Rebecca A. Carter

Innate Immune Defenses Mediated by Two ILC Subsets Are Critical for Protection against Acute Clostridium difficile Infection

Infection with the opportunistic enteric pathogen Clostridium difficile is an increasingly common clinical complication that follows antibiotic treatment-induced gut microbiota perturbation. Innate lymphoid cells (ILCs) are early responders to enteric pathogens; however, their role during C. difficile infection is undefined. To identify immune pathways that mediate recovery from C. difficile infection, we challenged C57BL/6, Rag1(-/-) (which lack T and B cells), and Rag2(-/-)Il2rg(-/-) (Ragγc(-/-)) mice (which additionally lack ILCs) with C. difficile. In contrast to Rag1(-/-) mice, ILC-deficient Ragγc(-/-) mice rapidly succumbed to infection. Rag1(-/-) but not Ragγc(-/-) mice upregulate expression of ILC1- or ILC3-associated proteins following C. difficile infection. Protection against infection was restored by transferring ILCs into Ragγc(-/-) mice. While ILC3s made a minor contribution to resistance, loss of IFN-γ or T-bet-expressing ILC1s in Rag1(-/-) mice increased susceptibility to C. difficile. These data demonstrate a critical role for ILC1s in defense against C. difficile.

Loss of Microbiota-Mediated Colonization Resistance to Clostridium difficile Infection With Oral Vancomycin Compared With Metronidazole

Antibiotic administration disrupts the intestinal microbiota, increasing susceptibility to pathogens such as Clostridium difficile. Metronidazole or oral vancomycin can cure C. difficile infection, and administration of these agents to prevent C. difficile infection in high-risk patients, although not sanctioned by Infectious Disease Society of America guidelines, has been considered. The relative impacts of metronidazole and vancomycin on the intestinal microbiota and colonization resistance are unknown. We investigated the effect of brief treatment with metronidazole and/or oral vancomycin on susceptibility to C. difficile, vancomycin-resistant Enterococcus, carbapenem-resistant Klebsiella pneumoniae, and Escherichia coli infection in mice. Although metronidazole resulted in transient loss of colonization resistance, oral vancomycin markedly disrupted the microbiota, leading to prolonged loss of colonization resistance to C. difficile infection and dense colonization by vancomycin-resistant Enterococcus, K. pneumoniae, and E. coli. Our results demonstrate that vancomycin, and to a lesser extent metronidazole, are associated with marked intestinal microbiota destruction and greater risk of colonization by nosocomial pathogens.

Distinct but Spatially Overlapping Intestinal Niches for Vancomycin-Resistant Enterococcus faecium and Carbapenem-Resistant Klebsiella pneumoniae

Antibiotic resistance among enterococci and γ-proteobacteria is an increasing problem in healthcare settings. Dense colonization of the gut by antibiotic-resistant bacteria facilitates their spread between patients and also leads to bloodstream and other systemic infections. Antibiotic-mediated destruction of the intestinal microbiota and consequent loss of colonization resistance are critical factors leading to persistence and spread of antibiotic-resistant bacteria. The mechanisms underlying microbiota-mediated colonization resistance remain incompletely defined and are likely distinct for different antibiotic-resistant bacterial species. It is unclear whether enterococci or γ-proteobacteria, upon expanding to high density in the gut, confer colonization resistance against competing bacterial species. Herein, we demonstrate that dense intestinal colonization with vancomycin-resistant Enterococcus faecium (VRE) does not reduce in vivo growth of carbapenem-resistant Klebsiella pneumoniae. Reciprocally, K. pneumoniae does not impair intestinal colonization by VRE. In contrast, transplantation of a diverse fecal microbiota eliminates both VRE and K. pneumoniae from the gut. Fluorescence in situ hybridization demonstrates that VRE and K. pneumoniae localize to the same regions in the colon but differ with respect to stimulation and invasion of the colonic mucus layer. While VRE and K. pneumoniae occupy the same three-dimensional space within the gut lumen, their independent growth and persistence in the gut suggests that they reside in distinct niches that satisfy their specific in vivo metabolic needs.

