Comparison of Clostridioides difficile nucleic acid amplification test (NAAT) results using fresh and frozen stool specimens and rectal swabs.

IMPORTANCE: Nucleic acid amplification tests (NAATs) are frequently used in Clostridioides difficile research and diagnostic testing, but the effect of freezing specimens on C. difficile NAAT performance is not well characterized. This study evaluated the concordance of NAAT results between fresh and frozen specimens (fecal and rectal swabs) and found it to be very good to excellent. The results indicate that frozen fecal and rectal swab specimens may be used for C. difficile NAAT testing in research when fresh specimens are not available.

Gut microbiome correlates of recurrent urinary tract infection: a longitudinal, multi-center study.

Background: Urinary tract infections (UTI) affect approximately 250 million people annually worldwide. Patients often experience a cycle of antimicrobial treatment and recurrent UTI (rUTI) that is thought to be facilitated by a gut reservoir of uropathogenic Escherichia coli (UPEC). Methods: 125 patients with UTI caused by an antibiotic-resistant organism (ARO) were enrolled from July 2016 to May 2019 in a longitudinal, multi-center cohort study. Multivariate statistical models were used to assess the relationship between uropathogen colonization and recurrent UTI (rUTI), controlling for clinical characteristics. 644 stool samples and 895 UPEC isolates were interrogated for taxonomic composition, antimicrobial resistance genes, and phenotypic resistance. Cohort UTI gut microbiome profiles were compared against published healthy and UTI reference microbiomes, as well as assessed within-cohort for timepoint- and recurrence-specific differences. Findings: Risk of rUTI was not independently associated with clinical characteristics. The UTI gut microbiome was distinct from healthy reference microbiomes in both taxonomic composition and antimicrobial resistance gene (ARG) burden, with 11 differentially abundant taxa at the genus level. rUTI and non-rUTI gut microbiomes in the cohort did not generally differ, but gut microbiomes from urinary tract colonized patients were elevated in E. coli abundance 7-14 days post-antimicrobial treatment. Corresponding UPEC gut isolates from urinary tract colonizing lineages showed elevated phenotypic resistance against 11 of 23 tested drugs compared to non-colonizing lineages. Interpretation: The gut microbiome is implicated in UPEC urinary tract colonization during rUTI, serving as an ARG-enriched reservoir for UPEC. UPEC can asymptomatically colonize the gut and urinary tract, and post-antimicrobial blooms of gut E. coli among urinary tract colonized patients suggest that cross-habitat migration of UPEC is an important mechanism of rUTI. Thus, treatment duration and UPEC populations in both the urinary and gastrointestinal tract should be considered in treating rUTI and developing novel therapeutics. Funding: This work was supported in part by awards from the U.S. Centers for Disease Control and Prevention Epicenter Prevention Program (grant U54CK000482; principal investigator, V.J.F.); to J.H.K. from the Longer Life Foundation (an RGA/Washington University partnership), the National Center for Advancing Translational Sciences (grants KL2TR002346 and UL1TR002345), and the National Institute of Allergy and Infectious Diseases (NIAID) (grant K23A1137321) of the National Institutes of Health (NIH); and to G.D. from NIAID (grant R01AI123394) and the Eunice Kennedy Shriver National Institute of Child Health and Human Development (grant R01HD092414) of NIH. R.T.'s research was funded by the Deutsche Forschungsgemeinschaft (DFG; German Research Foundation; grant 402733540). REDCap is Supported by Clinical and Translational Science Award (CTSA) Grant UL1 TR002345 and Siteman Comprehensive Cancer Center and NCI Cancer Center Support Grant P30 CA091842. The content is solely the responsibility of the authors and does not necessarily represent the official views of the funding agencies.

Persisting uropathogenic Escherichia coli lineages show signatures of niche-specific within-host adaptation mediated by mobile genetic elements.

Large-scale genomic studies have identified within-host adaptation as a hallmark of bacterial infections. However, the impact of physiological, metabolic, and immunological differences between distinct niches on the pathoadaptation of opportunistic pathogens remains elusive. Here, we profile the within-host adaptation and evolutionary trajectories of 976 isolates representing 119 lineages of uropathogenic Escherichia coli (UPEC) sampled longitudinally from both the gastrointestinal and urinary tracts of 123 patients with urinary tract infections. We show that lineages persisting in both niches within a patient exhibit increased allelic diversity. Habitat-specific selection results in niche-specific adaptive mutations and genes, putatively mediating fitness in either environment. Within-lineage inter-habitat genomic plasticity mediated by mobile genetic elements (MGEs) provides the opportunistic pathogen with a mechanism to adapt to the physiological conditions of either habitat, and reduced MGE richness is associated with recurrence in gut-adapted UPEC lineages. Collectively, our results establish niche-specific adaptation as a driver of UPEC within-host evolution.

Randomized Controlled Trial of Oral Vancomycin Treatment in Clostridioides difficile-Colonized Patients.

Clostridioides difficile infection (CDI) is most commonly diagnosed using nucleic acid amplification tests (NAAT); the low positive predictive value of these assays results in patients colonized with C. difficile unnecessarily receiving CDI treatment antibiotics. The risks and benefits of antibiotic treatment in individuals with such cases are unknown. Fecal samples of NAAT-positive, toxin enzyme immunoassay (EIA)-negative patients were collected before, during, and after randomization to vancomycin (n = 8) or placebo (n = 7). C. difficile and antibiotic-resistant organisms (AROs) were selectively cultured from fecal and environmental samples. Shotgun metagenomics and comparative isolate genomics were used to understand the impact of oral vancomycin on the microbiome and environmental contamination. Overall, 80% of placebo patients and 71% of vancomycin patients were colonized with C. difficile posttreatment. One person randomized to placebo subsequently received treatment for CDI. In the vancomycin-treated group, beta-diversity (P = 0.0059) and macrolide-lincosamide-streptogramin (MLS) resistance genes (P = 0.037) increased after treatment; C. difficile and vancomycin-resistant enterococci (VRE) environmental contamination was found in 53% of patients and 26% of patients, respectively. We found that vancomycin alters the gut microbiota, does not permanently clear C. difficile, and is associated with VRE colonization/environmental contamination. (This study has been registered at ClinicalTrials.gov under registration no. NCT03388268.)IMPORTANCE A gold standard diagnostic for Clostridioides difficile infection (CDI) does not exist. An area of controversy is how to manage patients whose stool tests positive by nucleic acid amplification tests but negative by toxin enzyme immunoassay. Existing data suggest most of these patients do not have CDI, but most are treated with oral vancomycin. Potential benefits to treatment include a decreased risk for adverse outcomes if the patient does have CDI and the potential to decrease C. difficile shedding/transmission. However, oral vancomycin perturbs the intestinal microbiota and promotes antibiotic-resistant organism colonization/transmission. We conducted a double-blinded randomized controlled trial to assess the risk-benefit of oral vancomycin treatment in this population. Oral vancomycin did not result in long-term clearance of C. difficile, perturbed the microbiota, and was associated with colonization/shedding of vancomycin-resistant enterococci. This work underscores the need to better understand this population of patients in the context of C. difficile/ARO-related outcomes and transmission.