tcdB Genes Research Articles

High Prevalence of Multidrug-Resistant Clostridioides difficile Following Extensive Use of Antimicrobials in Hospitalized Patients in Kenya.

Introduction Clostridioides difficile is a neglected pathogen in many African countries as it is generally not regarded as one of the major contributors toward the diarrheal disease burden in the continent. However, several studies have suggested that C. difficile infection (CDI) may be underreported in many African settings. The aim of this study was to determine the prevalence of CDI in hospitalized patients, evaluate antimicrobial exposure, and detect toxin and antimicrobial resistance profiles of the isolated C. difficile strains.MethodsIn this cross-sectional study, 333 hospitalized patients with hospital-onset diarrhoea were selected. The stool samples were collected and cultured on cycloserine-cefoxitin egg yolk agar (CCEY). Isolates were presumptively identified by phenotypic characteristics and Gram stain and confirmed by singleplex real-time PCR (qPCR) assays detecting the species-specific tpi gene, toxin A (tcdA) gene, toxin B (tcdB) gene, and the binary toxin (cdtA/cdtB) genes. Confirmed C. difficile isolates were tested against a panel of eight antimicrobials (vancomycin, metronidazole, rifampicin, ciprofloxacin, tetracycline, clindamycin, erythromycin, and ceftriaxone) using E-test strips.Results C. difficile was detected in 57 (25%) of diarrheal patients over the age of two, 56 (98.2%) of whom received antimicrobials before the diarrheal episode. Amongst the 71 confirmed isolates, 69 (97.1%) harbored at least one toxin gene. More than half of the toxigenic isolates harbored a truncated tcdA gene. All isolates were sensitive to vancomycin, while three isolates (2.1%) were resistant to metronidazole (MIC >32 mg/L). High levels of resistance were observed to rifampicin (65/71, 91.5%), erythromycin (63/71, 88.7%), ciprofloxacin (59/71, 83.1%), clindamycin (57/71, 80.3%), and ceftriaxone (36/71, 50.7.8%). Among the resistant isolates, 61 (85.9%) were multidrug-resistant.ConclusionMultidrug-resistant C. difficile strains were a significant cause of healthcare facility-onset C. difficile infections in patients with prior antimicrobial exposure in this Kenyan hospital.

Phylogenomics of 8,839 Clostridioides difficile genomes reveals recombination-driven evolution and diversification of toxin A and B.

Clostridioides difficile is the major worldwide cause of antibiotic-associated gastrointestinal infection. A pathogenicity locus (PaLoc) encoding one or two homologous toxins, toxin A (TcdA) and toxin B (TcdB), is essential for C. difficile pathogenicity. However, toxin sequence variation poses major challenges for the development of diagnostic assays, therapeutics, and vaccines. Here, we present a comprehensive phylogenomic analysis of 8,839 C. difficile strains and their toxins including 6,492 genomes that we assembled from the NCBI short read archive. A total of 5,175 tcdA and 8,022 tcdB genes clustered into 7 (A1-A7) and 12 (B1-B12) distinct subtypes, which form the basis of a new method for toxin-based subtyping of C. difficile. We developed a haplotype coloring algorithm to visualize amino acid variation across all toxin sequences, which revealed that TcdB has diversified through extensive homologous recombination throughout its entire sequence, and formed new subtypes through distinct recombination events. In contrast, TcdA varies mainly in the number of repeats in its C-terminal repetitive region, suggesting that recombination-mediated diversification of TcdB provides a selective advantage in C. difficile evolution. The application of toxin subtyping is then validated by classifying 351 C. difficile clinical isolates from Brigham and Women’s Hospital in Boston, demonstrating its clinical utility. Subtyping partitions TcdB into binary functional and antigenic groups generated by intragenic recombinations, including two distinct cell-rounding phenotypes, whether recognizing frizzled proteins as receptors, and whether it can be efficiently neutralized by monoclonal antibody bezlotoxumab, the only FDA-approved therapeutic antibody. Our analysis also identifies eight universally conserved surface patches across the TcdB structure, representing ideal targets for developing broad-spectrum therapeutics. Finally, we established an open online database (DiffBase) as a central hub for collection and classification of C. difficile toxins, which will help clinicians decide on therapeutic strategies targeting specific toxin variants, and allow researchers to monitor the ongoing evolution and diversification of C. difficile.

