Complete Genome Sequence of Lactobacillus johnsonii MR1, Isolated from a BALB/c Mouse Cecum.

Lactobacillus johnsonii strain MR1, which was isolated from the cecum of a BALB/cJ mouse in an airway allergy model, can decrease allergic airway inflammation in the model upon oral administration. Long-read sequencing of this isolate, which was performed using a MinION sequencer, yielded a single, closed genome of 1,953,837 bp, with a GC content of 34.67%.

Crohn's disease therapeutic dietary intervention (CD-TDI): study protocol for a randomised controlled trial.

INTRODUCTION: Dietary patterns that might induce remission in patients with active Crohn's disease (CD) are of interest to patients, but studies are limited in the published literature. We aim to explore the efficacy of the CD therapeutic dietary intervention (CD-TDI), a novel dietary approach developed from best practices and current evidence, to induce clinical and biomarker remission in adult patients with active CD. METHODS AND ANALYSIS: This study is a 13-week, multicentre, randomised controlled trial in patients with mild-to-moderate active CD at baseline. One hundred and two patients will be block randomised, by sex, 2:1 to the intervention (CD-TDI) or conventional management. Coprimary outcomes are clinical and biomarker remission, defined as a Harvey Bradshaw Index of <5 and a faecal calprotectin of <250 µg/g, respectively.Secondary outcomes include gut microbiota diversity and composition, faecal short-chain fatty acids, regulatory macrophage function, serum and faecal metabolomics, C reactive protein, peripheral blood mononuclear cell gene expression profiles, quality of life, sedentary time and physical activity at 7 and/or 13 weeks. Predictive models of clinical response to a CD-TDI will be investigated. ETHICS AND DISSEMINATION: The research protocol was approved by the Conjoint Health Research Ethics Board at the University of Calgary (REB19-0402) and the Health Research Ethics Board-Biomedical Panel at the University of Alberta (Pro00090772). Study findings will be presented at national and international conferences, submitted for publication in abstracts and manuscripts, shared on social media and disseminated through patient-education materials. TRIAL REGISTRATION NUMBER: NCT04596566.

Antibiotic-induced acceleration of type 1 diabetes alters maturation of innate intestinal immunity.

The early-life intestinal microbiota plays a key role in shaping host immune system development. We found that a single early-life antibiotic course (1PAT) accelerated type 1 diabetes (T1D) development in male NOD mice. The single course had deep and persistent effects on the intestinal microbiome, leading to altered cecal, hepatic, and serum metabolites. The exposure elicited sex-specific effects on chromatin states in the ileum and liver and perturbed ileal gene expression, altering normal maturational patterns. The global signature changes included specific genes controlling both innate and adaptive immunity. Microbiome analysis revealed four taxa each that potentially protect against or accelerate T1D onset, that were linked in a network model to specific differences in ileal gene expression. This simplified animal model reveals multiple potential pathways to understand pathogenesis by which early-life gut microbiome perturbations alter a global suite of intestinal responses, contributing to the accelerated and enhanced T1D development.

Delayed gut microbiota development in high-risk for asthma infants is temporarily modifiable by Lactobacillus supplementation.

Gut microbiota dysbiosis and metabolic dysfunction in infancy precedes childhood atopy and asthma development. Here we examined gut microbiota maturation over the first year of life in infants at high risk for asthma (HR), and whether it is modifiable by early-life Lactobacillus supplementation. We performed a longitudinal comparison of stool samples collected from HR infants randomized to daily oral Lactobacillus rhamnosus GG (HRLGG) or placebo (HRP) for 6 months, and healthy (HC) infants. Meconium microbiota of HRP participants is distinct, follows a delayed developmental trajectory, and is primarily glycolytic and depleted of a range of anti-inflammatory lipids at 6 months of age. These deficits are partly rescued in HRLGG infants, but this effect was lost at 12 months of age, 6 months after cessation of supplementation. Thus we show that early-life gut microbial development is distinct, but plastic, in HR infants. Our findings offer a novel strategy for early-life preventative interventions.

Pheromone killing of multidrug-resistant Enterococcus faecalis V583 by native commensal strains.

Multidrug-resistant Enterococcus faecalis possess numerous mobile elements that encode virulence and antibiotic resistance traits as well as new metabolic pathways, often constituting over one-quarter of the genome. It was of interest to determine how this large accretion of mobile elements affects competitive growth in the gastrointestinal (GI) tract consortium. We unexpectedly observed that the prototype clinical isolate strain V583 was actively killed by GI tract flora, whereas commensal enterococci flourished. It was found that killing of V583 resulted from lethal cross-talk between accumulated mobile elements and that this cross-talk was induced by a heptapeptide pheromone produced by native E. faecalis present in the fecal consortium. These results highlight two important aspects of the evolution of multidrug-resistant enterococci: (i) the accretion of mobile elements in E. faecalis V583 renders it incompatible with commensal strains, and (ii) because of this incompatibility, multidrug-resistant strains sharing features found in V583 cannot coexist with commensal strains. The accumulation of mobile elements in hospital isolates of enterococci can include those that are inherently incompatible with native flora, highlighting the importance of maintaining commensal populations as means of preventing colonization and subsequent infection by multidrug-resistant strains.

