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Clostridioides difficile infection (CDI) is a major cause of healthcare-associated diarrhea, despite the widespread implementation of contact precautions for patients with CDI. Here, we investigate strain contamination in a hospital setting and the genomic determinants of disease outcomes. Across two wards over 6 months, we selectively cultured C. difficile from patients ( n = 384) and their environments. Whole-genome sequencing (WGS) of 146 isolates revealed that most C. difficile isolates were from clade 1 (131/146, 89.7%), while only one isolate of the hypervirulent ST1 was recovered. Of culture-positive admissions ( n = 79), 19 (24%) patients were colonized with toxigenic C. difficile on admission to the hospital. We defined 25 strain networks at ≤2 core gene single nucleotide polymorphisms; two of these networks contain strains from different patients. Strain networks were temporally linked ( P < 0.0001). To understand the genomic correlates of the disease, we conducted WGS on an additional cohort of C. difficile ( n = 102 isolates) from the same hospital and confirmed that clade 1 isolates are responsible for most CDI cases. We found that while toxigenic C. difficile isolates are associated with the presence of cdtR , nontoxigenic isolates have an increased abundance of prophages. Our pangenomic analysis of clade 1 isolates suggests that while toxin genes ( tcdABER and cdtR ) were associated with CDI symptoms, they are dispensable for patient colonization. These data indicate that toxigenic and nontoxigenic C. difficile contamination persist in a hospital setting and highlight further investigation into how accessory genomic repertoires contribute to C. difficile colonization and disease., Importance: Clostridioides difficile infection remains a leading cause of hospital-associated diarrhea, despite increased antibiotic stewardship and transmission prevention strategies. This suggests a changing genomic landscape of C. difficile . Our study provides insight into the nature of prevalent C. difficile strains in a hospital setting and transmission patterns among carriers. Longitudinal sampling of surfaces and patient stool revealed that both toxigenic and nontoxigenic strains of C. difficile clade 1 dominate these two wards. Moreover, quantification of transmission in carriers of these clade 1 isolates underscores the need to revisit infection prevention measures in this patient group. We identified unique genetic signatures associated with virulence in this clade. Our data highlight the complexities of preventing transmission of this pathogen in a hospital setting and the need to investigate the mechanisms of in vivo persistence and virulence of prevalent lineages in the host gut microbiome., Competing Interests: The authors declare no conflict of interest.

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., Competing Interests: R.E.D., T.H., K.A.R., E.S., Z.H.I., C.C., and J.H.K. report no conflicts. C-A.D.B. is an employee of Pattern Bioscience. M.A.O. reports consulting fees from Pfizer. R.E.D. reports research support from Biomerieux. E.R.D. reports grants or contracts from Theriva Biologics and Ferring, and consulting fees from Pfizer, Glaxo-Smith Kline, Merck, Seres, Ferring, Summit, Abbott, and AztraZeneca.

Clostridioides difficile infection (CDI) imposes a substantial burden on the health care system in the United States. Understanding the biological basis for the spectrum of C. difficile -related disease manifestations is imperative to improving treatment and prevention of CDI. Here, we investigate the correlates of asymptomatic C. difficile colonization using a multi-omics approach. We compared the fecal microbiome and metabolome profiles of patients with CDI versus asymptomatically colonized patients, integrating clinical and pathogen factors into our analysis. We found that CDI patients were more likely to be colonized by strains with the binary toxin (CDT) locus or strains of ribotype 027, which are often hypervirulent. We find that microbiomes of asymptomatically colonized patients are significantly enriched for species in the class Clostridia relative to those of symptomatic patients. Relative to CDI microbiomes, asymptomatically colonized patient microbiomes were enriched with sucrose degradation pathways encoded by commensal Clostridia, in addition to glycoside hydrolases putatively involved in starch and sucrose degradation. Fecal metabolomics corroborates the carbohydrate degradation signature: we identify carbohydrate compounds enriched in asymptomatically colonized patients relative to CDI patients. Further, we reveal that across C. difficile isolates, the carbohydrates sucrose, rhamnose, and lactulose do not serve as robust growth substrates in vitro, consistent with their enriched detection in our metagenomic and metabolite profiling of asymptomatically colonized individuals. We conclude that pathogen genetic variation may be strongly related to disease outcome. More interestingly, we hypothesize that in asymptomatically colonized individuals, carbohydrate metabolism by other commensal Clostridia may prevent CDI by inhibiting C. difficile proliferation. These insights into C. difficile colonization and putative commensal competition suggest novel avenues to develop probiotic or prebiotic therapeutics against CDI., Competing Interests: SF, JR, TH, KR, EN, CB, JH, GD No competing interests declared, ED E.R.D. has received research support from Synthetic Biologics, Pfizer and Ferring, and has been a consultant for Summit, Merck, Ferring, Pfizer and Seres Therapeutics, all unrelated to this study, (© 2022, Fishbein et al.)

