Tunable control of B. infantis abundance and gut metabolites by co-administration of human milk oligosaccharides.

Precision engineering of the gut microbiome holds promise as an effective therapeutic approach for diseases associated with a disruption in this microbial community. Engrafting a live biotherapeutic product (LBP) in a predictable, controllable manner is key to the consistent success of this approach and has remained a challenge for most LBPs under development. We recently demonstrated high-level engraftment of Bifidobacterium longum subsp. infantis (B. infantis) in adults when co-dosed with a specific prebiotic, human milk oligosaccharides (HMO). Here, we present a cellular kinetic-pharmacodynamic approach, analogous to pharmacokinetic-pharmacodynamic-based analyses of small molecule- and biologic-based drugs, to establish how HMO controls expansion, abundance, and metabolic output of B. infantis in a human microbiota-based model in gnotobiotic mice. Our data demonstrate that the HMO dose controls steady-state abundance of B. infantis in the microbiome, and that B. infantis together with HMO impacts gut metabolite levels in a targeted, HMO-dependent manner. We also found that HMO creates a privileged niche for B. infantis expansion across a 5-log range of bacterial inocula. These results demonstrate remarkable control of both B. infantis levels and the microbiome community metabolic outputs using this synbiotic approach, and pave the way for precision engineering of desirable microbes and metabolites to treat a range of diseases.

Precision modulation of dysbiotic adult microbiomes with a human-milk-derived synbiotic reshapes gut microbial composition and metabolites.

Manipulation of the gut microbiome using live biotherapeutic products shows promise for clinical applications but remains challenging to achieve. Here, we induced dysbiosis in 56 healthy volunteers using antibiotics to test a synbiotic comprising the infant gut microbe, Bifidobacterium longum subspecies infantis (B. infantis), and human milk oligosaccharides (HMOs). B. infantis engrafted in 76% of subjects in an HMO-dependent manner, reaching a relative abundance of up to 81%. Changes in microbiome composition and gut metabolites reflect altered recovery of engrafted subjects compared with controls. Engraftment associates with increases in lactate-consuming Veillonella, faster acetate recovery, and changes in indolelactate and p-cresol sulfate, metabolites that impact host inflammatory status. Furthermore, Veillonella co-cultured in vitro and in vivo with B. infantis and HMO converts lactate produced by B. infantis to propionate, an important mediator of host physiology. These results suggest that the synbiotic reproducibly and predictably modulates recovery of a dysbiotic microbiome.

Dosing a synbiotic of human milk oligosaccharides and B. infantis leads to reversible engraftment in healthy adult microbiomes without antibiotics.

Predictable and sustainable engraftment of live biotherapeutic products into the human gut microbiome is being explored as a promising way to modulate the human gut microbiome. We utilize a synbiotic approach pairing the infant gut microbe Bifidobacterium longum subspecies infantis (B. infantis) and human milk oligosaccharides (HMO). B. infantis, which is typically absent in adults, engrafts into healthy adult microbiomes in an HMO-dependent manner at a relative abundance of up to 25% of the bacterial population without antibiotic pretreatment or adverse effects. Corresponding changes in metabolites are detected. Germ-free mice transplanted with dysbiotic human microbiomes also successfully engraft with B. infantis in an HMO-dependent manner, and the synbiotic augments butyrate levels both in this in vivo model and in in vitro cocultures of the synbiotic with specific Firmicutes species. Finally, the synbiotic inhibits the growth of enteropathogens in vitro. Our findings point to a potential safe mechanism for ameliorating dysbioses characteristic of numerous human diseases.

Vaccine Hyporesponse Induced by Individual Antibiotic Treatment in Mice and Non-Human Primates Is Diminished upon Recovery of the Gut Microbiome.

