Segmented Filamentous Bacteria Prevent and Cure Rotavirus Infection.

Rotavirus (RV) encounters intestinal epithelial cells amidst diverse microbiota, opening possibilities of microbes influencing RV infection. Although RV clearance typically requires adaptive immunity, we unintentionally generated RV-resistant immunodeficient mice, which, we hypothesized, reflected select microbes protecting against RV. Accordingly, such RV resistance was transferred by co-housing and fecal transplant. RV-protecting microbiota were interrogated by heat, filtration, and antimicrobial agents, followed by limiting dilution transplant to germ-free mice and microbiome analysis. This approach revealed that segmented filamentous bacteria (SFB) were sufficient to protect mice against RV infection and associated diarrhea. Such protection was independent of previously defined RV-impeding factors, including interferon, IL-17, and IL-22. Colonization of the ileum by SFB induced changes in host gene expression and accelerated epithelial cell turnover. Incubation of RV with SFB-containing feces reduced infectivity in vitro, suggesting direct neutralization of RV. Thus, independent of immune cells, SFB confer protection against certain enteric viral infections and associated diarrheal disease.

The stepwise assembly of the neonatal virome is modulated by breastfeeding.

The gut of healthy human neonates is usually devoid of viruses at birth, but quickly becomes colonized, which-in some cases-leads to gastrointestinal disorders1-4. Here we show that the assembly of the viral community in neonates takes place in distinct steps. Fluorescent staining of virus-like particles purified from infant meconium or early stool samples shows few or no particles, but by one month of life particle numbers increase to 109 per gram, and these numbers seem to persist throughout life5-7. We investigated the origin of these viral populations using shotgun metagenomic sequencing of virus-enriched preparations and whole microbial communities, followed by targeted microbiological analyses. Results indicate that, early after birth, pioneer bacteria colonize the infant gut and by one month prophages induced from these bacteria provide the predominant population of virus-like particles. By four months of life, identifiable viruses that replicate in human cells become more prominent. Multiple human viruses were more abundant in stool samples from babies who were exclusively fed on formula milk compared with those fed partially or fully on breast milk, paralleling reports that breast milk can be protective against viral infections8-10. Bacteriophage populations also differed depending on whether or not the infant was breastfed. We show that the colonization of the infant gut is stepwise, first mainly by temperate bacteriophages induced from pioneer bacteria, and later by viruses that replicate in human cells; this second phase is modulated by breastfeeding.

Persistence of the Probiotic Lacticaseibacillus rhamnosus Strain GG (LGG) in an In Vitro Model of the Gut Microbiome.

The consumption of probiotics is widely encouraged due to reports of their positive effects on human health. In particular, Lacticaseibacillus rhamnosus strain GG (LGG) is an approved probiotic that has been reported to improve health outcomes, especially for gastrointestinal disorders. However, how LGG cooperates with the gut microbiome has not been fully explored. To understand the interaction between LGG and its ability to survive and grow within the gut microbiome, this study introduced LGG into established microbial communities using an in vitro model of the colon. LGG was inoculated into the simulated ascending colon and its persistence in, and transit through the subsequent transverse and descending colon regions was monitored over two weeks. The impact of LGG on the existing bacterial communities was investigated using 16S rRNA sequencing and short-chain fatty acid analysis. LGG was able to engraft and proliferate in the ascending region for at least 10 days but was diminished in the transverse and descending colon regions with little effect on short-chain fatty acid abundance. These data suggest that the health benefits of the probiotic LGG rely on its ability to transiently engraft and modulate the host microbial community.

Comparative analysis of the gut microbiota cultured in vitro using a single colon versus a 3-stage colon experimental design.