Changes in the Association Between Diagnostic Testing Method, Polymerase Chain Reaction Ribotype, and Clinical Outcomes From Clostridioides difficile Infection: One Institution's Experience.

In Clostridioides difficile infection (CDI), the relationship between clinical, microbial, and temporal/epidemiological trends, disease severity and adverse outcomes is incompletely understood. In a follow-up to our study from 2010-2013, we evaluate stool toxin levels and C. difficile polymerase chain reaction (PCR) ribotypes. We hypothesized that elevated stool toxins and infection with ribotype 027 associate with adverse outcomes. In 565 subjects at the University of Michigan with CDI diagnosed by positive testing for toxins A/B by enzyme immunoassay (EIA) or PCR for the tcdB gene, we quantified stool toxin levels via a modified cell cytotoxicity assay (CCA), isolated C. difficile by anaerobic culture, and performed PCR ribotyping. Severe CDI was defined by Infectious Diseases Society of America (IDSA) criteria, and primary outcomes were all-cause 30-day mortality and a composite of colectomy, intensive care unit admission, and/or death attributable to CDI within 30 days. Analyses included bivariable tests and logistic regression. 199 samples were diagnosed by EIA; 447 were diagnosed by PCR. Toxin positivity associated with IDSA severity but not primary outcomes. In 2016, compared with 2010-2013, ribotype 106 newly emerged, accounting for 10.6% of strains, ribotype 027 fell from 16.5% to 9.3%, and ribotype 014-027 remained stable at 18.9%. Ribotype 014-020 associated with IDSA severity and 30-day mortality (P = .001). Toxin positivity by EIA and CCA associated with IDSA severity but not with subsequent adverse outcomes. The molecular epidemiology of C. difficile has shifted, which may have implications for the optimal diagnostic strategy for and clinical severity of CDI.

Quantitative characterization of Clostridioides difficile population in the gut microbiome of patients with C. difficile infection and their association with clinical factors

Objective was to analyse bacterial composition and abundance of Clostridioides difficile in gut microbiome of patients with C. difficile infection (CDI) in association with clinical characteristics. Whole metagenome sequencing of gut microbiome of 26 CDI patients was performed, and the relative abundance of C. difficile and its toxin genes was measured. Clinical characteristics of the patients were obtained through medical records. A strong correlation between the abundance of C. difficile and tcdB genes in CDI patients was found. The relative abundance of C. difficile in the gut microbiome ranged from undetectable to 2.8% (median 0.089). Patients with fever exhibited low abundance of C. difficile in their gut, and patients with fewer C. difficile organisms required long-term anti-CDI treatment. Abundance of Bifidobacterium and Bacteroides negatively correlated with that of C. difficile at the genus level. CDI patients were clustered using the bacterial composition of the gut: one with high population of Enterococcus (cluster 1, n = 12) and another of Bacteroides or Lactobacillus (cluster 2, n = 14). Cluster1 showed significantly lower bacterial diversity and clinical cure at the end of treatment. Additionally, patients with CDI exhibited increased ARGs; notably, blaTEM, blaSHV and blaCTX-M were enriched. C. difficile existed in variable proportion of the gut microbiome in CDI patients. CDI patients with Enterococcus-rich microbiome in the gut had lower bacterial diversity and poorer clinical cure.

Cost Consequences for the NHS of Using a Two-Step Testing Method for the Detection of Clostridium difficile with a Point of Care, Polymerase Chain Reaction Test as the First Step.

Clostridium difficile infection (CDI) is a common healthcare-associated infection. Current practice for diagnosing CDI in the Newcastle upon Tyne Hospitals NHS Foundation Trust involves a three-step, laboratory testing strategy using glutamate dehydrogenase (GDH) enzyme immunoassay (EIA), followed by a polymerase chain reaction (PCR) test then a toxin EIA. However, a PCR point of care test (POCT) for the C. difficile tcdB gene for screening suspected CDI cases, may provide a more efficient way of facilitating an equally effective, two-step, testing strategy with a toxin EIA. This study evaluated the cost consequences of changing from the three-step to a two-step testing strategy. A cost-consequences model was developed to compare the costs and consequences of the two strategies. Uncertainties in the model inputs were investigated with one- and two-way sensitivity analysis. The two-step, POCT strategy was estimated to save £283,282 per 1000 hospitalized NHS patients with suspected infectious diarrhea. Sensitivity analysis indicated that the turnaround time for the POCT was the largest driver for cost savings. Providing the POCT has sufficiently high diagnostic accuracy for detecting C. difficile, the two-step, POCT strategy for CDI identification is likely to be cost saving for NHS hospitals with an offsite laboratory.