Emerging evidence demonstrates a connection between microbiome composition and suboptimal response to vaccines (vaccine hyporesponse). Harnessing the interaction between microbes and the immune system could provide novel therapeutic strategies for improving vaccine response. Currently we do not fully understand the mechanisms and dynamics by which the microbiome influences vaccine response. Using both mouse and non-human primate models, we report that short-term oral treatment with a single antibiotic (vancomycin) results in the disruption of the gut microbiome and this correlates with a decrease in systemic levels of antigen-specific IgG upon subsequent parenteral vaccination. We further show that recovery of microbial diversity before vaccination prevents antibiotic-induced vaccine hyporesponse, and that the antigen specific IgG response correlates with the recovery of microbiome diversity. RNA sequencing analysis of small intestine, spleen, whole blood, and secondary lymphoid organs from antibiotic treated mice revealed a dramatic impact on the immune system, and a muted inflammatory signature is correlated with loss of bacteria from Lachnospiraceae, Ruminococcaceae, and Clostridiaceae. These results suggest that microbially modulated immune pathways may be leveraged to promote vaccine response and will inform future vaccine design and development strategies.

Immunomodulation by the Commensal Microbiome During Immune-Targeted Interventions: Focus on Cancer Immune Checkpoint Inhibitor Therapy and Vaccination.

Emerging evidence in clinical and preclinical studies indicates that success of immunotherapies can be impacted by the state of the microbiome. Understanding the role of the microbiome during immune-targeted interventions could help us understand heterogeneity of treatment success, predict outcomes, and develop additional strategies to improve efficacy. In this review, we discuss key studies that reveal reciprocal interactions between the microbiome, the immune system, and the outcome of immune interventions. We focus on cancer immune checkpoint inhibitor treatment and vaccination as two crucial therapeutic areas with strong potential for immunomodulation by the microbiota. By juxtaposing studies across both therapeutic areas, we highlight three factors prominently involved in microbial immunomodulation: short-chain fatty acids, microbe-associate molecular patterns (MAMPs), and inflammatory cytokines. Continued interrogation of these models and pathways may reveal critical mechanistic synergies between the microbiome and the immune system, resulting in novel approaches designed to influence the efficacy of immune-targeted interventions.

Salmonella enterica Requires Lipid Metabolism Genes To Replicate in Proinflammatory Macrophages and Mice.

To survive and replicate during infection, pathogens utilize different carbon and energy sources depending on the nutritional landscape of their host microenvironment. Salmonella enterica serovar Typhimurium is an intracellular bacterial pathogen that occupies diverse cellular niches. While it is clear that Salmonella Typhimurium requires access to glucose during systemic infection, data on the need for lipid metabolism are mixed. We report that Salmonella Typhimurium strains lacking lipid metabolism genes were defective for systemic infection of mice. Bacterial lipid import, ß-oxidation, and glyoxylate shunt genes were required for tissue colonization upon oral or intraperitoneal inoculation. In cultured macrophages, lipid import and ß-oxidation genes were required for bacterial replication and/or survival only when the cell culture medium was supplemented with nonessential amino acids. Removal of glucose from tissue culture medium further enhanced these phenotypes and, in addition, conferred a requirement for glyoxylate shunt genes. We also observed that Salmonella Typhimurium needs lipid metabolism genes in proinflammatory but not anti-inflammatory macrophages. These results suggest that during systemic infection, the Salmonella Typhimurium that relies upon host lipids to replicate is within proinflammatory macrophages that have access to amino acids but not glucose. An improved understanding of the host microenvironments in which pathogens have specific metabolic requirements may facilitate the development of targeted approaches to treatment.

Autophagy Induction by a Small Molecule Inhibits Salmonella Survival in Macrophages and Mice.