TLR-7 activation enhances IL-22–mediated colonization resistance against vancomycin-resistant enterococcus

A synthetic TLR-7 agonist restores immune defenses to an antibiotic-resistant pathogen lost due to antibiotic use. Combating antibiotic resistance Antibiotics are the front line of defense against many bacterial infections, but antibiotics also disrupt the intestinal microbiota, which provides a barrier against colonization by other pathogens, such as vancomycin-resistant Enterococcus faecium (VRE). Abt et al. now find that a synthetic ligand for Toll-like receptor 7 stimulates antiviral innate immune responses in the intestine similar to viral infection, restoring colonization resistance to VRE. Thus, this orally bioavailable therapy may serve to limit infection by intestinal pathogens in antibiotic-exposed, susceptible individuals. Antibiotic administration can disrupt the intestinal microbiota and down-regulate innate immune defenses, compromising colonization resistance against orally acquired bacterial pathogens. Vancomycin-resistant Enterococcus faecium (VRE), a major cause of antibiotic-resistant infections in hospitalized patients, thrives in the intestine when colonization resistance is compromised, achieving extremely high densities that can lead to bloodstream invasion and sepsis. Viral infections, by mechanisms that remain incompletely defined, can stimulate resistance against invading bacterial pathogens. We report that murine norovirus infection correlates with reduced density of VRE in the intestinal tract of mice with antibiotic-induced loss of colonization resistance. Resiquimod (R848), a synthetic ligand for Toll-like receptor 7 (TLR-7) that stimulates antiviral innate immune defenses, restores expression of the antimicrobial peptide Reg3γ and reestablishes colonization resistance against VRE in antibiotic-treated mice. Orally administered R848 triggers TLR-7 on CD11c+ dendritic cells, inducing interleukin-23 (IL-23) expression followed by a burst of IL-22 secretion by innate lymphoid cells, leading to Reg3γ expression and restoration of colonization resistance against VRE. Our findings reveal that an orally bioavailable TLR-7 ligand that stimulates innate antiviral immune pathways in the intestine restores colonization resistance against a highly antibiotic-resistant bacterial pathogen.

Commensal microbes provide first line defense against Listeria monocytogenes infection.

Listeria monocytogenes is a foodborne pathogen that causes septicemia, meningitis and chorioamnionitis and is associated with high mortality. Immunocompetent humans and animals, however, can tolerate high doses of L. monocytogenes without developing systemic disease. The intestinal microbiota provides colonization resistance against many orally acquired pathogens, and antibiotic-mediated depletion of the microbiota reduces host resistance to infection. Here we show that a diverse microbiota markedly reduces Listeria monocytogenes colonization of the gut lumen and prevents systemic dissemination. Antibiotic administration to mice before low dose oral inoculation increases L. monocytogenes growth in the intestine. In immunodeficient or chemotherapy-treated mice, the intestinal microbiota provides nonredundant defense against lethal, disseminated infection. We have assembled a consortium of commensal bacteria belonging to the Clostridiales order, which exerts in vitro antilisterial activity and confers in vivo resistance upon transfer into germ free mice. Thus, we demonstrate a defensive role of the gut microbiota against Listeria monocytogenes infection and identify intestinal commensal species that, by enhancing resistance against this pathogen, represent potential probiotics.

Bile acid sensitivity and in vivo virulence of clinical Clostridium difficile isolates

Clostridium difficile is an anaerobic bacterium that causes diarrheal illnesses. Disease onset is linked with exposure to oral antibiotics and consequent depletion of secondary bile acids. Here we investigate the relationship between in vitro secondary bile acid tolerance and in vivo disease scores of diverse C. difficile strains in mice.

