High-resolution analyses of associations between medications, microbiome, and mortality in cancer patients.

Discerning the effect of pharmacological exposures on intestinal bacterial communities in cancer patients is challenging. Here, we deconvoluted the relationship between drug exposures and changes in microbial composition by developing and applying a new computational method, PARADIGM (parameters associated with dynamics of gut microbiota), to a large set of longitudinal fecal microbiome profiles with detailed medication-administration records from patients undergoing allogeneic hematopoietic cell transplantation. We observed that several non-antibiotic drugs, including laxatives, antiemetics, and opioids, are associated with increased Enterococcus relative abundance and decreased alpha diversity. Shotgun metagenomic sequencing further demonstrated subspecies competition, leading to increased dominant-strain genetic convergence during allo-HCT that is significantly associated with antibiotic exposures. We integrated drug-microbiome associations to predict clinical outcomes in two validation cohorts on the basis of drug exposures alone, suggesting that this approach can generate biologically and clinically relevant insights into how pharmacological exposures can perturb or preserve microbiota composition. The application of a computational method called PARADIGM to a large dataset of cancer patients' longitudinal fecal specimens and detailed daily medication records reveals associations between drug exposures and the intestinal microbiota that recapitulate in vitro findings and are also predictive of clinical outcomes.

Microbiota-mediated inflammation and antimicrobial defense in the intestine.

The diverse microbial populations constituting the intestinal microbiota promote immune development and differentiation, but because of their complex metabolic requirements and the consequent difficulty culturing them, they remained, until recently, largely uncharacterized and mysterious. In the last decade, deep nucleic acid sequencing platforms, new computational and bioinformatics tools, and full-genome characterization of several hundred commensal bacterial species facilitated studies of the microbiota and revealed that differences in microbiota composition can be associated with inflammatory, metabolic, and infectious diseases, that each human is colonized by a distinct bacterial flora, and that the microbiota can be manipulated to reduce and even cure some diseases. Different bacterial species induce distinct immune cell populations that can play pro- and anti-inflammatory roles, and thus the composition of the microbiota determines, in part, the level of resistance to infection and susceptibility to inflammatory diseases. This review summarizes recent work characterizing commensal microbes that contribute to the antimicrobial defense/inflammation axis.

Innate Lymphocyte/Ly6C(hi) Monocyte Crosstalk Promotes Klebsiella Pneumoniae Clearance.

Increasing antibiotic resistance among bacterial pathogens has rendered some infections untreatable with available antibiotics. Klebsiella pneumoniae, a bacterial pathogen that has acquired high-level antibiotic resistance, is a common cause of pulmonary infections. Optimal clearance of K. pneumoniae from the host lung requires TNF and IL-17A. Herein, we demonstrate that inflammatory monocytes are rapidly recruited to the lungs of K. pneumoniae-infected mice and produce TNF, which markedly increases the frequency of IL-17-producing innate lymphoid cells. While pulmonary clearance of K. pneumoniae is preserved in neutrophil-depleted mice, monocyte depletion or TNF deficiency impairs IL-17A-dependent resolution of pneumonia. Monocyte-mediated bacterial uptake and killing is enhanced by ILC production of IL-17A, indicating that innate lymphocytes engage in a positive-feedback loop with monocytes that promotes clearance of pneumonia. Innate immune defense against a highly antibiotic-resistant bacterial pathogen depends on crosstalk between inflammatory monocytes and innate lymphocytes that is mediated by TNF and IL-17A.

From Hype to Hope: The Gut Microbiota in Enteric Infectious Disease.

One of the clearest functions of the gut microbiota in humans is resistance to colonization by enteric bacterial pathogens. Reconstitution of the microbiota offers an exciting therapeutic approach, but great challenges must be overcome.

Questioning the fetal microbiome illustrates pitfalls of low-biomass microbial studies.