Background: Once antibiotic-resistant bacteria become established within the gut microbiota, they can cause infections in the host and be transmitted to other people and the environment. Currently, there are no effective modalities for decreasing or preventing colonization by antibiotic-resistant bacteria. Intestinal microbiota restoration can prevent Clostridioides difficile infection (CDI) recurrences. Another potential application of microbiota restoration is suppression of non-C. difficile multidrug-resistant bacteria and overall decrease in the abundance of antibiotic resistance genes (the resistome) within the gut microbiota. This study characterizes the effects of RBX2660, a microbiota-based investigational therapeutic, on the composition and abundance of the gut microbiota and resistome, as well as multidrug-resistant organism carriage, after delivery to patients suffering from recurrent CDI., Methods: An open-label, multi-center clinical trial in 11 centers in the USA for the safety and efficacy of RBX2660 on recurrent CDI was conducted. Fecal specimens from 29 of these subjects with recurrent CDI who received either one (N = 16) or two doses of RBX2660 (N = 13) were analyzed secondarily. Stool samples were collected prior to and at intervals up to 6 months post-therapy and analyzed in three ways: (1) 16S rRNA gene sequencing for microbiota taxonomic composition, (2) whole metagenome shotgun sequencing for functional pathways and antibiotic resistome content, and (3) selective and differential bacterial culturing followed by isolate genome sequencing to longitudinally track multidrug-resistant organisms., Results: Successful prevention of CDI recurrence with RBX2660 correlated with taxonomic convergence of patient microbiota to the donor microbiota as measured by weighted UniFrac distance. RBX2660 dramatically reduced the abundance of antibiotic-resistant Enterobacteriaceae in the 2 months after administration. Fecal antibiotic resistance gene carriage decreased in direct relationship to the degree to which donor microbiota engrafted., Conclusions: Microbiota-based therapeutics reduce resistance gene abundance and resistant organisms in the recipient gut microbiome. This approach could potentially reduce the risk of infections caused by resistant organisms within the patient and the transfer of resistance genes or pathogens to others., Trial Registration: ClinicalTrials.gov, NCT01925417 ; registered on August 19, 2013.

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., (Copyright © 2021 Fishbein et al.)

We performed an intervention evaluating the impact of an electronic hard-stop clinical decision support tool on repeat Clostridioides difficile (CD) toxin enzyme immunoassay (T-EIA) testing. The CD testing rate and number of admissions with repeat tests decreased significantly postintervention (P < .01 for both); the percentage of positive tests was unchanged (P = .27).

Clostridioides difficile infection (CDI) accounts for a substantial proportion of deaths attributable to antibiotic-resistant bacteria in the United States. Although C. difficile can be an asymptomatic colonizer, its pathogenic potential is most commonly manifested in patients with antibiotic-modified intestinal microbiomes. In a cohort of 186 hospitalized patients, we showed that host and microbe-associated shifts in fecal metabolomes had the potential to distinguish patients with CDI from those with non-C. difficile diarrhea and C. difficile colonization. Patients with CDI exhibited a chemical signature of Stickland amino acid fermentation that was distinct from those of uncolonized controls. This signature suggested that C. difficile preferentially catabolizes branched chain amino acids during CDI. Unexpectedly, we also identified a series of noncanonical, unsaturated bile acids that were depleted in patients with CDI. These bile acids may derive from an extended host-microbiome dehydroxylation network in uninfected patients. Bile acid composition and leucine fermentation defined a prototype metabolomic model with potential to distinguish clinical CDI from asymptomatic C. difficile colonization.

Objective: To determine the prevalence of Clostridium difficile colonization among patients who meet the 2017 IDSA/SHEA C. difficile infection (CDI) Clinical Guideline Update criteria for the preferred patient population for C. difficile testing., Design: Retrospective cohort., Setting: Tertiary-care hospital in St. Louis, Missouri.PatientsPatients whose diarrheal stool samples were submitted to the hospital's clinical microbiology laboratory for C. difficile testing (toxin EIA) from August 2014 to September 2016.InterventionsElectronic and manual chart review were used to determine whether patients tested for C. difficile toxin had clinically significant diarrhea and/or any alternate cause for diarrhea. Toxigenic C. difficile culture was performed on all stool specimens from patients with clinically significant diarrhea and no known alternate cause for their diarrhea., Results: A total of 8,931 patients with stool specimens submitted were evaluated: 570 stool specimens were EIA positive (+) and 8,361 stool specimens were EIA negative (-). Among the EIA+stool specimens, 107 (19% of total) were deemed eligible for culture. Among the EIA- stool specimens, 515 (6%) were eligible for culture. One EIA+stool specimen (1%) was toxigenic culture negative. Among the EIA- stool specimens that underwent culture, toxigenic C. difficile was isolated from 63 (12%)., Conclusions: Most patients tested for C. difficile do not have clinically significant diarrhea and/or potential alternate causes for diarrhea. The prevalence of toxigenic C. difficile colonization among EIA- patients who met the IDSA/SHEA CDI guideline criteria for preferred patient population for C. difficile testing was 12%.