Recurrent Clostridioides difficile infection can be predicted using inflammatory mediator and toxin activity levels

Background:Clostridioides difficile infection (CDI) frequently recurs after initial treatment. Predicting recurrent CDI (rCDI) early in the disease course can assist clinicians in their decision making and improve outcomes. However, predictions based on clinical criteria alone are not accurate and/or do not validate other results. Here, we tested the hypothesis that circulating and stool-derived inflammatory mediators predict rCDI. Methods: Consecutive subjects with available specimens at diagnosis were included if they tested positive for toxigenic C. difficile (+enzyme immunoassay [EIA] for glutamate dehydrogenase and toxins A/B, with reflex to PCR for the tcdB gene for discordants). Stool was thawed on ice, diluted 1:1 in PBS with protease inhibitor, centrifuged, and used immediately. A 17-plex panel of inflammatory mediators was run on a Luminex 200 machine using a custom antibody-linked bead array. Prior to analysis, all measurements were normalized and log-transformed. Stool toxin activity levels were quantified using a custom cell-culture assay. Recurrence was defined as a second episode of CDI within 100 days. Ordination characterized variation in the panel between outcomes, tested with a permutational, multivariate ANOVA. Machine learning via elastic net regression with 100 iterations of 5-fold cross validation selected the optimal model and the area under the receiver operator characteristic curve (AuROC) was computed. Sensitivity analyses excluding those that died and/or lived >100 km away were performed. Results: We included 186 subjects, with 95 women (51.1%) and average age of 55.9 years (±20). More patients were diagnosed by PCR than toxin EIA (170 vs 55, respectively). Death, rCDI, and no rCDI occurred in 32 (17.2%), 36 (19.4%), and 118 (63.4%) subjects, respectively. Ordination revealed that the serum panel was associated with rCDI (P = .007) but the stool panel was not. Serum procalcitonin, IL-8, IL-6, CCL5, and EGF were associated with recurrence. The machine-learning models using the serum panel predicted rCDI with AuROCs between 0.74 and 0.8 (Fig. 1). No stool inflammatory mediators independently predicted rCDI. However, stool IL-8 interacted with toxin activity to predict rCDI (Fig. 2). These results did not change significantly upon sensitivity analysis. Conclusions: A panel of serum inflammatory mediators predicted rCDI with up to 80% accuracy, but the stool panel alone was less successful. Incorporating toxin activity levels alongside inflammatory mediator measurements is a novel, promising approach to studying stool-derived biomarkers of rCDI. This approach revealed that stool IL-8 is a potential biomarker for rCDI. These results need to be confirmed both with a larger dataset and after adjustment for clinical covariates.Funding: NoneDisclosure: Vincent Young is a consultant for Bio-K+ International, Pantheryx, and Vedanta Biosciences.

Putative Conjugative Plasmids with tcdB and cdtAB Genes in Clostridioides difficile.

The major toxins of Clostridioides difficile (TcdA, TcdB, CDT) are chromosomally encoded in nearly all known strains. Following up on previous findings, we identified 5 examples of a family of putative conjugative plasmids with tcdB and cdtAB in clinical C. difficile isolates from multilocus sequence typing clades C-I, 2, and 4.

Mild or Malign: Clinical Characteristics and Outcomes of Clostridium difficile Infection in Thailand.

