Activity of Gut-Derived Nisin-like Lantibiotics against Human Gut Pathogens and Commensals.

Recent advances in sequencing techniques unveiled the vast potential of ribosomally synthesized and post-translationally modified peptides (RiPPs) encoded in microbiomes. Class I lantibiotics such as nisin A, widely used as a food preservative, have been investigated for their efficacy in killing pathogens. However, the impact of nisin and nisin-like class I lantibiotics on commensal bacteria residing in the human gut remains unclear. Here, we report six gut-derived class I lantibiotics that are close homologues of nisin, four of which are novel. We applied an improved lantibiotic expression platform to produce and purify these lantibiotics for antimicrobial assays. We determined their minimal inhibitory concentration (MIC) against both Gram-positive human pathogens and gut commensals and profiled the lantibiotic resistance genes in these pathogens and commensals. Structure-activity relationship (SAR) studies with analogs revealed key regions and residues that impact their antimicrobial properties. Our characterization and SAR studies of nisin-like lantibiotics against both pathogens and human gut commensals could shed light on the future development of lantibiotic-based therapeutics and food preservatives.

Intestinal carbapenem-resistant Klebsiella pneumoniae undergoes complex transcriptional reprogramming following immune activation.

Carbapenem-resistant Klebsiella pneumoniae (CR-Kp) is a significant threat to public health worldwide. The primary reservoir for CR-Kp is the intestinal tract. There, the bacterium is usually present at low density but can bloom following antibiotic treatment, mostly in hospital settings. The impact of disturbances in the intestinal environment on the fitness, survival, expansion, and drug susceptibility of this pathogen is not well-understood, yet it may be relevant to devise strategies to tackle CR-Kp colonization and infection. Here, we adopted an in vivo model to examine the transcriptional adaptation of a CR-Kp clinical isolate to immune activation in the intestine. We report that as early as 6 hours following host treatment with anti-CD3 antibody, CR-Kp underwent rapid transcriptional changes including downregulation of genes involved in sugar utilization and amino acid biosynthesis and upregulation of genes involved in amino acid uptake and catabolism, antibiotic resistance, and stress response. In agreement with these findings, treatment increased the concentration of oxidative species and amino acids in the mouse intestine. Genes encoding for proteins containing the domain of unknown function (DUF) 1471 were strongly upregulated, however their deletion did not impair CR-Kp fitness in vivo upon immune activation. Transcription factor enrichment analysis identified the global regulator cAMP-Receptor Protein, CRP, as a potential orchestrator of the observed transcriptional signature. In keeping with the recognized role of CRP in regulating utilization of alternative carbon sources, crp deletion in CR-Kp resulted in strongly impaired gut colonization, although this effect was not amplified by immune activation. Thus, following intestinal colonization, which occurs in a CRP-dependent manner, CR-Kp can rapidly respond to immune cues by implementing a well-defined and complex transcriptional program whose direct relevance toward bacterial fitness warrants further investigation. Additional analyses utilizing this model may identify key factors to tackle CR-Kp colonization of the intestine.

Comprehensive analyses of a large human gut Bacteroidales culture collection reveal species and strain level diversity and evolution.

Species of the Bacteroidales order are among the most abundant and stable bacterial members of the human gut microbiome with diverse impacts on human health. While Bacteroidales strains and species are genomically and functionally diverse, order-wide comparative analyses are lacking. We cultured and sequenced the genomes of 408 Bacteroidales isolates from healthy human donors representing nine genera and 35 species and performed comparative genomic, gene-specific, mobile gene, and metabolomic analyses. Families, genera, and species could be grouped based on many distinctive features. However, we also show extensive DNA transfer between diverse families, allowing for shared traits and strain evolution. Inter- and intra-specific diversity is also apparent in the metabolomic profiling studies. This highly characterized and diverse Bacteroidales culture collection with strain-resolved genomic and metabolomic analyses can serve as a resource to facilitate informed selection of strains for microbiome reconstitution.

Protection against Clostridioides difficile disease by a naturally avirulent C. difficile strain.

Clostridioides difficile (C. difficile) strains belonging to the epidemic BI/NAP1/027 (RT027) group have been associated with increased transmissibility and disease severity. In addition to the major toxin A and toxin B virulence factors, RT027 strains also encode the CDT binary toxin. Our lab previously identified a toxigenic RT027 isolate, ST1-75, that is avirulent in mice despite densely colonizing the colon. Here, we show that coinfecting mice with the avirulent ST1-75 and virulent R20291 strains protects mice from colitis due to rapid clearance of the virulent strain and persistence of the avirulent strain. Although avirulence of ST1-75 is due to a mutation in the cdtR gene, which encodes a response regulator that modulates the production of all three C. difficile toxins, the ability of ST1-75 to protect against acute colitis is not directly attributable to the cdtR mutation. Metabolomic analyses indicate that the ST1-75 strain depletes amino acids more rapidly than the R20291 strain and supplementation with amino acids ablates ST1-75's competitive advantage, suggesting that the ST1-75 strain limits the growth of virulent R20291 bacteria by amino acid depletion. Since the germination kinetics and sensitivity to the co-germinant glycine are similar for the ST1-75 and R20291 strains, our results identify the rapidity of in vivo nutrient depletion as a mechanism providing strain-specific, virulence-independent competitive advantages to different BI/NAP1/027 strains. They also suggest that the ST1-75 strain may, as a biotherapeutic agent, enhance resistance to CDI in high-risk patients.