The objective of this study was to evaluate the clinical characteristics and outcomes of hospitalized patients tested for Clostridium difficile and determine the correlation between pretest probability for C. difficile infection (CDI) and assay results. Patients with testing ordered for C. difficile were enrolled and assigned a high, medium, or low pretest probability of CDI based on clinical evaluation, laboratory, and imaging results. Stool was tested for C. difficile by toxin enzyme immunoassay (EIA) and toxigenic culture (TC). Chi-square analyses and the log rank test were utilized. Among the 111 patients enrolled, stool samples from nine were TC positive and four were EIA positive. Sixty-one (55%) patients had clinically significant diarrhea, 19 (17%) patients did not, and clinically significant diarrhea could not be determined for 31 (28%) patients. Seventy-two (65%) patients were assessed as having a low pretest probability of having CDI, 34 (31%) as having a medium probability, and 5 (5%) as having a high probability. None of the patients with low pretest probabilities had a positive EIA, but four were TC positive. None of the seven patients with a positive TC but a negative index EIA developed CDI within 30 days after the index test or died within 90 days after the index toxin EIA date. Pretest probability for CDI should be considered prior to ordering C. difficile testing and must be taken into account when interpreting test results. CDI is a clinical diagnosis supported by laboratory data, and the detection of toxigenic C. difficile in stool does not necessarily confirm the diagnosis of CDI., (Copyright © 2017 American Society for Microbiology.)

Asymptomatic colonization may contribute to Clostridium difficile transmission. Few data identify which patients are at risk for colonization. We performed a prospective cohort study of C. difficile colonization and risk factors for C. difficile acquisition and loss in hospitalized patients. Patients admitted to medical or surgical wards at a tertiary care hospital were enrolled; interviews and chart review were performed to determine patient demographics, C. difficile infection (CDI) history, medications, and health care exposures. Stool samples/rectal swabs were collected at enrollment and discharge; stool samples from clinical laboratory tests were also included. Samples were cultured for C. difficile, and the isolates were tested for toxins A and B and ribotyped. Chi-square tests and univariate logistic regression were used for the analyses. Two hundred thirty-five patients were enrolled. Of the patients, 21% were colonized with C. difficile (toxigenic and nontoxigenic) at admission and 24% at discharge. Ribotype 027 accounted for 6% of the strains at admission and 12% at discharge. Of the patients colonized at admission, 78% were also colonized at discharge. Cephalosporin use was associated with C. difficile acquisition (47% of patients who acquired C. difficile versus 25% of patients who did not; P = 0.03). β-lactam-β-lactamase inhibitor combinations were associated with a loss of C. difficile colonization (36% of patients who lost C. difficile colonization versus 8% of patients colonized at both admission and discharge; P = 0.04), as was metronidazole (27% versus 3%; P = 0.03). Antibiotic use affects the epidemiology of asymptomatic C. difficile colonization, including acquisition and loss, and it requires additional study., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Clostridium difficile infections (CDI) are a growing concern in North America, because of their increasing incidence and severity. Using integrated approaches, we correlated pathogen genotypes and host clinical characteristics for 46 C. difficile infections in a tertiary care medical center during a 6-month interval from January to June 2010. Multilocus sequence typing (MLST) demonstrated 21 known and 2 novel sequence types (STs), suggesting that the institution's C. difficile strains are genetically diverse. ST-1 (which corresponds to pulsed-field gel electrophoresis strain type NAP1/ribotype 027) was the most prevalent (32.6%); 43.5% of the isolates were binary toxin gene positive, of which 75% were ST-1. All strains were ciprofloxacin resistant and metronidazole susceptible, and 8.3% and 13.0% of the isolates were resistant to clindamycin and tetracycline, respectively. The corresponding resistance loci, including potential novel mutations, were identified from the whole-genome sequencing (WGS) of the resistant strains. Core genome single nucleotide polymorphisms (SNPs) determining the phylogenetic relatedness of the 46 strains recapitulated MLST types and provided greater interstrain differentiation. The disease severity was greatest in patients infected with ST-1 and/or binary gene-positive strains, but genome-wide SNP analysis failed to provide additional associations with CDI severity within the same STs. We conclude that MLST and core genome SNP typing result in the same phylogenetic grouping of the 46 C. difficile strains collected in a single hospital. WGS also has the capacity to differentiate those strains within STs and allows the comparison of strains at the individual gene level and at the whole-genome level., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Background: Clostridium difficile infection (CDI) incidence has increased dramatically over the last decade. Recent studies suggest that asymptomatic carriers may be an important reservoir of C. difficile in healthcare settings. We sought to identify the prevalence and risk factors for asymptomatic C. difficile carriage on admission to the hospital., Methods: Patients admitted to Barnes-Jewish Hospital without diarrhea were enrolled from June 2010 through October 2011. Demographic information and healthcare and medication exposures 90 days prior to admission were collected. Stool specimens or rectal swabs were collected within 48 hours of admission and stored at -30°C until cultured. Clostridium difficile isolates were typed and compared with isolates from patients with CDI., Results: A stool/swab specimen was obtained for 259 enrolled subjects on admission. Two hundred four (79%) were not colonized, 40 (15%) had toxigenic C. difficile (TCD), and 15 (6%) had nontoxigenic C. difficile. There were no differences between TCD-colonized and -uncolonized subjects for age (mean, 56 vs 58 years; P = .46), comorbidities, admission from another healthcare facility (33% vs 24%; P = .23), or recent hospitalization (50% vs 50%; P = .43). There were no differences in antimicrobial exposures in the 90 days prior to admission (55% vs 56%; P = .91). Asymptomatic carriers were colonized with strains similar to strains from patients with CDI, but the relative proportions were different., Conclusions: There was a high prevalence of TCD colonization on admission. In contrast to past studies, TCD colonization was not associated with recent antimicrobial or healthcare exposures. Additional investigation is needed to determine the role of asymptomatic TCD carriers on hospital-onset CDI incidence., (© The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

