Intestinal microbiota containing Barnesiella species cures vancomycin-resistant Enterococcus faecium colonization.

Bacteria causing infections in hospitalized patients are increasingly antibiotic resistant. Classical infection control practices are only partially effective at preventing spread of antibiotic-resistant bacteria within hospitals. Because the density of intestinal colonization by the highly antibiotic-resistant bacterium vancomycin-resistant Enterococcus (VRE) can exceed 10(9) organisms per gram of feces, even optimally implemented hygiene protocols often fail. Decreasing the density of intestinal colonization, therefore, represents an important approach to limit VRE transmission. We demonstrate that reintroduction of a diverse intestinal microbiota to densely VRE-colonized mice eliminates VRE from the intestinal tract. While oxygen-tolerant members of the microbiota are ineffective at eliminating VRE, administration of obligate anaerobic commensal bacteria to mice results in a billionfold reduction in the density of intestinal VRE colonization. 16S rRNA gene sequence analysis of intestinal bacterial populations isolated from mice that cleared VRE following microbiota reconstitution revealed that recolonization with a microbiota that contains Barnesiella correlates with VRE elimination. Characterization of the fecal microbiota of patients undergoing allogeneic hematopoietic stem cell transplantation demonstrated that intestinal colonization with Barnesiella confers resistance to intestinal domination and bloodstream infection with VRE. Our studies indicate that obligate anaerobic bacteria belonging to the Barnesiella genus enable clearance of intestinal VRE colonization and may provide novel approaches to prevent the spread of highly antibiotic-resistant bacteria.

Familial transmission rather than defective innate immunity shapes the distinct intestinal microbiota of TLR-deficient mice.

The intestinal microbiota contributes to the development of the immune system, and conversely, the immune system influences the composition of the microbiota. Toll-like receptors (TLRs) in the gut recognize bacterial ligands. Although TLR signaling represents a major arm of the innate immune system, the extent to which TLRs influence the composition of the intestinal microbiota remains unclear. We performed deep 16S ribosomal RNA sequencing to characterize the complex bacterial populations inhabiting the ileum and cecum of TLR- and MyD88-deficient mice. The microbiota of MyD88- and TLR-deficient mouse colonies differed markedly, with each colony harboring distinct and distinguishable bacterial populations in the small and large intestine. Comparison of MyD88-, TLR2-, TLR4-, TLR5-, and TLR9-deficient mice and their respective wild-type (WT) littermates demonstrated that the impact of TLR deficiency on the composition of the intestinal microbiota is minimal under homeostatic conditions and after recovery from antibiotic treatment. Thus, differences between TLR-deficient mouse colonies reflected long-term divergence of the microbiota after extended husbandry in isolation from each other. Long-term breeding of isolated mouse colonies results in changes of the intestinal microbiota that are communicated to offspring by maternal transmission, which account for marked compositional differences between WT and mutant mouse strains.

Critical role for MyD88-mediated neutrophil recruitment during Clostridium difficile colitis.

Clostridium difficile can infect the large intestine and cause colitis when the normal intestinal microbiota is altered by antibiotic administration. Little is known about the innate immune signaling pathways that marshal inflammatory responses to C. difficile infection and whether protective and pathogenic inflammatory responses can be dissociated. Toll-like receptors predominantly signal via the MyD88 adaptor protein and are important mediators of innate immune signaling in the intestinal mucosa. Here, we demonstrate that MyD88-mediated signals trigger neutrophil and CCR2-dependent Ly6C(hi) monocyte recruitment to the colonic lamina propria (cLP) during infection, which prevent dissemination of bystander bacteria to deeper tissues. Mortality is markedly increased in MyD88-deficient mice following C. difficile infection, as are parameters of mucosal tissue damage and inflammation. Antibody-mediated depletion of neutrophils markedly increases mortality, while attenuated recruitment of Ly6C(hi) monocytes in CCR2-deficient mice does not alter the course of C. difficile infection. Expression of CXCL1, a neutrophil-recruiting chemokine, is impaired in the cLP of MyD88(-/-) mice. Our studies suggest that MyD88-mediated signals promote neutrophil recruitment by inducing expression of CXCL1, thereby providing critical early defense against C. difficile-mediated colitis.

Periodontal disease and the oral microbiota in new-onset rheumatoid arthritis.

OBJECTIVE: To profile the abundance and diversity of subgingival oral microbiota in patients with never-treated, new-onset rheumatoid arthritis (RA). METHODS: Periodontal disease (PD) status, clinical activity, and sociodemographic factors were determined in patients with new-onset RA, patients with chronic RA, and healthy subjects. Multiplexed-454 pyrosequencing was used to compare the composition of subgingival microbiota and establish correlations between the presence/abundance of bacteria and disease phenotypes. Anti-Porphyromonas gingivalis antibody testing was performed to assess prior exposure to the bacterial pathogen P gingivalis. RESULTS: The more advanced forms of periodontitis were already present at disease onset in patients with new-onset RA. The subgingival microbiota observed in patients with new-onset RA was distinct from that found in healthy controls. In most cases, however, these microbial differences could be attributed to the severity of PD and were not inherent to RA. The presence and abundance of P gingivalis were also directly associated with the severity of PD and were not unique to RA. The presence of P gingivalis was not correlated with anti-citrullinated protein antibody (ACPA) titers. Overall exposure to P gingivalis was similar between patients with new-onset RA and controls, observed in 78% of patients and 83% of controls. The presence and abundance of Anaeroglobus geminatus correlated with the presence of ACPAs/rheumatoid factor. Prevotella and Leptotrichia species were the only characteristic taxa observed in patients with new-onset RA irrespective of PD status. CONCLUSION: Patients with new-onset RA exhibited a high prevalence of PD at disease onset, despite their young age and paucity of smoking history. The subgingival microbiota profile in patients with new-onset RA was similar to that in patients with chronic RA and healthy subjects whose PD was of comparable severity. Although colonization with P gingivalis correlated with the severity of PD, overall exposure to P gingivalis was similar among the groups. The role of A geminatus and Prevotella/Leptotrichia species in this process merits further study.