Little is known about the clinical characteristics of Clostridium difficile infection (CDI) in Asia in general, and Thailand specifically, with a few studies suggesting that the disease may be milder than elsewhere. This study aimed to describe CDI in Thailand, evaluate treatment options and their outcomes, and explore possible protective factors responsible for any unique disease characteristics. From 2015 to 2018, 469 patients were included in the study. All patients had their stools tested for the tcdB gene by direct PCR and detection of toxigenic C. difficile by culture. C. difficile isolates were subjected to toxin gene profiling and ribotyping, and patient medical records were reviewed retrospectively. There were 248 and 221 patients included in CDI and control groups, respectively. The CDI group had a higher overall 30-day mortality rate than the control group (21% versus 14%, P = 0.046), but only 2 deaths (1%) were directly attributable to CDI. Metronidazole treatment was not inferior to vancomycin in this population, and vancomycin was associated with a higher 30-day mortality rate (P = 0.047). The prevalence of severe CDI and disease outcomes were not different between patients infected with A-B+ C. difficile and A+B+ C. difficile strains or between patients with and without colonization by nontoxigenic C. difficile Besides C. difficile-specific tests, neither a single laboratory result nor a combination of results was predictive of CDI. In conclusion, CDI in Thailand was relatively mild, and metronidazole remained an effective treatment option for these mild infections.

Clinical and microbial characterization of toxigenic Clostridium difficile isolated from antibiotic associated diarrhea in Egypt.

Background and Objectives:Clostridium difficile infection (CDI) has become a significant healthcare-associated infection throughout the world and is particularly important in developing countries. This study aimed to investigate clinical characterization and risk factors related to toxigenic C. difficile infection in adult and pediatric patients, antimicrobial susceptibility pattern. Also, to evaluate different diagnostic methods for rapid detection of C. difficile associated diarrhea (CDAD) in Egypt.Materials and Methods:Stool samples were collected from 95 pediatric patients and 37 adult patients suffering from antibiotic associated diarrhea and were subjected to direct toxin immunoassay and culture on cycloserine/cefoxitin/fructose agar. The presence of tcdA and tcdB genes was tested by PCR.Results:Toxigenic C. difficile was isolated from pediatric and adult patients at a rate of 17.89% (17/95) and 27% (10/37) respectively. The sensitivity and specificity of direct PCR from stool are (100%, 100% and 82.4%, 100%) in adult and pediatric samples respectively. The susceptibility of C. difficile to vancomycin and metronidazole were found to be 66.7% and 48.2% respectively.Conclusion:Diabetes mellitus, prior antibiotic treatment, hematological malignancy on chemotherapy, malnutrition, neutropenia and Ryle feeding are risk factors for development of CDAD. Tight restriction of unnecessary antibiotic uses is necessary in our locality. Direct detection of toxin genes in stool by PCR is sensitive and specific method for early detection of C. difficile.

Subtyping analysis reveals new variants and accelerated evolution of Clostridioides difficile toxin B

Clostridioides difficile toxins (TcdA and TcdB) are major exotoxins responsible for C. difficile infection (CDI) associated diseases. The previously reported TcdB variants showed distinct biological features, immunoactivities, and potential pathogenicity in disease progression. Here, we performed global comparisons of amino acid sequences of both TcdA and TcdB from 3,269 C. difficile genomes and clustered them according to the evolutionary relatedness. We found that TcdB was much diverse and could be divided into eight subtypes, of which four were first described. Further analysis indicates that the tcdB gene undergoes accelerated evolution to maximize diversity. By tracing TcdB subtypes back to their original isolates, we found that the distribution of TcdB subtypes was not completely aligned with the phylogeny of C. difficile. These findings suggest that the tcdB genes not only frequently mutate, but also continuously transfer and exchange among C. difficile strains.

Microbiota-associated Risk Factors for Clostridioides difficile Acquisition in Hospitalized Patients: A Prospective, Multicentric Study.

Asymptomatic C. difficile colonization is believed to predispose to subsequent C. difficile infection (CDI). While emerging insights into the role of the commensal microbiota in mediating colonization resistance against C. difficile have associated CDI with specific microbial components, corresponding prospectively collected data on colonization with C. difficile are largely unavailable. C. difficile status was assessed by GDH EIA and real-time PCR targeting the toxin A (tcdA) and B (tcdB) genes. 16S V3 and V4 gene sequencing results from fecal samples of patients tested positive for C. difficile were analyzed by assessing alpha and beta diversity, LefSe, and the Piphillin functional inference approach to estimate functional capacity. 1506 patients were recruited into a prospective observational study (DRKS00005335) upon admission into one of five academic hospitals. 936 of them provided fecal samples on admission and at discharge and were thus available for longitudinal analysis. Upon hospital admission, 5.5% (83/1506) and 3.7% (56/1506) of patients were colonized with toxigenic (TCD) and non-toxigenic C. difficile (NTCD), respectively. During hospitalization, 1.7% (16/936) acquired TCD. Risk factors for acquisition of TCD included pre-existing lung diseases, lower GI endoscopy and antibiotics. Species protecting against hospital-related C. difficile acquisition included Gemmiger spp., Odoribacter splanchnicus, Ruminococcus bromii and other Ruminococcus spp. Metagenomic pathway analysis identified steroid biosynthesis as the most underrepresented metabolic pathway in patients who later acquire C. difficile colonization. Gemmiger spp., Odoribacter splanchnicus, Ruminococcus bromii and other Ruminococci were associated with a decreased risk of C. difficile acquisition. DRKS00005335.