Salmonella enterica are natural bacterial pathogens of humans and animals that cause systemic infection or gastroenteritis. During systemic infection, Salmonella generally reside within professional phagocytes, typically macrophages, whereas gastroenteritis is caused by infection of epithelial cells. We are only beginning to understand which host pathways contribute to Salmonella survival in particular cell types. We therefore sought to identify compounds that perturb Salmonella-host interactions using a chemical genetics approach. We found one small molecule, D61, that reduces Salmonella load in cell-line and primary macrophages but has no effect on Salmonella growth in epithelial cells or rich medium. We determined that in macrophages D61 induces LC3II, a marker of the autophagy pathway, and promotes aggregation of LC3II near Salmonella We found that D61 antibacterial activity depends on the VPS34 complex and on ATG5. D61 also reduced Salmonella load in the spleens and livers of infected mice. Lastly, we demonstrate that D61 antibacterial activity in macrophages is synergistic with the antibiotic chloramphenicol, but that this synergy is largely independent of the known autophagy-stimulating activity of chloramphenicol. Thus, a small molecule has anti-bacterial activity specifically in macrophages and mice based on the promotion of bacterial degradation by autophagy.Importance Autophagy is a conserved cellular response to metabolic stress and to invading pathogens. For many pathogens, including Salmonella, autophagy can play a detrimental or beneficial role during infection depending on the cellular context. We combined chemical genetics with single cell analyses and murine infection to dissect host-pathogen interactions. We identified a small molecule that reduces bacterial load in macrophages by increasing autophagic flux. This compound also reduces bacterial colonization of tissues in infected mice. These observations demonstrate the potential therapeutic utility of stimulating autophagy in cells and animals to curb infection.

A cell-based infection assay identifies efflux pump modulators that reduce bacterial intracellular load.

Bacterial efflux pumps transport small molecules from the cytoplasm or periplasm outside the cell. Efflux pump activity is typically increased in multi-drug resistant (MDR) pathogens; chemicals that inhibit efflux pumps may have potential for antibiotic development. Using an in-cell screen, we identified three efflux pump modulators (EPMs) from a drug diversity library. The screening platform uses macrophages infected with the human Gram-negative pathogen Salmonella enterica (Salmonella) to identify small molecules that prevent bacterial replication or survival within the host environment. A secondary screen for hit compounds that increase the accumulation of an efflux pump substrate, Hoechst 33342, identified three small molecules with activity comparable to the known efflux pump inhibitor PAßN (Phe-Arg ß-naphthylamide). The three putative EPMs demonstrated significant antibacterial activity against Salmonella within primary and cell culture macrophages and within a human epithelial cell line. Unlike traditional antibiotics, the three compounds did not inhibit bacterial growth in standard microbiological media. The three compounds prevented energy-dependent efflux pump activity in Salmonella and bound the AcrB subunit of the AcrAB-TolC efflux system with KDs in the micromolar range. Moreover, the EPMs display antibacterial synergy with antimicrobial peptides, a class of host innate immune defense molecules present in body fluids and cells. The EPMs also had synergistic activity with antibiotics exported by AcrAB-TolC in broth and in macrophages and inhibited efflux pump activity in MDR Gram-negative ESKAPE clinical isolates. Thus, an in-cell screening approach identified EPMs that synergize with innate immunity to kill bacteria and have potential for development as adjuvants to antibiotics.

Molecular analysis of single room humidifier bacteriology.

Portable, single-room humidifiers are commonly used in homes for comfort and health benefits, but also create habitats for microbiology. Currently there is no information on home humidifier microbiology aside from anecdotal evidence of infection with opportunistic pathogens and irritation from endotoxin exposure. To obtain a broader perspective on humidifier microbiology, DNAs were isolated from tap source waters, tank waters, and biofilm samples associated with 26 humidifiers of ultrasonic and boiling modes of operation in the Front Range of Colorado. Humidifiers sampled included units operated by individuals in their homes, display models continuously operated by a retail store, and new humidifiers operated in a controlled laboratory study. The V1V2 region of the rRNA gene was amplified and sequenced to determine the taxonomic composition of humidifier samples. Communities encountered were generally low in richness and diversity and were dominated by Sphingomonadales, Rhizobiales, and Burkholderiales of the Proteobacteria, and MLE1-12, a presumably non-photosynthetic representative of the cyanobacterial phylum. Very few sequences of potential health concern were detected. The bacteriology encountered in source waters sampled here was similar to that encountered in previous studies of municipal drinking waters. Source water bacteriology was found to have the greatest effect on tank water and biofilm bacteriology, an effect confirmed by a controlled study comparing ultrasonic and boiler humidifiers fed with tap vs. treated (deionized, reverse osmosis, 0.2 µm filtered) water over a period of two months.