Enhancing mucosal immunity by transient microbiota depletion.

Tissue resident memory CD8+ T cells (Trm) are poised for immediate reactivation at sites of pathogen entry and provide optimal protection of mucosal surfaces. The intestinal tract represents a portal of entry for many infectious agents; however, to date specific strategies to enhance Trm responses at this site are lacking. Here, we present TMDI (Transient Microbiota Depletion-boosted Immunization), an approach that leverages antibiotic treatment to temporarily restrain microbiota-mediated colonization resistance, and favor intestinal expansion to high densities of an orally-delivered Listeria monocytogenes strain carrying an antigen of choice. By augmenting the local chemotactic gradient as well as the antigenic load, this procedure generates a highly expanded pool of functional, antigen-specific intestinal Trm, ultimately enhancing protection against infectious re-challenge in mice. We propose that TMDI is a useful model to dissect the requirements for optimal Trm responses in the intestine, and also a potential platform to devise novel mucosal vaccination approaches.

Functional and Genomic Variation between Human-Derived Isolates of Lachnospiraceae Reveals Inter- and Intra-Species Diversity.

Bacteria belonging to the Lachnospiraceae family are abundant, obligate anaerobic members of the microbiota in healthy humans. Lachnospiraceae impact their hosts by producing short-chain fatty acids, converting primary to secondary bile acids, and facilitating colonization resistance against intestinal pathogens. To increase our understanding of genomic and functional diversity between members of this family, we cultured 273 Lachnospiraceae isolates representing 11 genera and 27 species from human donors and performed whole-genome sequencing assembly and annotation. This analysis revealed substantial inter- and intra-species diversity in pathways that likely influence an isolate's ability to impact host health. These differences are likely to impact colonization resistance through lantibiotic expression or intestinal acidification, influence host mucosal immune cells and enterocytes via butyrate production, or contribute to synergism within a consortium by heterogenous polysaccharide metabolism. Identification of these specific functions could facilitate development of probiotic bacterial consortia that drive and/or restore in vivo microbiome functions.

Impact of Antibiotic-Resistant Bacteria on Immune Activation and Clostridioides difficile Infection in the Mouse Intestine.

Antibiotic treatment of patients undergoing complex medical treatments can deplete commensal bacterial strains from the intestinal microbiota, thereby reducing colonization resistance against a wide range of antibiotic-resistant pathogens. Loss of colonization resistance can lead to marked expansion of vancomycin-resistant Enterococcus faecium (VRE), Klebsiella pneumoniae, and Escherichia coli in the intestinal lumen, predisposing patients to bloodstream invasion and sepsis. The impact of intestinal domination by these antibiotic-resistant pathogens on mucosal immune defenses and epithelial and mucin-mediated barrier integrity is unclear. We used a mouse model to study the impact of intestinal domination by antibiotic-resistant bacterial species and strains on the colonic mucosa. Intestinal colonization with K. pneumoniae, Proteus mirabilis, or Enterobacter cloacae promoted greater recruitment of neutrophils to the colonic mucosa. To test the hypothesis that the residual microbiota influences the severity of colitis caused by infection with Clostridioides difficile, we coinfected mice that were colonized with ampicillin-resistant bacteria with a virulent strain of C. difficile and monitored colonization and pathogenesis. Despite the compositional differences in the gut microbiota, the severity of C. difficile infection (CDI) and mortality did not differ significantly between mice colonized with different ampicillin-resistant bacterial species. Our results suggest that the virulence mechanisms enabling CDI and epithelial destruction outweigh the relatively minor impact of less-virulent antibiotic-resistant intestinal bacteria on the outcome of CDI.

Microbiota-derived lantibiotic restores resistance against vancomycin-resistant Enterococcus.

Intestinal commensal bacteria can inhibit dense colonization of the gut by vancomycin-resistant Enterococcus faecium (VRE), a leading cause of hospital-acquired infections1,2. A four-strained consortium of commensal bacteria that contains Blautia producta BPSCSK can reverse antibiotic-induced susceptibility to VRE infection3. Here we show that BPSCSK reduces growth of VRE by secreting a lantibiotic that is similar to the nisin-A produced by Lactococcus lactis. Although the growth of VRE is inhibited by BPSCSK and L. lactis in vitro, only BPSCSK colonizes the colon and reduces VRE density in vivo. In comparison to nisin-A, the BPSCSK lantibiotic has reduced activity against intestinal commensal bacteria. In patients at high risk of VRE infection, high abundance of the lantibiotic gene is associated with reduced density of E. faecium. In germ-free mice transplanted with patient-derived faeces, resistance to VRE colonization correlates with abundance of the lantibiotic gene. Lantibiotic-producing commensal strains of the gastrointestinal tract reduce colonization by VRE and represent potential probiotic agents to re-establish resistance to VRE.

Roles of Polycomb group proteins Enhancer of zeste (E(z)) and Polycomb (Pc) during metamorphosis and larval leg regeneration in the flour beetle Tribolium castaneum.