Whether the human fetus and the prenatal intrauterine environment (amniotic fluid and placenta) are stably colonized by microbial communities in a healthy pregnancy remains a subject of debate. Here we evaluate recent studies that characterized microbial populations in human fetuses from the perspectives of reproductive biology, microbial ecology, bioinformatics, immunology, clinical microbiology and gnotobiology, and assess possible mechanisms by which the fetus might interact with microorganisms. Our analysis indicates that the detected microbial signals are likely the result of contamination during the clinical procedures to obtain fetal samples or during DNA extraction and DNA sequencing. Furthermore, the existence of live and replicating microbial populations in healthy fetal tissues is not compatible with fundamental concepts of immunology, clinical microbiology and the derivation of germ-free mammals. These conclusions are important to our understanding of human immune development and illustrate common pitfalls in the microbial analyses of many other low-biomass environments. The pursuit of a fetal microbiome serves as a cautionary example of the challenges of sequence-based microbiome studies when biomass is low or absent, and emphasizes the need for a trans-disciplinary approach that goes beyond contamination controls by also incorporating biological, ecological and mechanistic concepts.

Control of T cell antigen reactivity via programmed TCR downregulation.

The T cell antigen receptor (TCR) is unique in that its affinity for ligand is unknown before encounter and can vary by orders of magnitude. How the immune system regulates individual T cells that display very different reactivity to antigen remains unclear. Here we found that activated CD4(+) T cells, at the peak of clonal expansion, persistently downregulated their TCR expression in proportion to the strength of the initial antigen recognition. This programmed response increased the threshold for cytokine production and recall proliferation in a clone-specific manner and ultimately excluded clones with the highest antigen reactivity. Thus, programmed downregulation of TCR expression represents a negative feedback mechanism for constraining T cell effector function with a suitable time delay to thereby allow pathogen control while avoiding excess inflammatory damage.

Radical host-specific therapies for TB.

Although proinflammatory cytokines such as TNF are critical for containment of tuberculosis, they can also exacerbate disease when produced at high levels. In this issue of Cell, Roca and Ramakrishnan demonstrate that high TNF production induces reactive oxygen species in infected macrophages, ultimately leading to macrophage necrosis and bacterial dissemination.

Translating Microbiome Research From and To the Clinic.

Extensive research has elucidated the influence of the gut microbiota on human health and disease susceptibility and resistance. We review recent clinical and laboratory-based experimental studies associating the gut microbiota with certain human diseases. We also highlight ongoing translational advances that manipulate the gut microbiota to treat human diseases and discuss opportunities and challenges in translating microbiome research from and to the bedside.

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.

TAM mediates adaptation of carbapenem-resistant Klebsiella pneumoniae to antimicrobial stress during host colonization and infection.

Gram-negative pathogens, such as Klebsiella pneumoniae, remodel their outer membrane (OM) in response to stress to maintain its integrity as an effective barrier and thus to promote their survival in the host. The emergence of carbapenem-resistant K. pneumoniae (CR-Kp) strains that are resistant to virtually all antibiotics is an increasing clinical problem and OM impermeability has limited development of antimicrobial agents because higher molecular weight antibiotics cannot access sites of activity. Here, we demonstrate that TAM (translocation and assembly module) deletion increases CR-Kp OM permeability under stress conditions and enhances sensitivity to high-molecular weight antimicrobials. SILAC-based proteomic analyses revealed mis-localization of membrane proteins in the TAM deficient strain. Stress-induced sensitization enhances clearance of TAM-deficient CR-Kp from the gut lumen following fecal microbiota transplantation and from infection sites following pulmonary or systemic infection. Our study suggests that TAM, as a regulator of OM permeability, represents a potential target for development of agents that enhance the effectiveness of existing antibiotics.

Fecal microbiota diversity disruption and clinical outcomes after auto-HCT: a multicenter observational study.