The importance of the gut microbiota in human health and disease progression makes it a target for research in both the biomedical and nutritional fields. To date, a number of in vitro systems have been designed to recapitulate the gut microbiota of the colon ranging in complexity from the application of a single vessel to cultivate the community in its entirety, to multi-stage systems that mimic the distinct regional microbial communities that reside longitudinally through the colon. While these disparate types of in vitro designs have been employed previously, information regarding similarities and differences between the communities that develop within was less defined. Here, a comparative analysis of the population dynamics and functional production of short-chain fatty acids (SCFAs) was performed using the gut microbiota of the same donor cultured using a single vessel and a 3-stage colon system. The results found that the single vessel communities maintained alpha diversity at a level comparable to the distal regions of the 3-stage colon system. Yet, there was a marked difference in the type and abundance of taxa, particularly between families Enterobacteriaceae, Bacteroidaceae, Synergistaceae, and Fusobacteriaceae. Functionally, the single vessel community produced significantly less SCFAs compared to the 3-stage colon system. These results provide valuable information on how culturing technique effects gut microbial composition and function, which may impact studies relying on the application of an in vitro strategy. This data can be used to justify experimental strategy and provides insight on the application of a simplified versus complex study design. KEY POINTS : • A mature gut microbiota community can be developed in vitro using different methods. • Beta diversity metrics are affected by the in vitro culturing method applied. • The type and amount of short-chain fatty acids differed between culturing methods.

Whole-Genome Sequencing To Identify Drivers of Carbapenem-Resistant Klebsiella pneumoniae Transmission within and between Regional Long-Term Acute-Care Hospitals.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) is an antibiotic resistance threat of the highest priority. Given the limited treatment options for this multidrug-resistant organism (MDRO), there is an urgent need for targeted strategies to prevent transmission. Here, we applied whole-genome sequencing to a comprehensive collection of clinical isolates to reconstruct regional transmission pathways and analyzed this transmission network in the context of statewide patient transfer data and patient-level clinical data to identify drivers of regional transmission. We found that high regional CRKP burdens were due to a small number of regional introductions, with subsequent regional proliferation occurring via patient transfers among health care facilities. While CRKP was predicted to have been imported into each facility multiple times, there was substantial variation in the ratio of intrafacility transmission events per importation, indicating that amplification occurs unevenly across regional facilities. While myriad factors likely influence intrafacility transmission rates, an understudied one is the potential for clinical characteristics of colonized and infected patients to influence their propensity for transmission. Supporting the contribution of high-risk patients to elevated transmission rates, we observed that patients colonized and infected with CRKP in high-transmission facilities had higher rates of carbapenem use, malnutrition, and dialysis and were older. This report highlights the potential for regional infection prevention efforts that are grounded in genomic epidemiology to identify the patients and facilities that make the greatest contribution to regional MDRO prevalence, thereby facilitating the design of precision interventions of maximal impact.

In vivo requirement for Atg5 in antigen presentation by dendritic cells.

Autophagy is known to be important in presentation of cytosolic antigens on MHC class II (MHC II). However, the role of autophagic process in antigen presentation in vivo is unclear. Mice with dendritic cell (DC)-conditional deletion in Atg5, a key autophagy gene, showed impaired CD4(+) T cell priming after herpes simplex virus infection and succumbed to rapid disease. The most pronounced defect of Atg5(-/-) DCs was the processing and presentation of phagocytosed antigens containing Toll-like receptor stimuli for MHC class II. In contrast, cross-presentation of peptides on MHC I was intact in the absence of Atg5. Although induction of metabolic autophagy did not enhance MHC II presentation, autophagic machinery was required for optimal phagosome-to-lysosome fusion and subsequent processing of antigen for MHC II loading. Thus, our study revealed that DCs utilize autophagic machinery to optimally process and present extracellular microbial antigens for MHC II presentation.

Toxin-positive Clostridium difficile latently infect mouse colonies and protect against highly pathogenic C. difficile.