Fecal metabolite profiling identifies liver transplant recipients at risk for postoperative infection.

Metabolites produced by the intestinal microbiome modulate mucosal immune defenses and optimize epithelial barrier function. Intestinal dysbiosis, including loss of intestinal microbiome diversity and expansion of antibiotic-resistant pathobionts, is accompanied by changes in fecal metabolite concentrations and increased incidence of systemic infection. Laboratory tests that quantify intestinal dysbiosis, however, have yet to be incorporated into clinical practice. We quantified fecal metabolites in 107 patients undergoing liver transplantation (LT) and correlated these with fecal microbiome compositions, pathobiont expansion, and postoperative infections. Consistent with experimental studies implicating microbiome-derived metabolites with host-mediated antimicrobial defenses, reduced fecal concentrations of short- and branched-chain fatty acids, secondary bile acids, and tryptophan metabolites correlate with compositional microbiome dysbiosis in LT patients and the relative risk of postoperative infection. Our findings demonstrate that fecal metabolite profiling can identify LT patients at increased risk of postoperative infection and may provide guideposts for microbiome-targeted therapies.

Dietary- and host-derived metabolites are used by diverse gut bacteria for anaerobic respiration.

Respiratory reductases enable microorganisms to use molecules present in anaerobic ecosystems as energy-generating respiratory electron acceptors. Here we identify three taxonomically distinct families of human gut bacteria (Burkholderiaceae, Eggerthellaceae and Erysipelotrichaceae) that encode large arsenals of tens to hundreds of respiratory-like reductases per genome. Screening species from each family (Sutterella wadsworthensis, Eggerthella lenta and Holdemania filiformis), we discover 22 metabolites used as respiratory electron acceptors in a species-specific manner. Identified reactions transform multiple classes of dietary- and host-derived metabolites, including bioactive molecules resveratrol and itaconate. Products of identified respiratory metabolisms highlight poorly characterized compounds, such as the itaconate-derived 2-methylsuccinate. Reductase substrate profiling defines enzyme-substrate pairs and reveals a complex picture of reductase evolution, providing evidence that reductases with specificities for related cinnamate substrates independently emerged at least four times. These studies thus establish an exceptionally versatile form of anaerobic respiration that directly links microbial energy metabolism to the gut metabolome.

An evolution-based framework for describing human gut bacteria.

The human gut microbiome contains many bacterial strains of the same species ('strain-level variants'). Describing strains in a biologically meaningful way rather than purely taxonomically is an important goal but challenging due to the genetic complexity of strain-level variation. Here, we measured patterns of co-evolution across >7,000 strains spanning the bacterial tree-of-life. Using these patterns as a prior for studying hundreds of gut commensal strains that we isolated, sequenced, and metabolically profiled revealed widespread structure beneath the phylogenetic level of species. Defining strains by their co-evolutionary signatures enabled predicting their metabolic phenotypes and engineering consortia from strain genome content alone. Our findings demonstrate a biologically relevant organization to strain-level variation and motivate a new schema for describing bacterial strains based on their evolutionary history.

Exposure and resistance to lantibiotics impact microbiota composition and function.

The intestinal microbiota is composed of hundreds of distinct microbial species that interact with each other and their mammalian host. Antibiotic exposure dramatically impacts microbiota compositions and leads to acquisition of antibiotic-resistance genes. Lantibiotics are ribosomally synthesized and post-translationally modified peptides produced by some bacterial strains to inhibit the growth of competing bacteria. Nisin A is a lantibiotic produced by Lactococcus lactis that is commonly added to food products to reduce contamination with Gram-positive pathogens. Little is known, however, about lantibiotic-resistance of commensal bacteria inhabiting the human intestine. Herein, we demonstrate that Nisin A administration to mice alters fecal microbiome compositions and the concentration of taurine-conjugated primary bile acids. Lantibiotic Resistance System genes (LRS) are encoded by lantibiotic-producing bacterial strains but, we show, are also prevalent in microbiomes across human cohorts spanning vastly different lifestyles and 5 continents. Bacterial strains encoding LRS have enhanced in vivo fitness upon dietary exposure to Nisin A but reduced fitness in the absence of lantibiotic pressure. Differential binding of host derived, secreted IgA contributes to fitness discordance between bacterial strains encoding or lacking LRS. Although LRS are associated with mobile genetic elements, sequence comparisons of LRS encoded by distinct bacterial species suggest they have been long-term components of their respective genomes. Our study reveals the prevalence, abundance and physiologic significance of an underappreciated subset of antimicrobial resistance genes encoded by commensal bacterial species constituting the human gut microbiome, and provides insights that will guide development of microbiome augmenting strategies.