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.

Outbreaks of Typhlocolitis Caused by Hypervirulent Group ST1 Clostridioides difficile in Highly Immunocompromised Strains of Mice.

Clostridioides difficile is an enteric pathogen that can cause significant clinical disease in both humans and animals. However, clinical disease arises most commonly after treatment with broad-spectrum antibiotics. The organism's ability to cause naturally occurring disease in mice is rare, and little is known about its clinical significance in highly immunocompromised mice. We report on 2 outbreaks of diarrhea associated with C. difficile in mice. In outbreak 1, 182 of approximately 2, 400 NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) and related strains of mice became clinically ill after cessation of a 14-d course of 0.12% amoxicillin feed to control an increase in clinical signs associated with Corynebacterium bovis infection. Most mice had been engrafted with human tumors; the remainder were experimentally naïve. Affected animals exhibited 1 of 3 clinical syndromes: 1) peracute death; 2) severe diarrhea leading to euthanasia or death; or 3) mild to moderate diarrhea followed by recovery. A given cage could contain both affected and unaffected mice. Outbreak 2 involved a small breeding colony (approximately 50 mice) of NOD. CB17-Prkdcscid/NCrCrl (NOD-scid) mice that had not received antibiotics or experimental manipulations. In both outbreaks, C. difficile was isolated, and toxins A and B were detected in intestinal content or feces. Histopathologic lesions highly suggestive of C. difficile enterotoxemia included fibrinonecrotizing and neutrophilic typhlocolitis with characteristic 'volcano' erosions or pseudomembrane formation. Genomic analysis of 4 isolates (3 from outbreak 1 and 1 from outbreak 2) revealed that these isolates were closely related to a pathogenic human isolate, CD 196. To our knowledge, this report is the first to describe naturally occurring outbreaks of C. difficile-associated typhlocolitis with significant morbidity and mortality in highly immunocompromised strains of mice.

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.

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.

Distinct behavior of myelomonocytic cells and CD8 T cells underlies the hepatic response to Listeria monocytogenes.

Background: The immune response to Listeria monocytogenes (LM) is characterized by formation of leukocyte rich foci of infection in liver and spleen.  Although much has been gained in our understanding of immune response through the study of LM, little is known about spatio-temporal regulation of immune response to Listeria in liver. Methods: We utilize a combination of molecular, genetic and intravital microscopic approaches to gain insight into the dynamics of foci and leukocyte behavior during hepatic Listeriosis.  Results: LM foci efficiently exclude blood flow, indicating the presence of a barrier separating the foci and healthy tissue.  Despite this barrier, sinusoidal myelomonocytic cells readily enter or transiently interact with cells at the edge of foci of infection.  Next, utilizing L9.6 transgenic CD8 + T cells specific for an endogenously processed LM antigen, p60 217-225, along with LM deficient in this epitope, we define the role of TCR in T cell migratory behavior in infected liver.  Surprisingly, T cell behavior varies with micro-anatomic locale.  Near foci, non-specific adhesion mechanisms dominate lymphocyte behavior.  Antigen specific effects on motility became detectable only distal to foci.  Conclusions: These data suggest that LM antigens act in a paracrine manner to mediate protection from Listeriosis in the liver.

Inflammatory Monocytes Promote Perineural Invasion via CCL2-Mediated Recruitment and Cathepsin B Expression.

Perineural invasion (PNI) is an ominous event strongly linked to poor clinical outcome. Cells residing within peripheral nerves collaborate with cancer cells to enable PNI, but the contributing conditions within the tumor microenvironment are not well understood. Here, we show that CCR2-expressing inflammatory monocytes (IM) are preferentially recruited to sites of PNI, where they differentiate into macrophages and potentiate nerve invasion through a cathepsin B-mediated process. A series of adoptive transfer experiments with genetically engineered donors and recipients demonstrated that IM recruitment to nerves was driven by CCL2 released from Schwann cells at the site of PNI, but not CCL7, an alternate ligand for CCR2. Interruption of either CCL2-CCR2 signaling or cathepsin B function significantly impaired PNI in vivo Correlative studies in human specimens demonstrated that cathepsin B-producing macrophages were enriched in invaded nerves, which was associated with increased local tumor recurrence. These findings deepen our understanding of PNI pathogenesis and illuminate how PNI is driven in part by corruption of a nerve repair program. Further, they support the exploration of inhibiting IM recruitment and function as a targeted therapy for PNI. Cancer Res; 77(22); 6400-14. ©2017 AACR.

