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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.

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. Transfer of a host’s microbiota by faecal microbiota transplantation has shown benefit in the context of recurrent Clostridioides difficle infection. Here the authors shows the inflammatory status of the recipient can impact on engraftment and the efficacy of the introduced microbiota in a model of C.difficile infection.

Background Historically, the human womb has been thought to be sterile in healthy pregnancies, but this idea has been challenged by recent studies using DNA sequence-based methods, which have suggested that the womb is colonized with bacteria. For example, analysis of DNA from placenta samples yielded small proportions of microbial sequences which were proposed to represent normal bacterial colonization. However, an analysis by our group showed no distinction between background negative controls and placenta samples. Also supporting the idea that the womb is sterile is the observation that germ-free mammals can be generated by sterile delivery of neonates into a sterile isolator, after which neonates remain germ-free, which would seem to provide strong data in support of sterility of the womb. Results To probe this further and to investigate possible placental colonization associated with spontaneous preterm birth, we carried out another study comparing microbiota in placenta samples from 20 term and 20 spontaneous preterm deliveries. Both 16S rRNA marker gene sequencing and shotgun metagenomic sequencing were used to characterize placenta and control samples. We first quantified absolute amounts of bacterial 16S rRNA gene sequences using 16S rRNA gene quantitative PCR (qPCR). As in our previous study, levels were found to be low in the placenta samples and indistinguishable from negative controls. Analysis by DNA sequencing did not yield a placenta microbiome distinct from negative controls, either using marker gene sequencing as in our previous work, or with shotgun metagenomic sequencing. Several types of artifacts, including erroneous read classifications and barcode misattribution, needed to be identified and removed from the data to clarify this point. Conclusions Our findings do not support the existence of a consistent placental microbiome, in either placenta from term deliveries or spontaneous preterm births.

The gut of healthy human neonates is usually devoid of viruses at birth, but quickly becomes colonized, which—in some cases—leads to gastrointestinal disorders 1 – 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 10 9 per gram, and these numbers seem to persist throughout life 5 – 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 infections 8 – 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. The infant gut is colonized first by temperate bacteriophages induced from pioneer bacteria and later by viruses that replicate in human cells, the populations of which are modulated by breastfeeding.

Mycobacterium chelonae is a rapidly growing nontuberculous mycobacterium that is a common cause of nosocomial infections. Here we describe investigation of a possible nosocomial transmission of M . chelonae at the Hospital of the University of Pennsylvania (HUP). M . chelonae strains with similar high-level antibiotic resistance patterns were isolated from two patients who developed post-operative infections at HUP in 2017, suggesting a possible point source infection. The isolates, along with other clinical isolates from other patients, were sequenced using the Illumina and Oxford Nanopore technologies. The resulting short and long reads were hybrid assembled into draft genomes. The genomes were compared by quantifying single nucleotide variants in the core genome and assessed using a control dataset to quantify error rates in comparisons of identical genomes. We show that all M . chelonae isolates tested were highly dissimilar, as indicated by high pairwise SNV values, consistent with environmental acquisition and not a nosocomial point source. Our control dataset determined a threshold for evaluating identity between strains while controlling for sequencing error. Finally, antibiotic resistance genes were predicted for our isolates, and several single nucleotide variants were identified that have the potential to modulated drug resistance.

The recent ban of the antimicrobial compound triclosan from use in consumer soaps followed research that showcased the risk it poses to the environment and to human health. Triclosan has been found in human plasma, urine and milk, demonstrating that it is present in human tissues. Previous work has also demonstrated that consumption of triclosan disrupts the gut microbial community of mice and zebrafish. Due to the widespread use of triclosan and ubiquity in the environment, it is imperative to understand the impact this chemical has on the human body and its symbiotic resident microbes. To that end, this study is the first to explore how triclosan impacts the human gut microbial community in vitro both during and after treatment. Through our in vitro system simulating three regions of the human gut; the ascending colon, transverse colon, and descending colon regions, we found that treatment with triclosan significantly impacted the community structure in terms of reduced population, diversity, and metabolite production, most notably in the ascending colon region. Given a 2 week recovery period, most of the population levels, community structure, and diversity levels were recovered for all colon regions. Our results demonstrate that the human gut microbial community diversity and population size is significantly impacted by triclosan at a high dose in vitro, and that the community is recoverable within this system.

Research on the human microbiome has yielded numerous insights into health and disease, but also has resulted in a wealth of experimental artifacts. Here, we present suggestions for optimizing experimental design and avoiding known pitfalls, organized in the typical order in which studies are carried out. We first review best practices in experimental design and introduce common confounders such as age, diet, antibiotic use, pet ownership, longitudinal instability, and microbial sharing during cohousing in animal studies. Typically, samples will need to be stored, so we provide data on best practices for several sample types. We then discuss design and analysis of positive and negative controls, which should always be run with experimental samples. We introduce a convenient set of non-biological DNA sequences that can be useful as positive controls for high-volume analysis. Careful analysis of negative and positive controls is particularly important in studies of samples with low microbial biomass, where contamination can comprise most or all of a sample. Lastly, we summarize approaches to enhancing experimental robustness by careful control of multiple comparisons and to comparing discovery and validation cohorts. We hope the experimental tactics summarized here will help researchers in this exciting field advance their studies efficiently while avoiding errors.

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.

ObjectiveClostridium 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.DesignMice 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.ResultsWhole-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.ConclusionsThese 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.

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.