Molecular Characterization and Diagnosis of Nosocomial Clostridium difficile Infection in Hospitalized Patients

Background: Toxigenic Clostridium difficile is one of the prevalent diarrheagenic pathogens in hospitalized patients. Objectives: The study assessed the ability of three diagnostic methods in identifying C. difficile strains. The genotyping of the isolates was done, as well. Methods: Stool samples were subjected to three different diagnostic methods including direct stool culture, glutamate dehydroge-nase enzyme immunoassay (GDH-EIA), and direct stool PCR for the detection of the tcdA and tcdB genes. The sensitivity and specificity of the tests were evaluated. The genotyping was done by the PFGE method. Results: Of 120 samples, 20 (16) were positive for C. difficile based on PCR, while 15 (12.5) and 12 (10) were positive according to GDH-EIA and direct stool culture. Among patients with C. difficile-associated diseases (CDAD), 11 (61) were more than 65-years-old. The specificity of PCR, GDH-EIA, and direct culture was almost similar and equal to 100, but their sensitivity was 90, 70, and 60, respectively. The positive predictive value (PPV) was lower for GDH-EIA than for the other two methods, and the highest negative predictive value (NPV) was related to the PCR method. The results showed a high similarity between the isolates, and only were three pulsotypes differentiated among the isolates. Conclusions: The specificity and sensitivity of the direct stool PCR method were higher than those of the other two methods. Al-though PCR inhibitors may reduce its ability for the correct diagnosis of negative samples, it seems to be a reliable method for the detection of C. difficile infection. The weakness of the GDH-EIA method was its lower PPV, which can cause false-positive results. Toxin patterns and pulsotypes of C. difficile isolates revealed a high similarity between the strains isolated from the same units. © 2020, Author(s).

Colonization of Toxigenic Clostridium difficile Among Intensive Care Unit Patients: A Multi-Centre Cross-Sectional Study.

Background: Clostridium difficile (CD) is a major cause of healthcare-associated infections and antibiotic-associated diarrhea in hospitalized patients worldwide. Carriers of toxigenic CD (tCD) have a higher risk of developing CD infections and can transmit CD to the environment and susceptible patients. However, little is known regarding the carriers and transmission of tCD in China.Methods: A multi-center cross-sectional study of tCD colonization (tCDC) was conducted from October 24 to 31, 2014, at 33 hospitals in Shanghai, China. Rectal swabs or stool samples were collected and tested, and the clinical and demographic status, epidemiological data, and blood parameters of 531 participants were recorded. The status of tCDC was defined by a positive result on the nucleic acid amplification test for the tcdA (toxin A), tcdB (toxin B), and cdtAB (toxin CDT) genes after positive bacterial culture.Results: The overall prevalence of CD colonization (CDC) was 19.02%, tCDC accounted for 92.08%, and A+B+CDT– was the dominant genotype (87.13%). The CD infection (CDI) prevalence was 1.51%. Potential tCDC-associated factors were admission to secondary grade hospitals, a body mass index <18.5, hospitalization during the previous 30 days, underlying diseases (including hypertension, diabetes mellitus, coronary heart disease, and respiratory failure), diarrhea during the previous 7 days, and exposure to fluoroquinolones or lansoprazole.Conclusions: This study reveals the prevalence of CDC and tCDC in Shanghai, elucidates several associated factors, contributes to the awareness of the current epidemiology in parts of eastern China and provides new insights for further study and infection control practices.