Pathogenicity Locus, Core Genome, and Accessory Gene Contributions to Clostridium difficile Virulence

ABSTRACT Clostridium difficile is a spore-forming anaerobic bacterium that causes colitis in patients with disrupted colonic microbiota. While some individuals are asymptomatic C. difficile carriers, symptomatic disease ranges from mild diarrhea to potentially lethal toxic megacolon. The wide disease spectrum has been attributed to the infected host’s age, underlying diseases, immune status, and microbiome composition. However, strain-specific differences in C. difficile virulence have also been implicated in determining colitis severity. Because patients infected with C. difficile are unique in terms of medical history, microbiome composition, and immune competence, determining the relative contribution of C. difficile virulence to disease severity has been challenging, and conclusions regarding the virulence of specific strains have been inconsistent. To address this, we used a mouse model to test 33 clinical C. difficile strains isolated from patients with disease severities ranging from asymptomatic carriage to severe colitis, and we determined their relative in vivo virulence in genetically identical, antibiotic-pretreated mice. We found that murine infections with C. difficile clade 2 strains (including multilocus sequence type 1/ribotype 027) were associated with higher lethality and that C. difficile strains associated with greater human disease severity caused more severe disease in mice. While toxin production was not strongly correlated with in vivo colonic pathology, the ability of C. difficile strains to grow in the presence of secondary bile acids was associated with greater disease severity. Whole-genome sequencing and identification of core and accessory genes identified a subset of accessory genes that distinguish high-virulence from lower-virulence C. difficile strains. IMPORTANCE Clostridium difficile is an important cause of hospital-associated intestinal infections, and recent years have seen an increase in the number and severity of cases in the United States. A patient’s antibiotic history, immune status, and medical comorbidities determine, in part, the severity of C. difficile infection. The relative virulence of different clinical C. difficile strains, although postulated to determine disease severity in patients, has been more difficult to consistently associate with mild versus severe colitis. We tested 33 distinct clinical C. difficile isolates for their ability to cause disease in genetically identical mice and found that C. difficile strains belonging to clade 2 were associated with higher mortality. Differences in survival were not attributed to differences in toxin production but likely resulted from the distinct gene content in the various clinical isolates. IMPORTANCE Clostridium difficile is an important cause of hospital-associated intestinal infections, and recent years have seen an increase in the number and severity of cases in the United States. A patient’s antibiotic history, immune status, and medical comorbidities determine, in part, the severity of C. difficile infection. The relative virulence of different clinical C. difficile strains, although postulated to determine disease severity in patients, has been more difficult to consistently associate with mild versus severe colitis. We tested 33 distinct clinical C. difficile isolates for their ability to cause disease in genetically identical mice and found that C. difficile strains belonging to clade 2 were associated with higher mortality. Differences in survival were not attributed to differences in toxin production but likely resulted from the distinct gene content in the various clinical isolates.

Abstract A082: Identification of commensal bacterial strains that provide resistance to L. monocytogenes infection