Breast-fed and bottle-fed infant rhesus macaques develop distinct gut microbiotas and immune systems.

Diet has a strong influence on the intestinal microbiota in both humans and animal models. It is well established that microbial colonization is required for normal development of the immune system and that specific microbial constituents prompt the differentiation or expansion of certain immune cell subsets. Nonetheless, it has been unclear how profoundly diet might shape the primate immune system or how durable the influence might be. We show that breast-fed and bottle-fed infant rhesus macaques develop markedly different immune systems, which remain different 6 months after weaning when the animals begin receiving identical diets. In particular, breast-fed infants develop robust populations of memory T cells as well as T helper 17 (TH17) cells within the memory pool, whereas bottle-fed infants do not. These findings may partly explain the variation in human susceptibility to conditions with an immune basis, as well as the variable protection against certain infectious diseases.

Effects of early-life exposure to allergens and bacteria on recurrent wheeze and atopy in urban children.

BACKGROUND: Wheezing illnesses cause major morbidity in infants and are frequent precursors to asthma. OBJECTIVE: We sought to examine environmental factors associated with recurrent wheezing in inner-city environments. METHODS: The Urban Environment and Childhood Asthma study examined a birth cohort at high risk for asthma (n = 560) in Baltimore, Boston, New York, and St Louis. Environmental assessments included allergen exposure and, in a nested case-control study of 104 children, the bacterial content of house dust collected in the first year of life. Associations were determined among environmental factors, aeroallergen sensitization, and recurrent wheezing at age 3 years. RESULTS: Cumulative allergen exposure over the first 3 years was associated with allergic sensitization, and sensitization at age 3 years was related to recurrent wheeze. In contrast, first-year exposure to cockroach, mouse, and cat allergens was negatively associated with recurrent wheeze (odds ratio, 0.60, 0.65, and 0.75, respectively; P ≤ .01). Differences in house dust bacterial content in the first year, especially reduced exposure to specific Firmicutes and Bacteriodetes, was associated with atopy and atopic wheeze. Exposure to high levels of both allergens and this subset of bacteria in the first year of life was most common among children without atopy or wheeze. CONCLUSIONS: In inner-city environments children with the highest exposure to specific allergens and bacteria during their first year were least likely to have recurrent wheeze and allergic sensitization. These findings suggest that concomitant exposure to high levels of certain allergens and bacteria in early life might be beneficial and suggest new preventive strategies for wheezing and allergic diseases.

House dust exposure mediates gut microbiome Lactobacillus enrichment and airway immune defense against allergens and virus infection.

Exposure to dogs in early infancy has been shown to reduce the risk of childhood allergic disease development, and dog ownership is associated with a distinct house dust microbial exposure. Here, we demonstrate, using murine models, that exposure of mice to dog-associated house dust protects against ovalbumin or cockroach allergen-mediated airway pathology. Protected animals exhibited significant reduction in the total number of airway T cells, down-regulation of Th2-related airway responses, as well as mucin secretion. Following dog-associated dust exposure, the cecal microbiome of protected animals was extensively restructured with significant enrichment of, amongst others, Lactobacillus johnsonii. Supplementation of wild-type animals with L. johnsonii protected them against both airway allergen challenge or infection with respiratory syncytial virus. L. johnsonii-mediated protection was associated with significant reductions in the total number and proportion of activated CD11c(+)/CD11b(+) and CD11c(+)/CD8(+) cells, as well as significantly reduced airway Th2 cytokine expression. Our results reveal that exposure to dog-associated household dust results in protection against airway allergen challenge and a distinct gastrointestinal microbiome composition. Moreover, the study identifies L. johnsonii as a pivotal species within the gastrointestinal tract capable of influencing adaptive immunity at remote mucosal surfaces in a manner that is protective against a variety of respiratory insults.

Development of a standardized approach for environmental microbiota investigations related to asthma development in children.

Standardized studies examining environmental microbial exposure in populations at risk for asthma are necessary to improve our understanding of the role this factor plays in disease development. Here we describe studies aimed at developing guidelines for high-resolution culture-independent microbiome profiling, using a phylogenetic microarray (PhyloChip), of house dust samples in a cohort collected as part of the NIH-funded Inner City Asthma Consortium (ICAC). We demonstrate that though extracted DNA concentrations varied across dust samples, the majority produced sufficient 16S rRNA to be profiled by the array. Comparison of array and 454-pyrosequencing performed in parallel on a subset of samples, illustrated that increasingly deeper sequencing efforts validated greater numbers of array-detected taxa. Community composition agreement across samples exhibited a hierarchy in concordance, with the highest level of agreement in replicate array profiles followed by samples collected from adjacent 1×1 m(2) sites in the same room, adjacent sites with different sized sampling quadrants (1×1 and 2×2 m(2)), different sites within homes (living and bedroom) to lowest in living room samples collected from different homes. The guidelines for sample collection and processing in this pilot study extend beyond PhyloChip based studies of house-associated microbiota, and bear relevance for other microbiome profiling approaches such as next-generation sequencing.