Conventional and molecular diagnosis of clostridium difficile infections in a tertiary care hospital

Introduction: Indiscriminate use of broad-spectrum antibiotics has dramatically increased the incidence of Clostridium difficile-associated diarrhea (CDAD) in recent years. It is the most common cause of antibiotic-associated diarrhea (AAD) responsible for one-third of AAD cases. Aim: The aim was to study C. difficile in AAD. Objectives: The objectives were to study the prevalence of CDAD, to isolate C. difficile from AAD, and to study the molecular detection of toxin-producing strains of C. difficile. Materials and Methods: A total of 222 patients of AAD were assessed for C. difficile over a period of 2 years. Anaerobic culture for C. difficile was done on cycloserine-cefoxitin-fructose agar and brain–heart infusion agar. Enzyme-linked immunosorbent assay (ELISA) for toxin A and toxin B was used for detecting toxigenic strains of C. difficile. Identification of C. difficile and toxin-producing strains was done with the help of polymerase chain reaction (PCR). Observation and Results: Out of the total 222 cases of AAD, 20 (9%) were positive by culture and 70 (31.53%) were found to be toxin-producing C. difficile by ELISA. C. difficile was positive by PCR in 32 (14.41%); of these, 18 (56.25%) isolates were toxigenic, i.e., they possessed either the tcdA or the tcdB gene or both. Among the toxigenic isolates, 10 (31.25%) possessed both of the toxigenic genes (tcdA and tcdB) and the remaining 8 (25%) had one of the toxin genes. Only the toxin A (tcdA+ tcdB-) gene was found in 4 (12.5%) and only the toxin B (tcdA- tcdB+) gene in 4 (12.5%) of the toxigenic isolates. Conclusion: CDAD is an emerging nosocomial infection. Frequent and indiscriminate use of antibiotics has increased the prevalence of C. difficile infection. Conventional and molecular diagnosis can help to accurate diagnosis of these infections.

Polymerase chain reaction for Clostridioides difficile infection detection: Necessity or redundancy? – A pilot study in a tertiary health-care centre in Central Kerala

INTRODUCTION: Clostridioides difficile infection (CDI) is one of the hospital-acquired infections and the most common cause for antibiotic-associated diarrhoea. Documentation of CDI is difficult, and interpretation of diagnostic results often requires consultation with clinical microbiologists. The purpose of this study was to compare the results of the combination of glutamate dehydrogenase enzyme (GDH) and toxin assay with the polymerase chain reaction (PCR) results, in order to find if the combination could substitute for the expensive molecular tests. MATERIALS AND METHODS: The sample size was statistically calculated to be 30, using an SPSS software. Both GDH and toxin assay were simultaneously tested in all the randomly selected stool samples, by simple random sampling, and irrespective of the results, they were also tested for tcdB gene by PCR in the present study. All the samples were also plated onto Brazier's C. difficile agar and incubated anaerobically. RESULTS: The sensitivity and specificity of GDH (using PCR as gold standard) were found to be 100% and 76.47%, respectively, and the sensitivity and specificity of toxin enzyme immunoassay (EIA) assay (using PCR as gold standard) were found to be 66.67% and 92.86%, respectively. However, when the toxin-equivocal results were also considered as positive, the sensitivity of toxin EIA was found to be 100%. The overall agreeability, using Cohen's Kappa statistic between GDH and toxin detection by enzyme-linked fluorescence assay, showed that they had moderate and substantial agreement, respectively, when compared to PCR. CONCLUSIONS: In this study, each of the toxin negatives and positives was also PCR negative and positive, respectively. All the toxin-equivocal samples tested positive on PCR, so it is our conclusion that in the settings where they cannot be taken for further molecular testing, those samples be considered as harbouring toxigenic C. difficile.