Listeria monocytogenes is an important cause of foodborne infections, particularly in patients with hematologic malignancies, causing septicemia and meningoencephalitis. Although most strains of L. monocytogenes are antibiotic sensitive, antibiotic treatment is frustratingly ineffective at curing highly immunocompromised patients, resulting in a mortality rate of approximately 30%. The gut microbiota, which consists of commensal bacterial populations that inhabit the intestine, normally confers protection against orally acquired pathogens by exerting colonization resistance. Changes in microbiota composition induced by disease, pharmacological or antibiotic therapies can lead to dysbiosis, thereby impairing commensal-mediated colonization resistance and predisposing to infection. Our preliminary data show that bacterial species represented in a healthy microbiota can efficiently eliminate Listeria monocytogenes from the gut lumen, thus preventing the pathogen from translocating across the intestinal epithelium and spreading systemically. This mechanism of protection is particularly important in the context of congenic or acquired (chemotherapy-driven) immune deficiencies. Indeed, mice lacking T and B lymphocyets as well as innate lymphoid cells (Rag2/Il2rg Double Knockout mice, or Raggc), are highly susceptible to oral doses of Listeria that are non-lethal in WT mice. Although generally implicated in susceptibility to Listeriosis, the adaptive immune system is not involved in the in vivo clearance of the pathogen from the intestine, and the increased susceptibility to infection of these mice mainly results from the absence of innate immune cells producing interferon gamma at early stages, which leads to bacterial dissemination. Interestingly, Raggc mice are capable of surviving Listeria infection if challenged with low doses, which likely to mimicks the bacterial dosage acquired by immunocompromised patients. However, Raggc mice pre-treated with antibiotics experience uncontrolled intestinal growth of Listeria and subsequent systemic spread thus succumbing to low inoculum infection. This phenotype could be reproduced in mice treated with a combination of doxorubicin and cyclophosphamide, a cocktail often used in the treatment of cancer; although chemotherapy seemed to have an effect on the ability of the microbiota to restrict instestinal growth of Listeria, chemotherapy-treated mice became highly susceptible to low doses of Listeria only when pre-treated with antibiotics. Because antibiotics are often required for patients receiving cancer treatment, our these findings have potential clinical relevance. We are identifying specific commensal bacterial taxa and molecular mechanisms that mediate protection against L. monocytogenes. Our data indicate that Lactobacillus in the small intestine and Clostridium in the large intestine restrict Listeria growth in the gut lumen, thus lowering the bacterial burden and systemic spread. Germ-free (GF) mice reconstituted with a consortium of putative protective bacteria have greater resistance to infection than untreated GF mice or GF mice reconstituted with microbiota of antibiotic-treated mice, suggesting that gut commensals are independent and crucial contributors to host defense against Listeria. Our ultimate goal is to identify bacterial species that can be developed as probiotics for cancer patients to prevent intestinal L. monocytogenes carriage and infection. Combined shotgun sequencing and ex vivo assays on stool samples from cancer patients will be used to correlate loss of specific commensal bacteria with a predisposition to L. monocytogenes infection. Ultimately, this work will 1.) provide a novel approach to identifying subjects at high risk for L. monocytogenes infection and 2.) identify and characterize probiotic bacterial species to prevent and/or treat L. monocytogenes infections in cancer patients. Citation Format: Simone Becattini, Sohn G. Kim, Rebecca A. Carter, Lilan Ling, Ingrid M. Leiner, Eric G. Pamer. Identification of commensal bacterial strains that provide resistance to L. monocytogenes infection [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A082.

Abstract A100: Cooperative defense against acute Clostridium difficile infection is mediated by two innate lymphoid cell subsets

Clostridium difficile, an opportunistic pathogen that infects the gastrointestinal tract following perturbation of the microbiota, damages the intestinal epithelium and can cause debilitating and potentially fatal colitis. Antibiotic treatment of C. difficile is often ineffective and whether innate immune defenses can be exploited to promote C. difficile clearance is unknown. Innate lymphoid cells (ILCs) are early responders to enteric infection, however, their role during C. difficile infection is undefined. To identify immune pathways that mediate recovery from C. difficile infection, we compared C57BL/6, Rag1-/-, and Rag2-/-Il2rg-/- (Ragγc-/-) mice challenged with C. difficile. In contrast to C57BL/6 and Rag1-/- mice, ILC-deficient Ragγc-/- mice rapidly succumbed to infection. Protection against infection was restored by transferring ILCs into Ragγc-/- mice. While ILC3s made a minor contribution to resistance, loss of T-bet-expressing ILC1s or IFN-γ in Rag1-/- mice increased susceptibility to C. difficile. These data demonstrate a critical role for ILC1s in defense against C. difficile. Citation Format: Michael C. Abt, Brittany B. Lewis, Lilan Ling, Rebecca A. Carter, Boj Susac, Eric G. Pamer. Cooperative defense against acute Clostridium difficile infection is mediated by two innate lymphoid cell subsets. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A100.