We previously described clinically relevant reductions in fecal microbiota diversity in patients undergoing allogeneic hematopoietic cell transplantation (allo-HCT). Recipients of high-dose chemotherapy and autologous HCT (auto-HCT) incur similar antibiotic exposures and nutritional alterations. To characterize the fecal microbiota in the auto-HCT population, we analyzed 1161 fecal samples collected from 534 adult recipients of auto-HCT for lymphoma, myeloma, and amyloidosis in an observational study conducted at 2 transplantation centers in the United States. By using 16S ribosomal gene sequencing, we assessed fecal microbiota composition and diversity, as measured by the inverse Simpson index. At both centers, the diversity of early pretransplant fecal microbiota was lower in patients than in healthy controls and decreased further during the course of transplantation. Loss of diversity and domination by specific bacterial taxa occurred during auto-HCT in patterns similar to those with allo-HCT. Above-median fecal intestinal diversity in the periengraftment period was associated with decreased risk of death or progression (progression-free survival hazard ratio, 0.46; 95% confidence interval, 0.26-0.82; P = .008), adjusting for disease and disease status. This suggests that further investigation into the health of the intestinal microbiota in auto-HCT patients and posttransplant outcomes should be undertaken.

Microbiota-Based Therapies for Clostridium difficile and Antibiotic-Resistant Enteric Infections.

Bacterial pathogens are increasingly antibiotic resistant, and development of clinically effective antibiotics is lagging. Curing infections increasingly requires antimicrobials that are broader spectrum, more toxic, and more expensive, and mortality attributable to antibiotic-resistant pathogens is rising. The commensal microbiota, comprising microbes that colonize the mammalian gastrointestinal tract, can provide high levels of resistance to infection, and the contributions of specific bacterial species to resistance are being discovered and characterized. Microbiota-mediated mechanisms of colonization resistance and pathogen clearance include bactericidal activity, nutrient depletion, immune activation, and manipulation of the gut's chemical environment. Current research is focusing on development of microbiota-based therapies to reduce intestinal colonization with antibiotic-resistant pathogens, with the goal of reducing pathogen transmission and systemic dissemination.

The microbe-derived short-chain fatty acids butyrate and propionate are associated with protection from chronic GVHD.

Studies of the relationship between the gastrointestinal microbiota and outcomes in allogeneic hematopoietic stem cell transplantation (allo-HCT) have thus far largely focused on early complications, predominantly infection and acute graft-versus-host disease (GVHD). We examined the potential relationship of the microbiome with chronic GVHD (cGVHD) by analyzing stool and plasma samples collected late after allo-HCT using a case-control study design. We found lower circulating concentrations of the microbe-derived short-chain fatty acids (SCFAs) propionate and butyrate in day 100 plasma samples from patients who developed cGVHD, compared with those who remained free of this complication, in the initial case-control cohort of transplant patients and in a further cross-sectional cohort from an independent transplant center. An additional cross-sectional patient cohort from a third transplant center was analyzed; however, serum (rather than plasma) was available, and the differences in SCFAs observed in the plasma samples were not recapitulated. In sum, our findings from the primary case-control cohort and 1 of 2 cross-sectional cohorts explored suggest that the gastrointestinal microbiome may exert immunomodulatory effects in allo-HCT patients at least in part due to control of systemic concentrations of microbe-derived SCFAs.

Compositional Flux Within the Intestinal Microbiota and Risk for Bloodstream Infection With Gram-negative Bacteria.