2372. PCR Ribotype and Antimicrobial Susceptibility of Clostridioides (Formerly Clostridium) difficile in Korea

Background Clostridioides difficile infection is a leading cause of healthcare-associated diarrhea. The epidemiology and characteristics of C. difficile vary geographically. We performed toxin enzyme immunoassay (EIA), toxigenic gene analysis, antimicrobial susceptibility tests (AST), and PCR ribotyping to elucidate the characteristics of C. difficile in Korea.MethodsBetween July 2017 and June 2018, C. difficile was prospectively isolated in 128 specimens from the culture of 1,182 unduplicated specimens. Seventy-five stool specimens with a positive toxin EIA between July 2016 and June 2017 were also included. We performed PCR for the tcdA and tcdB genes on these isolates, and AST and PCR ribotyping on the isolates with a positive toxin EIA.ResultsOlder patients tended to have a higher rate of positive toxin EIA and positive cultures than did younger patients. Ribotype 018 was predominantly identified (48.6%), followed by ribotype 014/020 (9.9%), and ribotype 002 (8.3%). All of A-B+ isolates were either ribotype 017 or B-2. Ribotypes 017, 018, and B-2 showed high resistance to various antibiotics. In contrast, ribotypes 002, 014/020 and C-4 demonstrated low resistance rates, except that to moxifloxacin in ribotype 002. Clindamycin and erythromycin showed a positive correlation. Most of the isolates resistant to rifampicin or tetracycline showed a high MIC to both erythromycin and clindamycin.ConclusionRibotype 018, which is highly transmissible and resistant to various antimicrobial agents, is predominant in Korea. Ribotype 002 has also been increasing in prevalence in Korea.Disclosures All authors: No reported disclosures.

2410. Molecular Characteristics of Environmental Clostridioides difficile From a Large Texas Hospital

Background Clostridioides difficile is an anaerobic spore-forming, toxin-producing Gram-positive bacillus listed by the CDC as an “urgent threat” pathogen. Epidemiologic studies using whole-genome sequencing (WGS) have found that genetically distinct lineages infections occur in hospitalized patients, in addition to the fact that C. difficile spores persist on hospital surfaces after disinfection. The purposes of this study were to isolate and characterize C. difficile from the environment of a large Texas hospital.MethodsWe collected 330 swab samples of hospital environmental surfaces using sterile cotton gauze. The samples were then anaerobically enriched in brain heart infusion broth for 48–72 hours and plated onto cycloserine–cefoxitin fructose agar (CCFA). Suspected colonies were then genetically characterized using PCR (for tcdA, tcdB, cdtA, cdtB and tpi genes) and genotyped using fluorescent PCR ribotyping techniques.ResultsA total of 90/330 (27.3%) environmental samples were culture positive for C. difficile, of which 75/90 (82.1%) tested were toxigenic C. difficile by the presence of tcdA, tcdB, cdtA or cdtB. A total of 16 distinct ribotypes were identified from 41 C. difficile isolates tested using a fluorescent-ribotyping method. The predominant ribotypes isolated were F078–126 (8/41), F002 (5/41), F106 (4/41), F255 (4/41), and F014-020 (3/41).ConclusionWe found a diversity of C. difficile strain types in various hospital high-touch surface environment in addition to ribotype F027 and F078, suggesting the hospital environment a reservoir and significant source C. difficile infections.Disclosures All authors: No reported disclosures.

2355. The Association Between Diagnostic Testing Method and Clostridium difficile Infection Severity

BackgroundThe optimal diagnostic strategy for Clostridium difficile infection (CDI) is not known, and no test is shown to clearly differentiate colonization from symptomatic infection. We hypothesized that detection and/or quantification of stool toxins would associate with severe disease and adverse outcomes.MethodsWe conducted a retrospective cohort study among subjects with CDI diagnosed in 2016 at the University of Michigan. The clinical microbiology laboratory tested for glutamate dehydrogenase antigen and toxins A/B by enzyme immunoassay (EIA). Discordant results reflexed to PCR for the tcdB gene. Stool toxin levels were quantified via a modified cell cytotoxicity assay (CCA). C. difficile was isolated by anaerobic culture and ribotyped. Severe CDI was defined by the IDSA criteria: white blood cell count >15,000 cells/µL or a 1.5-fold increase in serum creatinine above baseline. The primary outcomes were all-cause 30-day mortality and a composite of colectomy, ICU admission, and/or death attributable to CDI within 30 days. Analysis included standard bivariable tests and adjusted models via logistic regression.ResultsFrom 565 adult patients, we obtained 646 samples; 199 (30.8%) contained toxins by EIA. Toxin positivity associated with IDSA severity (Table 1), but not our primary outcomes on unadjusted analysis. After adjustment for putative confounders, we still did not observe an association between toxin positivity and our primary outcomes. Stool toxin levels by CCA >6.4 ng/mL associated with IDSA severity (Table 1), but not the primary outcomes. Compared with the period from 2010 to 2013, the circulating ribotypes of C. difficile at our institution changed in 2016. Notably ribotype 106 newly emerged, accounting for 10.6% of strains, and ribotype 027 fell to 9.3% (Table 2). The incidence of ribotype 014-027 has remained stable at 18.9%, but this strain was associated with both IDSA severity and 30-day mortality (OR = 3.32; P = 0.001).ConclusionToxin detection by EIA/CCA associated with IDSA severity, but this study was unable to confirm an association with subsequent adverse outcomes. The molecular epidemiology of C. difficile has shifted, and this may have implications for the optimal diagnostic strategy for CDI. Disclosures All authors: No reported disclosures.