Regulation of intestinal inflammation by microbiota following allogeneic bone marrow transplantation.

Despite a growing understanding of the link between intestinal inflammation and resident gut microbes, longitudinal studies of human flora before initial onset of intestinal inflammation have not been reported. Here, we demonstrate in murine and human recipients of allogeneic bone marrow transplantation (BMT) that intestinal inflammation secondary to graft-versus-host disease (GVHD) is associated with major shifts in the composition of the intestinal microbiota. The microbiota, in turn, can modulate the severity of intestinal inflammation. In mouse models of GVHD, we observed loss of overall diversity and expansion of Lactobacillales and loss of Clostridiales. Eliminating Lactobacillales from the flora of mice before BMT aggravated GVHD, whereas reintroducing the predominant species of Lactobacillus mediated significant protection against GVHD. We then characterized gut flora of patients during onset of intestinal inflammation caused by GVHD and found patterns mirroring those in mice. We also identified increased microbial chaos early after allogeneic BMT as a potential risk factor for subsequent GVHD. Together, these data demonstrate regulation of flora by intestinal inflammation and suggest that flora manipulation may reduce intestinal inflammation and improve outcomes for allogeneic BMT recipients.

Profound alterations of intestinal microbiota following a single dose of clindamycin results in sustained susceptibility to Clostridium difficile-induced colitis.

Antibiotic-induced changes in the intestinal microbiota predispose mammalian hosts to infection with antibiotic-resistant pathogens. Clostridium difficile is a Gram-positive intestinal pathogen that causes colitis and diarrhea in patients following antibiotic treatment. Clindamycin predisposes patients to C. difficile colitis. Here, we have used Roche-454 16S rRNA gene pyrosequencing to longitudinally characterize the intestinal microbiota of mice following clindamycin treatment in the presence or absence of C. difficile infection. We show that a single dose of clindamycin markedly reduces the diversity of the intestinal microbiota for at least 28 days, with an enduring loss of ca. 90% of normal microbial taxa from the cecum. Loss of microbial complexity results in dramatic sequential expansion and contraction of a subset of bacterial taxa that are minor contributors to the microbial consortium prior to antibiotic treatment. Inoculation of clindamycin-treated mice with C. difficile (VPI 10463) spores results in rapid development of diarrhea and colitis, with a 4- to 5-day period of profound weight loss and an associated 40 to 50% mortality rate. Recovering mice resolve diarrhea and regain weight but remain highly infected with toxin-producing vegetative C. difficile bacteria and, in comparison to the acute stage of infection, have persistent, albeit ameliorated cecal and colonic inflammation. The microbiota of "recovered" mice remains highly restricted, and mice remain susceptible to C. difficile infection at least 10 days following clindamycin, suggesting that resolution of diarrhea and weight gain may result from the activation of mucosal immune defenses.

Ly6G+ neutrophils are dispensable for defense against systemic Listeria monocytogenes infection.

Listeria monocytogenes is a facultative intracellular bacterium that causes systemic infections in immunocompromised hosts. Early recruitment of myeloid cells, including inflammatory monocytes and neutrophils, to sites of L. monocytogenes infection is essential for the control of infection and host survival. Because previous experimental studies used depleting or blocking Abs that affected both inflammatory monocytes and neutrophils, the relative contributions of these cell populations to defense against L. monocytogenes infection remain incompletely defined. In this article, we used highly selective depletion strategies to either deplete inflammatory monocytes or neutrophils from L. monocytogenes-infected mice and demonstrate that neutrophils are dispensable for early and late control of infection. In contrast, inflammatory monocytes are essential for bacterial clearance during the innate and adaptive phases of the immune response to L. monocytogenes infection.

Vancomycin-resistant Enterococcus domination of intestinal microbiota is enabled by antibiotic treatment in mice and precedes bloodstream invasion in humans.

Bloodstream infection by highly antibiotic-resistant bacteria, such as vancomycin-resistant Enterococcus (VRE), is a growing clinical problem that increasingly defies medical intervention. Identifying patients at high risk for bacterial sepsis remains an important clinical challenge. Recent studies have shown that antibiotics can alter microbial diversity in the intestine. Here, we characterized these effects using 16s rDNA pyrosequencing and demonstrated that antibiotic treatment of mice enabled exogenously administered VRE to efficiently and nearly completely displace the normal microbiota of the small and large intestine. In the clinical setting, we found that intestinal domination by VRE preceded bloodstream infection in patients undergoing allogeneic hematopoietic stem cell transplantation. Our results demonstrate that antibiotics perturb the normal commensal microbiota and set the stage for intestinal domination by bacteria associated with hospital-acquired infections. Thus, high-throughput DNA sequencing of the intestinal microbiota could identify patients at high risk of developing bacterial sepsis.