OBJECTIVE: Clostridium difficile is a toxin-producing bacterium and a leading cause of antibiotic-associated disease. The ability of C. difficile to form spores and infect antibiotic-treated persons at low multiplicity of infection (MOI) underlies its large disease burden. However, C. difficile-induced disease might also result from long-harboured C. difficile that blooms in individuals administered antibiotics. DESIGN: Mice purchased from multiple vendors and repeatedly testing negative for this pathogen by quantitative PCR bloomed C. difficile following antibiotic treatment. This endogenous C. difficile strain, herein termed LEM1, which formed spores and produced toxin, was compared with highly pathogenic C. difficile strain VPI10463. RESULTS: Whole-genome sequencing revealed that LEM1 and VPI10463 shared 95% of their genes, including all known virulence genes. In contrast to VPI10463, LEM1 did not induce overt disease when administered to antibiotic-treated or germ-free mice, even at high doses. Rather, blooms of LEM1 correlated with survival following VPI10463 inoculation, and exogenous administration of LEM1 before or shortly following VPI10463 inoculation prevented C. difficile-induced death. Accordingly, despite similar growth properties in vitro, LEM1 strongly outcompeted VPI10463 in mice even at 100-fold lower inocula. CONCLUSIONS: These results highlight the difficulty of determining whether individual cases of C. difficile infection resulted from a bloom of endogenous C. difficile or a new exposure to this pathogen. In addition to impacting the design of studies using mouse models of C. difficile-induced disease, this study identified, isolated and characterised an endogenous murine spore-forming C. difficile strain able to decrease colonisation, associated disease and death induced by a pathogenic C. difficile strain.

Toll-like receptor 9 in plasmacytoid dendritic cells fails to detect parvoviruses.

Toll-like receptor 9 (TLR9) recognizes genomes of double-stranded DNA (dsDNA) viruses in the endosome to stimulate plasmacytoid dendritic cells (pDCs). However, how and if viruses with single-stranded DNA (ssDNA) genomes are detected by pDCs remain unclear. Here we have shown that despite the ability of purified genomic DNA to stimulate TLR9 and despite the ability to enter TLR9 endosomes, ssDNA viruses of the Parvoviridae family failed to elicit an interferon (IFN) response in pDCs.

Recruited inflammatory monocytes stimulate antiviral Th1 immunity in infected tissue.

Monocytes patrol various tissues for signs of infection and inflammation. Inflammatory monocytes enter peripheral tissues at sites of microbial infection and differentiate into dendritic cells and macrophages. Here, we examined the importance of monocytes in primary mucosal infection with herpes simplex virus 2 (HSV-2), and demonstrate that monocyte-derived APCs are required to elicit IFN-γ secretion from effector Th1 cells to mediate antiviral protection. However, monocyte-derived APCs were dispensable for the generation of Th1 immunity and for the restimulation of memory Th1 cells during secondary viral challenge. These results demonstrate that distinct APC subsets are dedicated for CD4 T cell priming, elicitation, and memory recall responses to a given viral pathogen within the same mucosal tissue and reveal a specialized role for monocyte-derived APCs in the emergency response to infection.

CD4+ T cells support cytotoxic T lymphocyte priming by controlling lymph node input.

Rapid induction of CD8(+) cytotoxic T lymphocyte (CTL) responses is critical to combat acute infection with intracellular pathogens. CD4(+) T cells help prime antigen-specific CTLs in secondary lymphoid organs after infection in the periphery. Although the frequency of naïve precursors is very low, the immune system is able to efficiently screen for cognate CTLs through mechanisms that are not well understood. Here we examine the role of CD4(+) T cells in early phases of the immune response. We show that CD4(+) T cells help optimal CTL expansion by facilitating entry of naïve polyclonal CD8(+) T cells into the draining lymph node (dLN) early after infection or immunization. CD4(+) T cells also facilitate input of naïve B cells into reactive LNs. Such "help" involves expansion of the arteriole feeding the dLN and enlargement of the dLN through activation of dendritic cells. In an antigen- and CD40-dependent manner, CD4(+) T cells activate dendritic cells to support naïve lymphocyte recruitment to the dLN. Our results reveal a previously unappreciated mode of CD4(+) T-cell help, whereby they increase the input of naïve lymphocytes to the relevant LN for efficient screening of cognate CD8(+) T cells.