Probiotic manipulation of the gastrointestinal microbiota.

In a recent publication we examined whether high abundance of a probiotic species, Lactobacillus casei subsp. rhamnosus GG (LGG), impacted the overall composition of the gastrointestinal (GI) microbiota of six-month-old infants at high risk for asthma development. Profound GI microbiota restructuring and the establishment of significantly more even and putatively, functionally redundant consortia were characteristic of high LGG abundance. Here we discuss, in the context of more recently published data, support for the hypothesis that the beneficial effect of probiotic supplementation on human health lies in the formation of a stable and resilient gut ecosystem enriched for species that exert a concerted beneficial effect on the host immune system via direct and indirect mechanisms.

The commensal microbiology of the gastrointestinal tract.

The gastrointestinal (GI) tract is a dynamic environment and therefore the stability of the commensal community, or microbiota, is under constant challenge. Microscopic observations have revealed that the majority of bacteria present in the GI tract are not detected using standard culturing techniques, however with the application of culture-independent techniques it has been estimated that between 500 to 1000 bacterial species inhabit the human GI tract. Numerically predominant organisms in the microbiota belong to two eubacterial divisions, the Cytophaga-Flavobacterium-Bacteroides (CFB) and the Firmicutes, and fall into three main groups; Clostridium rRNA subcluster XIVa, Clostridium rRNA subcluster IV and Bacteroides. The prevalence and diversity of bacteria in different areas of the GI tract is influenced by the different conditions at these sites and thus the microbiota of the stomach and jejunum varies with that of the large intestine. Additionally, host genotype, age and diet have all been shown to affect microbial diversity in the GI tract. The distal intestine harbours the highest bacterial cell densities for any known ecosystem. Characterizing the species composition of the healthy microbiota may be a key step in identifying bacterial or associated physiological conditions that are present or absent in an unhealthy microbiota.

SigB-dependent in vitro transcription of prfA and some newly identified genes of Listeria monocytogenes whose expression is affected by PrfA in vivo.

Recent studies have identified several new genes in Listeria monocytogenes which are positively or negatively affected by PrfA and grouped into three classes (E. Milohanic et al., Mol. Microbiol. 47:1613-1625, 2003). In vitro transcription performed with promoters of some class III genes showed strict SigB-dependent but PrfA-independent transcription initiation. Transcription starting at the prfA promoter PprfA2 was also optimal with SigB-loaded RNA polymerase, suggesting a direct link between SigB- and PrfA-dependent gene expression.

In vitro transcription of the Listeria monocytogenes virulence genes inlC and mpl reveals overlapping PrfA-dependent and -independent promoters that are differentially activated by GTP.

Most known virulence genes of Listeria monocytogenes are regulated by the transcriptional factor PrfA. Using our recently established in vitro transcription system, we have studied the PrfA-dependent promoter (PinlC) regulating the expression of the small, secreted internalin C. PrfA-dependent and PrfA-independent transcription is observed starting from PinlC in vitro and in vivo, suggesting the presence of two apparently overlapping promoters both of which use the same -10 box. Although the PrfA-dependent transcription requires, as expected, the PrfA-box, PrfA-independent transcription depends on a -35 box located directly downstream of the PrfA-box. PrfA-independent transcription starts at A, 7 bp downstream of the common -10 box (A7), and is strongly inhibited by PrfA because of the close proximity of the PrfA binding site to the -35 box. PrfA-dependent transcription starts preferentially at G5 but, in the absence of this start nucleotide, alternative start sites at A positions 7 or 8 bp downstream of the -10 box can also be used. The -35 box of the PrfA-independent promoter can be functionally inactivated without affecting PrfA-dependent transcription as long as the distance between the PrfA-box and the -10 box remains fixed to 22 (or 23) bp. Vice versa, the PrfA-box can be deleted without affecting PrfA-independent transcription from PinlC, which is no longer inhibited by PrfA. The PrfA-dependent transcription initiation needs, in contrast to the PrfA-independent one, the presence of a high concentration of GTP (and ATP) but not of CTP and UTP. Overlapping PrfA-dependent and PrfA-independent promoter activity was also demonstrated for the mpl promoter (Pmpl). Again, PrfA-dependent transcription starting at Pmpl is dominant at high GTP concentration and PrfA-independent transcription at low GTP. Here too, the PrfA-dependent and the PrfA-independent promoters share the same -10 box characteristic of SigA-loaded RNA polymerase. High GTP concentration also appears to be necessary for transcription initiation at other PrfA-dependent promoters (Phly, PactA) but not at the PrfA-independent promoter PinlC-m8.