BACKGROUND: Gram-negative bloodstream infections (BSIs) represent a significant complication facing allogeneic hematopoietic cell transplant (allo-HCT) recipients, as a result of intestinal translocation during neutropenia. In this study we sought to better understand how the composition of the intestinal microbiota is connected to risk of gram-negative BSIs, expanding on our prior work in these patients. METHODS: Fecal specimens were collected from recipients of allo-HCT and analyzed using 16S ribosomal RNA gene sequencing. Samples and clinical data extending from the pretransplant conditioning period through stem cell engraftment were used in the analysis. Intestinal domination (relative abundance ≥ 30%) by gram-negative bacteria was used as predictor of gram-negative BSI using Cox proportional hazards modeling. Further analysis of microbiota composition was performed at the genus level. RESULTS: Seven hundred eight allo-HCT subjects were studied (7.5% developed gram-negative infection), with 4768 fecal samples for analysis. Gram-negative intestinal domination was associated with subsequent BSI, which was observed overall and individually at the genus level: Escherichia, Klebsiella, Enterobacter, Pseudomonas, and Stenotrophomonas. Fluoroquinolone prophylaxis was associated with decreased BSI and intestinal colonization by gram-negative microbes. In fluoroquinolone-prophylaxed patients, Escherichia coli was more frequently observed as breakthrough, both in terms of intestinal colonization and BSIs, compared with nonprophylaxed patients. Initial colonization by members of Ruminococcaceae and Bacteroidetes were associated with protection against gram-negative BSI. CONCLUSIONS: Gram-negative intestinal colonization is highly predictive of BSI in the setting of allo-HCT. Fluoroquinolones appear to reduce these infections by influencing gut colonization.

Interleukin 23 production by intestinal CD103(+)CD11b(+) dendritic cells in response to bacterial flagellin enhances mucosal innate immune defense.

Microbial penetration of the intestinal epithelial barrier triggers inflammatory responses that include induction of the bactericidal C-type lectin RegIIIγ. Systemic administration of flagellin, a bacterial protein that stimulates Toll-like receptor 5 (TLR5), induces epithelial expression of RegIIIγ and protects mice from intestinal colonization with antibiotic-resistant bacteria. Flagellin-induced RegIIIγ expression is IL-22 dependent, but how TLR signaling leads to IL-22 expression is incompletely defined. By using conditional depletion of lamina propria dendritic cell (LPDC) subsets, we demonstrated that CD103(+)CD11b(+) LPDCs, but not monocyte-derived CD103(-)CD11b(+) LPDCs, expressed high amounts of IL-23 after bacterial flagellin administration and drove IL-22-dependent RegIIIγ production. Maximal expression of IL-23 subunits IL-23p19 and IL-12p40 occurred within 60 min of exposure to flagellin. IL-23 subsequently induced a burst of IL-22 followed by sustained RegIIIγ expression. Thus, CD103(+)CD11b(+) LPDCs, in addition to promoting long-term tolerance to ingested antigens, also rapidly produce IL-23 in response to detection of flagellin in the lamina propria.

Precision microbiome reconstitution restores bile acid mediated resistance to Clostridium difficile.

The gastrointestinal tracts of mammals are colonized by hundreds of microbial species that contribute to health, including colonization resistance against intestinal pathogens. Many antibiotics destroy intestinal microbial communities and increase susceptibility to intestinal pathogens. Among these, Clostridium difficile, a major cause of antibiotic-induced diarrhoea, greatly increases morbidity and mortality in hospitalized patients. Which intestinal bacteria provide resistance to C. difficile infection and their in vivo inhibitory mechanisms remain unclear. Here we correlate loss of specific bacterial taxa with development of infection, by treating mice with different antibiotics that result in distinct microbiota changes and lead to varied susceptibility to C. difficile. Mathematical modelling augmented by analyses of the microbiota of hospitalized patients identifies resistance-associated bacteria common to mice and humans. Using these platforms, we determine that Clostridium scindens, a bile acid 7α-dehydroxylating intestinal bacterium, is associated with resistance to C. difficile infection and, upon administration, enhances resistance to infection in a secondary bile acid dependent fashion. Using a workflow involving mouse models, clinical studies, metagenomic analyses, and mathematical modelling, we identify a probiotic candidate that corrects a clinically relevant microbiome deficiency. These findings have implications for the rational design of targeted antimicrobials as well as microbiome-based diagnostics and therapeutics for individuals at risk of C. difficile infection.

The intestinal microbiota: Antibiotics, colonization resistance, and enteric pathogens.