Distinct community structures of the fungal microbiome and respiratory health in adults with cystic fibrosis.

BACKGROUND: The respiratory tract fungal microbiome in cystic fibrosis (CF) has been understudied despite increasing recognition of fungal pathogens in CF lung disease. We sought to better understand the fungal communities in adults with CF, and to define relationships between fungal profiles and clinical characteristics. METHODS: We enrolled 66 adults with CF and collected expectorated sputum, spirometry, Cystic Fibrosis Questionnaire-revised, and clinical data. Fungi were molecularly profiled by sequencing of the internal transcribed spacer (ITS) region. Total fungal abundance was measured by quantitative PCR. Relative abundance and qPCR-corrected abundances were determined. Selective fungus culture identified cultivable fungi. Alpha diversity and beta diversity were measured and relationships with clinical parameters were interrogated. RESULTS: Median age was 29 years and median FEV1 percent predicted 58%. Members of the Candida genus were the most frequent dominant taxa in CF sputum. Apiotrichum, Trichosporon, Saccharomyces cerevisiae, and Scedosporium were present in high relative abundance in few samples; whereas, Aspergillus species were detected at low levels. Higher FEV1% predicted and CFTR modulator use were associated with greater alpha-diversity. Chronic azithromycin use was associated with lower alpha-diversity. Patients with acute pulmonary had distinct fungal community composition compared to clinically stable subjects. Differing yeast species were mainly responsible for the community differences. CONCLUSION: The respiratory tract fungal microbiome in adults with CF is associated with lung function, pulmonary exacerbation status, macrolide use, and CFTR modulator use. Future work to better understand fungal diversity in the CF airway and its impact on lung health is necessary.

Lacticaseibacillus rhamnosus Strain GG (LGG) Regulate Gut Microbial Metabolites, an In Vitro Study Using Three Mature Human Gut Microbial Cultures in a Simulator of Human Intestinal Microbial Ecosystem (SHIME).

In the present research, we investigated changes in the gut metabolome that occurred in response to the administration of the Laticaseibacillus rhamnosus strain GG (LGG). The probiotics were added to the ascending colon region of mature microbial communities established in a human intestinal microbial ecosystem simulator. Shotgun metagenomic sequencing and metabolome analysis suggested that the changes in microbial community composition corresponded with changes to metabolic output, and we can infer linkages between some metabolites and microorganisms. The in vitro method permits a spatially-resolved view of metabolic transformations under human physiological conditions. By this method, we found that tryptophan and tyrosine were mainly produced in the ascending colon region, while their derivatives were detected in the transverse and descending regions, revealing sequential amino acid metabolic pathways along with the colonic tract. The addition of LGG appeared to promote the production of indole propionic acid, which is positively associated with human health. Furthermore, the microbial community responsible for the production of indole propionic acid may be broader than is currently known.

CD8+ T cell responses following replication-defective adenovirus serotype 5 immunization are dependent on CD11c+ dendritic cells but show redundancy in their requirement of TLR and nucleotide-binding oligomerization domain-like receptor signaling.

