Limited effects of long-term daily cranberry consumption on the gut microbiome in a placebo-controlled study of women with recurrent urinary tract infections.

BACKGROUND: Urinary tract infections (UTIs) affect 15 million women each year in the United States, with > 20% experiencing frequent recurrent UTIs. A recent placebo-controlled clinical trial found a 39% reduction in UTI symptoms among recurrent UTI sufferers who consumed a daily cranberry beverage for 24 weeks. Using metagenomic sequencing of stool from a subset of these trial participants, we assessed the impact of cranberry consumption on the gut microbiota, a reservoir for UTI-causing pathogens such as Escherichia coli, which causes > 80% of UTIs. RESULTS: The overall taxonomic composition, community diversity, carriage of functional pathways and gene families, and relative abundances of the vast majority of observed bacterial taxa, including E. coli, were not changed significantly by cranberry consumption. However, one unnamed Flavonifractor species (OTU41), which represented ≤1% of the overall metagenome, was significantly less abundant in cranberry consumers compared to placebo at trial completion. Given Flavonifractor's association with negative human health effects, we sought to determine OTU41 characteristic genes that may explain its differential abundance and/or relationship to key host functions. Using comparative genomic and metagenomic techniques, we identified genes in OTU41 related to transport and metabolism of various compounds, including tryptophan and cobalamin, which have been shown to play roles in host-microbe interactions. CONCLUSION: While our results indicated that cranberry juice consumption had little impact on global measures of the microbiome, we found one unnamed Flavonifractor species differed significantly between study arms. This suggests further studies are needed to assess the role of cranberry consumption and Flavonifractor in health and wellbeing in the context of recurrent UTI. TRIAL REGISTRATION: Clinical trial registration number: ClinicalTrials.gov NCT01776021 .

Chemical disarming of isoniazid resistance in Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) killed more people in 2017 than any other single infectious agent. This dangerous pathogen is able to withstand stresses imposed by the immune system and tolerate exposure to antibiotics, resulting in persistent infection. The global tuberculosis (TB) epidemic has been exacerbated by the emergence of mutant strains of Mtb that are resistant to frontline antibiotics. Thus, both phenotypic drug tolerance and genetic drug resistance are major obstacles to successful TB therapy. Using a chemical approach to identify compounds that block stress and drug tolerance, as opposed to traditional screens for compounds that kill Mtb, we identified a small molecule, C10, that blocks tolerance to oxidative stress, acid stress, and the frontline antibiotic isoniazid (INH). In addition, we found that C10 prevents the selection for INH-resistant mutants and restores INH sensitivity in otherwise INH-resistant Mtb strains harboring mutations in the katG gene, which encodes the enzyme that converts the prodrug INH to its active form. Through mechanistic studies, we discovered that C10 inhibits Mtb respiration, revealing a link between respiration homeostasis and INH sensitivity. Therefore, by using C10 to dissect Mtb persistence, we discovered that INH resistance is not absolute and can be reversed.

Structure-based discovery of glycomimetic FmlH ligands as inhibitors of bacterial adhesion during urinary tract infection.

Treatment of bacterial infections is becoming a serious clinical challenge due to the global dissemination of multidrug antibiotic resistance, necessitating the search for alternative treatments to disarm the virulence mechanisms underlying these infections. Uropathogenic Escherichia coli (UPEC) employs multiple chaperone-usher pathway pili tipped with adhesins with diverse receptor specificities to colonize various host tissues and habitats. For example, UPEC F9 pili specifically bind galactose or N-acetylgalactosamine epitopes on the kidney and inflamed bladder. Using X-ray structure-guided methods, virtual screening, and multiplex ELISA arrays, we rationally designed aryl galactosides and N-acetylgalactosaminosides that inhibit the F9 pilus adhesin FmlH. The lead compound, 29ß-NAc, is a biphenyl N-acetyl-ß-galactosaminoside with a Ki of ∼90 nM, representing a major advancement in potency relative to the characteristically weak nature of most carbohydrate-lectin interactions. 29ß-NAc binds tightly to FmlH by engaging the residues Y46 through edge-to-face π-stacking with its A-phenyl ring, R142 in a salt-bridge interaction with its carboxylate group, and K132 through water-mediated hydrogen bonding with its N-acetyl group. Administration of 29ß-NAc in a mouse urinary tract infection (UTI) model significantly reduced bladder and kidney bacterial burdens, and coadministration of 29ß-NAc and mannoside 4Z269, which targets the type 1 pilus adhesin FimH, resulted in greater elimination of bacteria from the urinary tract than either compound alone. Moreover, FmlH specifically binds healthy human kidney tissue in a 29ß-NAc-inhibitable manner, suggesting a key role for F9 pili in human kidney colonization. Thus, these glycoside antagonists of FmlH represent a rational antivirulence strategy for UPEC-mediated UTI treatment.

Antivirulence C-Mannosides as Antibiotic-Sparing, Oral Therapeutics for Urinary Tract Infections.

Gram-negative uropathogenic Escherichia coli (UPEC) bacteria are a causative pathogen of urinary tract infections (UTIs). Previously developed antivirulence inhibitors of the type 1 pilus adhesin, FimH, demonstrated oral activity in animal models of UTI but were found to have limited compound exposure due to the metabolic instability of the O-glycosidic bond (O-mannosides). Herein, we disclose that compounds having the O-glycosidic bond replaced with carbon linkages had improved stability and inhibitory activity against FimH. We report on the design, synthesis, and in vivo evaluation of this promising new class of carbon-linked C-mannosides that show improved pharmacokinetic (PK) properties relative to O-mannosides. Interestingly, we found that FimH binding is stereospecifically modulated by hydroxyl substitution on the methylene linker, where the R-hydroxy isomer has a 60-fold increase in potency. This new class of C-mannoside antagonists have significantly increased compound exposure and, as a result, enhanced efficacy in mouse models of acute and chronic UTI.

Molecular regulation of urothelial renewal and host defenses during infection with uropathogenic Escherichia coli.

Uropathogenic Escherichia coli (UPEC), the principal cause of urinary tract infection in women, attaches to the superficial facet cell layer of the bladder epithelium (urothelium) via its FimH adhesin. Attachment triggers exfoliation of bacteria-laden superficial facet cells, followed by rapid reconstitution of the urothelium through differentiation of underlying basal and intermediate cells. We have used DNA microarrays to define the molecular regulators of urothelial renewal and host defense expressed in adult C57Bl/6 female mice during the early phases of infection with isogenic virulent (FimH+) or avirulent (FimH-) UPEC strains. The temporal evolution and cellular origins of selected responses were then characterized by real time quantitative reverse transcriptase-PCR, in situ hybridization, and immunohistochemical analyses. Well before exfoliation is evident, FimH-mediated attachment suppresses transforming growth factor-beta (Bmp4) and Wnt5a/Ca(2+) signaling to promote subsequent differentiation of basal/intermediate cells. The early transcriptional responses to attachment also include induction of regulators of proliferation (e.g. epidermal growth factor family members), induction of the ETS transcription factor Elf3, which transactivates genes involved in epithelial differentiation and host defense (inducible nitric-oxide synthase), induction of modulators, and mediators of pro-inflammatory responses (e.g. Socs3, Cebp/delta, Bcl3, and CC/CXC chemokines), induction of modulators of apoptotic responses (A20), and induction of intermediate cell tight junction components (claudin-4). Both early and late phases of the host response exhibit remarkable specificity for the FimH+ strain and provide new insights about the molecular cascade mobilized to combat UPEC-associated urinary tract infection.