The human gastrointestinal tract hosts a diverse network of microorganisms, collectively known as the microbiota that plays an important role in health and disease. For instance, the intestinal microbiota can prevent invading microbes from colonizing the gastrointestinal tract, a phenomenon known as colonization resistance. Perturbations to the microbiota, such as antibiotic administration, can alter microbial composition and result in the loss of colonization resistance. Consequently, the host may be rendered susceptible to colonization by a pathogen. This is a particularly relevant concern in the hospital setting, where antibiotic use and antibiotic-resistant pathogen exposure are more frequent. Many nosocomial infections arise from gastrointestinal colonization. Due to their resistance to antibiotics, treatment is often very challenging. However, recent studies have demonstrated that manipulating the commensal microbiota can prevent and treat various infections in the intestine. In this review, we discuss the members of the microbiota, as well as the mechanisms, that govern colonization resistance against specific pathogens. We also review the effects of antibiotics on the microbiota, as well as the unique epidemiology of immunocompromised patients that renders them a particularly high-risk population to intestinal nosocomial infections.

Antibiotic Degradation by Commensal Microbes Shields Pathogens.

The complex bacterial populations that constitute the gut microbiota can harbor antibiotic resistance genes (ARGs), including those encoding ß-lactamase enzymes (BLA), which degrade commonly prescribed antibiotics such as ampicillin. The prevalence of such genes in commensal bacteria has been increased in recent years by the wide use of antibiotics in human populations and in livestock. While transfer of ARGs between bacterial species has well-established dramatic public health implications, these genes can also function in trans within bacterial consortia, where antibiotic-resistant bacteria can provide antibiotic-sensitive neighbors with leaky protection from drugs, as shown both in vitro and in vivo, in models of lung and subcutaneous coinfection. However, whether the expression of ARGs by harmless commensal bacterial species can destroy antibiotics in the intestinal lumen and shield antibiotic-sensitive pathogens is unknown. To address this question, we colonized germfree or wild-type mice with a model intestinal commensal strain of Escherichia coli that produces either functional or defective BLA. Mice were subsequently infected with Listeria monocytogenes or Clostridioides difficile, followed by treatment with oral ampicillin. The production of functional BLA by commensal E. coli markedly reduced clearance of these pathogens and enhanced systemic dissemination during ampicillin treatment. Pathogen resistance was independent of ARG acquisition via horizontal gene transfer but instead relied on antibiotic degradation in the intestinal lumen by BLA. We conclude that commensal bacteria that have acquired ARGs can mediate shielding of pathogens from the bactericidal effects of antibiotics.

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.

Impact of gut colonization with butyrate-producing microbiota on respiratory viral infection following allo-HCT.

Respiratory viral infections are frequent in patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HCT) and can potentially progress to lower respiratory tract infection (LRTI). The intestinal microbiota contributes to resistance against viral and bacterial pathogens in the lung. However, whether intestinal microbiota composition and associated changes in microbe-derived metabolites contribute to the risk of LRTI following upper respiratory tract viral infection remains unexplored in the setting of allo-HCT. Fecal samples from 360 allo-HCT patients were collected at the time of stem cell engraftment and subjected to deep, 16S ribosomal RNA gene sequencing to determine microbiota composition, and short-chain fatty acid levels were determined in a nested subset of fecal samples. The development of respiratory viral infections and LRTI was determined for 180 days following allo-HCT. Clinical and microbiota risk factors for LRTI were subsequently evaluated using survival analysis. Respiratory viral infection occurred in 149 (41.4%) patients. Of those, 47 (31.5%) developed LRTI. Patients with higher abundances of butyrate-producing bacteria were fivefold less likely to develop viral LRTI, independent of other factors (adjusted hazard ratio = 0.22, 95% confidence interval 0.04-0.69). Higher representation of butyrate-producing bacteria in the fecal microbiota is associated with increased resistance against respiratory viral infection with LRTI in allo-HCT patients.