Replication-defective adenovirus serotype 5 (rAd5) is the most potent recombinant vector for eliciting CD8 T cell responses in humans. In this study, the innate mechanisms that influence T cell responses following rAd5 immunization were assessed in mice. Using rAd5 expressing enhanced GFP (eGFP-rAd5), we show that rAd5 transfects CD11c(+) dendritic cells (DCs) in draining lymph nodes in vivo following s.c. or i.m. immunization. Among distinct DC subsets, eGFP expression was highest in CD11c(+)CD8(-)B220(-) with a lower frequency detected in CD11c(+)CD8(+)B220(-) and CD11c(+)B220(+) plasmacytoid DCs. CD11c(+) DCs but not CD11c(-) cells from mice immunized with rAd5 encoding the SIINFEKL peptide induced proliferation of naive OT-I CD8 T cells. Furthermore, CD11c(+)CD8(+)B220(-) was the most potent DC subset for eliciting naive OT-I CD8 T cell proliferation. Of note, mice with pre-existing immunity to rAd5 had a substantial decrease in eGFP expression in DCs, which was associated with approximately 2-fold decrease in Th1 and complete inhibition of CD8 responses. Thus, pre-existing rAd5 immunity has a greater influence on CD8 compared with CD4 T cell responses. In terms of how innate cytokines and signaling pathways influenced T cell immunity following rAd5 immunization, we show that the magnitude and quality of CD8 T cell responses are partially dependent on MyD88 but independent of IL-12, type I IFN, apoptosis-associated speck-like protein, nucleotide-binding oligomerization domain-like receptor protein 3, and IL-1. Taken together, these data demonstrate a critical role for CD11c(+) DCs for CD8 responses but striking redundancy for innate cytokines and signaling by TLR and nucleotide-binding oligomerization domain-like receptor pathways.

The impact of environmental pH on the gut microbiota community structure and short chain fatty acid production.

Environmental pH is a critical parameter for maintenance of the gut microbiota. Here, the impact of pH on the gut microbiota luminal and mucosal community structure and short chain fatty acid (SCFA) production was evaluated in vitro, and data compiled to reveal a donor-independent response to an increase or decrease in environmental pH. The results found that raising environmental pH significantly increased luminal community richness and decreased mucosal community evenness. This corresponded with an increased abundance of Ruminococcaceae Ruminococcus and Erysipelotrichaceae Erysipelatoclostridium, and a decreased abundance of Coriobacteriaceae Collinsella and Enterobacteriaceae Shigella for both the luminal and mucosal communities. Total SCFA levels were significantly higher, primarily due to an increase in acetic and 2-methylbutanoic acids. Lowering pH decreased luminal community evenness and decreased mucosal community evenness and richness. This corresponded with an increased abundance of Lachnospiraceae Enterocloster, Veillonellaceae Megasphaera, Veillonellaceae Sporomusa, Erysipelotrichaceae Eubacterium, and Alcaligenaceae Sutterella, and decreased abundance of Odoribacteraceae Butyricimonas, Fusobacteriaceae Fusobacterium, Veillonellaceae Phascolarctobacterium, and multiple Enterobacteriaceae species for both the luminal and mucosal communities. Total SCFA levels were significantly lower, with an observed drop in acetic and propionic acids, and increased butyric and valeric acids. Taken together, these results indicate that alterations to environmental pH can modulate the gut microbiota community structure and function, and some changes may occur in a donor-independent manner.

Preterm infants at low risk for early-onset sepsis differ in early fecal microbiome assembly.

Antibiotics are administered near-universally to very low birth weight (VLBW) infants after birth for suspected early-onset sepsis (EOS). We previously identified a phenotypic group of VLBW infants, referred to as low-risk for EOS (LRE), whose risk of EOS is low enough to avoid routine antibiotic initiation. In this cohort study, we compared 18 such infants with 30 infants categorized as non-LRE to determine if the lower risk of pathogen transmission at birth is accompanied by differences in microbiome acquisition and development. We did shotgun metagenomic sequencing of 361 fecal samples obtained serially. LRE infants had a higher human-to-bacterial DNA ratio than non-LRE infants in fecal samples on days 1-3 after birth, confirming lower bacterial acquisition among LRE infants. The microbial diversity and composition in samples from days 4-7 differed between the groups with a predominance of Staphylococcus epidermidis in LRE infants and Enterobacteriaceae sp. in non-LRE infants. Compositional differences were congruent with the distribution of virulence factors and antibiotic resistant genes. After the first week, the overall composition was similar, but changes in relative abundance for several taxa with increasing age differed between groups. Of the nine late-onset bacteremia episodes, eight occurred in non-LRE infants. Species isolated from the blood culture was detected in the pre-antibiotic fecal samples of the infant for all episodes, though these species were also found in infants without bacteremia. In conclusion, LRE infants present a distinct pattern of microbiome development that is aligned with their low risk for EOS. Further investigation to determine the impact of these differences on later outcomes such as late-onset bacteremia is warranted.

