Highlights d C. albicans intestinal colonization protects against C. albicans invasive infection d Systemic fungal-specific Th17 CD4 + T cell accumulation with intestinal colonization d Tonic neutrophil stimulation augments host defense against extracellular pathogens d Antimicrobial immunity balanced by susceptibility to allergic airway inflammation

Severe Clostridiodes difficile infection (CDI) is life-threatening and responds poorly to treatment. Obesity is associated with development of severe CDI. Therefore, to define the mechanisms that exacerbate disease severity, we examined CDI pathogenesis in high-fat diet (HFD)-fed obese mice. Compared to control mice, HFD-fed mice failed to clear C. difficile bacteria which resulted in protracted diarrhea, weight loss and colonic damage. After infection, HFD-induced obese mice had an intestinal bile acid (BA) pool that was dominated by primary BAs which are known promoters of C. difficile spore germination, and lacked secondary BAs that inhibit C. difficile growth. Concurrently, synthesis of primary BAs from liver was significantly increased in C. difficile-infected HFD-fed mice. A key pathway that regulates hepatic BA synthesis is via feedback inhibition from intestinal Farnesoid X receptors (FXRs). Our data reveal that the proportion of FXR agonist BAs to FXR antagonist BAs in the intestinal lumen was significantly reduced in HFD-fed mice after CDI. Treatment of HFD-fed mice with an FXR agonist Obeticholic acid, resulted in decreased primary BA synthesis, fewer C. difficile bacteria and better CDI outcomes. Thus, OCA treatment holds promise as a therapy for severe CDI.

Prevention of multidrug-resistant (MDR) bacterial infections relies on accurate detection of these organisms. We investigated shotgun metagenome sequencing for the detection of methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus (VRE), and MDR Enterobacteriaceae. Fecal metagenomes were analyzed from high-risk inpatients and compared to those of low-risk outpatients and controls with minimal risk for a MDR bacterial infection. Principal-component analysis clustered patient samples into distinct cohorts, confirming that the microbiome composition was significantly different between cohorts (P ‫؍‬ 0.006). Microbial diversity and relative anaerobe abundance were preserved in outpatients compared to those in controls. Relative anaerobe abundance was significantly reduced in inpatients compared to that in outpatients (P ‫؍‬ 0.006). Although the potential for MDR bacteria was increased in inpatients and outpatients compared to that in controls (P < 0.001), there was no difference between inpatients and outpatients. However, 9 (53%) inpatients had colonization with a MDR bacterium that was not identified by culture. Unlike culture, shotgun sequencing quantitatively characterizes the burdens of multiple MDR bacteria relative to all of the microbiota within the intestinal community. We propose consideration of key microbiome features, such as diversity and relative anaerobe abundance, in addition to the detection of MDR bacteria by shotgun metagenome sequencing as a novel method that might better identify patients who are at increased risk of a MDR infection. Multidrug-resistant (MDR) bacterial infections cause significant morbidity and mortality and are increasingly common in children (1-3). Clinically important MDR bacteria include methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus (VRE), and MDR Enterobacteriaceae with AmpC beta-lactamase resistance (AmpC), extended-spectrum beta-lactamase (ESBL) resistance, or carbapenem-resistant betalactamases (CRE). Antimicrobial exposures reduce the commensal gut anaerobes with compensatory increased Enterobacteriaceae and Enterococcus abundances as well as bacteriophages, allowing transmission of antibiotic resistance genes between species (4). This results in the loss of host resistance to abnormal fecal colonization and may select for MDR bacteria (5-7). Subsequently, the intestines serve as the primary reservoir of MDR bacteria (8, 9). Increased abundances of these organisms within the intestines precede the onset of an invasive bloodstream infection (BSI), and the risk of infection is related to the density of colonization (10-13).Timely and accurate detection of MDR bacteria is crucial to prevent the spread of MDR bacteria and to decrease rates of MDR infections (14). There is an urgent need for the development of new diagnostic methods, including metagenomics, to combat rising antibiotic resistance rates, which was explicitly stated in recent national action plans by the CDC and the White House (15, 16). D...

Clostridium difficile is the leading cause of nosocomial infections in the U.S. Clinical disease outcomes after C. difficile infection (CDI) are dependent on intensity of host inflammatory responses. Specifically, peak peripheral white blood cell (WBC) count >20×109/L is an indicator of adverse outcomes in CDI patients, and is associated with higher 30-day mortality. We show that homozygosity for a common single nucleotide polymorphism (Q to R mutation in leptin receptor that is present in up to 50% of people), significantly increases the risk of having peak peripheral WBC count >20×109/L (odds ratio=4.7; p = 0.0024) in CDI patients. In a murine model of CDI, we demonstrate that mice homozygous for the same SNP (RR mice) have more blood and tissue leukocytes (specifically neutrophils), exaggerated tissue inflammation and higher mortality as compared to control mice, despite similar pathogen burden. Further, we show that neutrophilia in RR mice is mediated by gut microbiota-directed expression of CXC chemokine receptor 2 (CXCR2) which promotes the release of neutrophils from bone marrow reservoir. Overall these studies provide novel mechanistic insights into the role of human genetic polymorphisms and gut microbiota in regulating the fundamental biological process of CDI-induced neutrophilia.