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

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.IMPORTANCEClostridium 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.

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.

Cooperating Commensals Restore Colonization Resistance to Vancomycin-Resistant Enterococcus faecium.

Antibiotic-mediated microbiota destruction and the consequent loss of colonization resistance can result in intestinal domination with vancomycin-resistant Enterococcus (VRE), leading to bloodstream infection in hospitalized patients. Clearance of VRE remains a challenging goal that, if achieved, would reduce systemic VRE infections and patient-to-patient transmission. Although obligate anaerobic commensal bacteria have been associated with colonization resistance to VRE, the specific bacterial species involved remain undefined. Herein, we demonstrate that a precisely defined consortium of commensal bacteria containing the Clostridium cluster XIVa species Blautia producta and Clostridium bolteae restores colonization resistance against VRE and clears VRE from the intestines of mice. While C. bolteae did not directly mediate VRE clearance, it enabled intestinal colonization with B. producta, which directly inhibited VRE growth. These findings suggest that therapeutic or prophylactic administration of defined bacterial consortia to individuals with compromised microbiota composition may reduce inter-patient transmission and intra-patient dissemination of highly antibiotic-resistant pathogens.

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.

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

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.

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.

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.

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.

Distinct Contributions of Neutrophils and CCR2+ Monocytes to Pulmonary Clearance of Different Klebsiella pneumoniae Strains.

Klebsiella pneumoniae is a common respiratory pathogen, with some strains having developed broad resistance to clinically available antibiotics. Humans can become infected with many different K. pneumoniae strains that vary in genetic background, antibiotic susceptibility, capsule composition, and mucoid phenotype. Genome comparisons have revealed differences between K. pneumoniae strains, but the impact of genomic variability on immune-mediated clearance of pneumonia remains unclear. Experimental studies of pneumonia in mice have used the rodent-adapted 43816 strain of K. pneumoniae and demonstrated that neutrophils are essential for optimal host defense. It remains unclear, however, whether CCR2(+) monocytes contribute to K. pneumoniae clearance from the lung. We selectively depleted neutrophils, CCR2(+) monocytes, or both from immunocompetent mice and determined susceptibility to infection by the 43816 strain and 4 newly isolated clinical K. pneumoniae strains. The clinical K. pneumoniae strains, including one carbapenem-resistant ST258 strain, are less virulent than 43816. Optimal clearance of each of the 5 strains required either neutrophils or CCR2(+) monocytes. Selective neutrophil depletion markedly worsened infection with K. pneumoniae strain 43816 and three clinical isolates but did not increase susceptibility of mice to infection with the carbapenem-resistant K. pneumoniae ST258 strain. Depletion of CCR2(+) monocytes delayed recovery from infection with each of the 5 K. pneumoniae strains, revealing a contribution of these cells to bacterial clearance from the lung. Our findings demonstrate strain-dependent variation in the contributions of neutrophils and CCR2(+) monocytes to clearance of K. pneumoniae pulmonary infection.

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.

Inflammatory monocytes orchestrate innate antifungal immunity in the lung.

Aspergillus fumigatus is an environmental fungus that causes invasive aspergillosis (IA) in immunocompromised patients. Although -CC-chemokine receptor-2 (CCR2) and Ly6C-expressing inflammatory monocytes (CCR2⁺Mo) and their derivatives initiate adaptive pulmonary immune responses, their role in coordinating innate immune responses in the lung remain poorly defined. Using conditional and antibody-mediated cell ablation strategies, we found that CCR2⁺Mo and monocyte-derived dendritic cells (Mo-DCs) are essential for innate defense against inhaled conidia. By harnessing fluorescent Aspergillus reporter (FLARE) conidia that report fungal cell association and viability in vivo, we identify two mechanisms by which CCR2⁺Mo and Mo-DCs exert innate antifungal activity. First, CCR2⁺Mo and Mo-DCs condition the lung inflammatory milieu to augment neutrophil conidiacidal activity. Second, conidial uptake by CCR2⁺Mo temporally coincided with their differentiation into Mo-DCs, a process that resulted in direct conidial killing. Our findings illustrate both indirect and direct functions for CCR2⁺Mo and their derivatives in innate antifungal immunity in the lung.