With the increasing prevalence of Clostridium difficile infection among hospitalized patients, a clear understanding of C. difficile epidemiology is needed to evaluate current prevention policies, and to create new and effective policies. To determine the epidemiology of C. difficile, the most sensitive methods for detection of C. difficile are required. The purpose of this study was to systematically assess multiple methods to determine the most sensitive method to recover C. difficile from stool and rectal swabs. Stool samples from healthy asymptomatic individuals that were collected and confirmed to be culture negative for C. difficile were spiked with known concentrations of C. difficile ranging from 10(4) CFU of C. difficile per ml of stool to 10 CFU per ml. Two sets of experiments (A and B) were performed and each involved multiple combinations of untreated and treated stool/rectal swab specimens and selective and non-selective broth and agar. Overall, recovery of C. difficile was increased with the use of an initial broth enrichment followed by plating to solid medium. The most sensitive method of C. difficile detection for both stools and swab specimens was heat shock prior to inoculation of cycloserine-cefoxitin mannitol broth with taurocholate lysozyme cysteine (CCMB, Anaerobe Systems, Morgan Hill, CA) followed by isolation from pre-reduced TSA II with 5% sheep blood (BAP, BBL BD and Co., Sparks, MD). Identifying the most sensitive method of recovery will allow for further study of asymptomatic C. difficile carriers and their role in the epidemiology of C. difficile., (Copyright © 2012. Published by Elsevier Ltd.)

Asymptomatic Clostridium difficile colonization is common in hospitalized patients. Existing C. difficile assay comparisons lack data on severity of diarrhea or patient outcomes, limiting the ability to interpret their results in regard to the diagnosis of C. difficile infection (CDI). The objective of this study was to measure how including patient presentation with the C. difficile assay result impacted assay performance to diagnose CDI. Stool specimens from 150 patients that met inclusion and exclusion criteria were selected. Nine methods to detect C. difficile in stool were evaluated. All patients were interviewed prospectively to assess diarrhea severity. We then assessed how different reference standards, with and without the inclusion of patient presentation, impact the sensitivity, specificity, and positive and negative predictive values of the assays to diagnose CDI. There were minimal changes in sensitivity; however, specificity was significantly lower for the assays Tox A/B II, C. diff Chek-60, BD GeneOhm Cdiff, Xpert C. difficile, and Illumigene C. difficile and for toxigenic culture (P was <0.01 for all except Tox A/B II from fresh stool, for which the P value was 0.016) when the reference standard was recovery of toxigenic C. difficile from stool plus the presence of clinically significant diarrhea compared to when the reference standard was having at least four assays positive while ignoring diarrhea severity. There were 15 patients whose assay result was reported as negative but subsequently found to be positive by at least four assays in the comparison. None suffered from any CDI-related adverse events. In conclusion, clinical presentation is important when interpreting C. difficile diagnostic assays.