Many organisms both undergo dramatic morphological changes during post-embryonic development and also regenerate lost structures, but the roles of epigenetic regulators in such processes are only beginning to be understood. In the present study, the functions of two histone modifiers were examined during metamorphosis and larval limb regeneration in the red flour beetle Tribolium castaneum. Polycomb (Pc), a member of Polycomb repressive complex 1 (PRC1), and Enhancer of zeste (E(z)), a member of Polycomb repressive complex 2 (PRC2), were silenced in larvae using RNA interference. In the absence of Pc, the head appendages of adults transformed into a leg-like morphology, and the legs and wings assumed a metathoracic identity, indicating that Pc acts to specify proper segmental identity. Similarly, silencing of E(z) led to homeotic transformation of legs and wings. Additional defects were also observed in limb patterning as well as eye morphogenesis, indicating that PcG proteins play critical roles in imaginal precursor cells. In addition, larval legs and antennae failed to re-differentiate when either Pc or E(z) was knocked down, indicating that histone modification is necessary for proper blastema growth and differentiation. These findings indicate that PcG proteins play extensive roles in postembryonic plasticity of imaginal precursor cells.

Genome-Wide Screening for Enteric Colonization Factors in Carbapenem-Resistant ST258 Klebsiella pneumoniae.

A diverse, antibiotic-naive microbiota prevents highly antibiotic-resistant microbes, including carbapenem-resistant Klebsiella pneumoniae (CR-Kp), from achieving dense colonization of the intestinal lumen. Antibiotic-mediated destruction of the microbiota leads to expansion of CR-Kp in the gut, markedly increasing the risk of bacteremia in vulnerable patients. While preventing dense colonization represents a rational approach to reduce intra- and interpatient dissemination of CR-Kp, little is known about pathogen-associated factors that enable dense growth and persistence in the intestinal lumen. To identify genetic factors essential for dense colonization of the gut by CR-Kp, we constructed a highly saturated transposon mutant library with >150,000 unique mutations in an ST258 strain of CR-Kp and screened for in vitro growth and in vivo intestinal colonization in antibiotic-treated mice. Stochastic and partially reversible fluctuations in the representation of different mutations during dense colonization revealed the dynamic nature of intestinal microbial populations. We identified genes that are crucial for early and late stages of dense gut colonization and confirmed their role by testing isogenic mutants in in vivo competition assays with wild-type CR-Kp Screening of the transposon library also identified mutations that enhanced in vivo CR-Kp growth. These newly identified colonization factors may provide novel therapeutic opportunities to reduce intestinal colonization by CR-KpIMPORTANCEKlebsiella pneumoniae is a common cause of bloodstream infections in immunocompromised and hospitalized patients, and over the last 2 decades, some strains have acquired resistance to nearly all available antibiotics, including broad-spectrum carbapenems. The U.S. Centers for Disease Control and Prevention has listed carbapenem-resistant K. pneumoniae (CR-Kp) as an urgent public health threat. Dense colonization of the intestine by CR-Kp and other antibiotic-resistant bacteria is associated with an increased risk of bacteremia. Reducing the density of gut colonization by CR-Kp is likely to reduce their transmission from patient to patient in health care facilities as well as systemic infections. How CR-Kp expands and persists in the gut lumen, however, is poorly understood. Herein, we generated a highly saturated mutant library in a multidrug-resistant K. pneumoniae strain and identified genetic factors that are associated with dense gut colonization by K. pneumoniae This study sheds light on host colonization by K. pneumoniae and identifies potential colonization factors that contribute to high-density persistence of K. pneumoniae in the intestine.

Inhibiting antibiotic-resistant Enterobacteriaceae by microbiota-mediated intracellular acidification.

Klebsiella pneumoniae, Escherichia coli, and other members of the Enterobacteriaceae family are common human pathogens that have acquired broad antibiotic resistance, rendering infection by some strains virtually untreatable. Enterobacteriaceae are intestinal residents, but generally represent <1% of the adult colonic microbiota. Antibiotic-mediated destruction of the microbiota enables Enterobacteriaceae to expand to high densities in the colon, markedly increasing the risk of bloodstream invasion, sepsis, and death. Here, we demonstrate that an antibiotic-naive microbiota suppresses growth of antibiotic-resistant clinical isolates of Klebsiella pneumoniae, Escherichia coli, and Proteus mirabilis by acidifying the proximal colon and triggering short chain fatty acid (SCFA)-mediated intracellular acidification. High concentrations of SCFAs and the acidic environment counter the competitive edge that O2 and NO3 respiration confer upon Enterobacteriaceae during expansion. Reestablishment of a microbiota that produces SCFAs enhances clearance of Klebsiella pneumoniae, Escherichia coli, and Proteus mirabilis from the intestinal lumen and represents a potential therapeutic approach to enhance clearance of antibiotic-resistant pathogens.