Modulation of the Gut Microbiota Structure and Function by Two Structurally Different Lemon Pectins.

Pectins are plant polysaccharides consumed as part of a diet containing fruits and vegetables. Inside the gastrointestinal tract, pectin cannot be metabolized by the mammalian cells but is fermented by the gut microbiota in the colon with the subsequent release of end products including short-chain fatty acids (SCFA). The prebiotic effects of pectin have been previously evaluated but reports are inconsistent, most likely due to differences in the pectin chemical structure which can vary by molecular weight (MW) and degree of esterification (DE). Here, the effects of two different MW lemon pectins with varying DEs on the gut microbiota of two donors were evaluated in vitro. The results demonstrated that low MW, high DE lemon pectin (LMW-HDE) altered community structure in a donor-dependent manner, whereas high MW, low DE lemon pectin (HMW-LDE) increased taxa within Lachnospiraceae in both donors. LMW-HDE and HMW-LDE lemon pectins both increased total SCFAs (1.49- and 1.46-fold, respectively) and increased acetic acid by 1.64-fold. Additionally, LMW-HDE lemon pectin led to an average 1.41-fold increase in butanoic acid. Together, these data provide valuable information linking chemical structure of pectin to its effect on the gut microbiota structure and function, which is important to understanding its prebiotic potential.

Host immunity modulates the efficacy of microbiota transplantation for treatment of Clostridioides difficile infection.

Fecal microbiota transplantation (FMT) is a successful therapeutic strategy for treating recurrent Clostridioides difficile infection. Despite remarkable efficacy, implementation of FMT therapy is limited and the mechanism of action remains poorly understood. Here, we demonstrate a critical role for the immune system in supporting FMT using a murine C. difficile infection system. Following FMT, Rag1 heterozygote mice resolve C. difficile while littermate Rag1-/- mice fail to clear the infection. Targeted ablation of adaptive immune cell subsets reveal a necessary role for CD4+ Foxp3+ T-regulatory cells, but not B cells or CD8+ T cells, in FMT-mediated resolution of C. difficile infection. FMT non-responsive mice exhibit exacerbated inflammation, impaired engraftment of the FMT bacterial community and failed restoration of commensal bacteria-derived secondary bile acid metabolites in the large intestine. These data demonstrate that the host's inflammatory immune status can limit the efficacy of microbiota-based therapeutics to treat C. difficile infection.

Effects of Vancomycin on Persistent Pain-Stimulated and Pain-Depressed Behaviors in Female Fischer Rats With or Without Voluntary Access to Running Wheels.

The present experiments determined the effects of the narrow-spectrum antibiotic vancomycin on inflammatory pain-stimulated and pain-depressed behaviors in rats. Persistent inflammatory pain was modeled using dilute formalin (0.5%). Two weeks of oral vancomycin administered in drinking water attenuated Phase II formalin pain-stimulated behavior, and prevented formalin pain-depressed wheel running. Fecal microbiota transplantation produced a non-significant trend toward reversal of the vancomycin effect on pain-stimulated behavior. Vancomycin depleted Firmicutes and Bacteroidetes populations in the gut while having a partial sparing effect on Lactobacillus species and Clostridiales. The vancomycin treatment effect was associated with an altered profile in amino acid concentrations in the gut with increases in arginine, glycine, alanine, proline, valine, leucine, and decreases in tyrosine and methionine. These results indicate that vancomycin may have therapeutic effects against persistent inflammatory pain conditions that are distal to the gut. PERSPECTIVE: The narrow-spectrum antibiotic vancomycin reduces pain-related behaviors in the formalin model of inflammatory pain. These data suggest that manipulation of the gut microbiome may be one method to attenuate inflammatory pain amplitude.