbiota and we demonstrate five distinct community pneumotypes. Four of these pneumotypes are dominated by the genera Staphylococcus, Streptococcus, Moraxella, and Corynebacterium, respectively. Furthermore, we show temporal pneumotype changes suggesting a rapid development towards maturation of the hypopharyngeal microbiota and a significant effect from older siblings. Despite an overall common trajectory towards maturation, individual infants' microbiota are more similar to their own, than to others, over time. CONCLUSIONS: Our findings demonstrate a consolidation of the population of indigenous bacteria in healthy airways and indicate distinct trajectories in the early development of the hypopharyngeal microbiota.