2403. Clostridium difficile ribotypes and human microbiota differ in Taiwan and the United States with respect to diarrheal patients

Background Clostridium difficile infection (CDI) has high incidence in the United States, but less so in East Asian countries such as Taiwan. The reason for this is not understood, but microbial studies could reveal important epidemiologic insights. We hypothesized that the circulating strains of C. difficile and the gut microbiota differ between the United States and Taiwan.MethodsPatients with diarrhea ± CDI from the University of Michigan and Chang Gung Memorial Hospital were included. CDI was defined by + enzyme immunoassay for the glutamate dehydrogenase gene and toxins A/B, with reflex to tcdB gene PCR for discordants. C. difficile was isolated by anaerobic culture and characterized by PCR ribotype. The fecal microbiota was assessed by sequence analysis of 16S rRNA-encoding gene amplicons targeting the V4 region. Amplicon sequences were processed using the mothur bioinformatics pipeline, with an operational taxonomic unit (OTU) defined by < = 3% sequence homology. Analysis was performed via logistic regression, principal coordinates (PCoA), and ANOVA.ResultsCommunity diversity by Shannon index of CDI- patients was lower (Figure 1); this difference was greater in Taiwan (P < .001, OR = 3.9 per unit Shannon). Taiwanese CDI- patients had lower Bacteroidetes relative abundance (RA) (Figure 2). The Taiwanese CDI- group also differed on PCoA and ANOVA (Figure 3, P < .001). OTU1 (genus Firmicutes) was depleted in CDI+ patients (P < .001, OR = 0.69 per 10% RA increase). Circulating ribotypes (Table 1) differ between countries, with no epidemic strains (R027/R078) present in Taiwan (P = .027). R027 and 014/020 comprised > 50% of US isolates while > 50% of Taiwanese isolates were R002.ConclusionTaiwan and US CDI+ patients differ in dominant ribotypes. It is overall difficult to differentiate diarrheal CDI+ and CDI- patients by the microbiome. Taiwanese CDI- patients are outliers, and possible reasons (e.g., differential burden of parasitic infection; diet) require further study. The increased diversity and lower Bacteroidetes in CDI+ vs. CDI- diarrheal patients contrast with prior studies that instead compared with CDI- non-diarrheal patients. Circulating strains in Taiwan include no epidemic variants; whether this explains the differential incidence needs further study. Disclosures All authors: No reported disclosures.

2236. Stool-Derived Inflammatory Mediators Serve as Biomarkers of Severity in Clostridium difficile Infection

Background Clostridium difficile infection (CDI) is a major public health concern and frequently results in severe disease, including death. Predicting subsequent complications early in the course can help optimize treatments and improve outcomes. However, models based on clinical criteria alone are not accurate and/or do not validate. We hypothesized that inflammatory mediators from the stool would be biomarkers for severity and complications.MethodsSubjects were included after testing positive for toxigenic C. difficile by the clinical microbiology laboratory via enzyme immunoassay (EIA) for glutamate dehydrogenase and toxins A/B, with reflex to tcdB gene PCR for discordants. Stool was thawed on ice, diluted 1:1 with PBS and protease inhibitor, centrifuged, and the supernatant was analyzed by a custom antibody-linked bead array with 17 inflammatory mediators. Measurements were normalized and log-transformed. IDSA severity was defined by serum white blood cell count > 15000 cells/µL or creatinine 1.5-fold above baseline. Primary 30-day outcomes were all-cause mortality and attributable disease-related complications (DRC): ICU admission, colectomy, and/or death. Analyses included principal components, permutational multivariate ANOVA (PERMANOVA), and logistic regression ± L1 regularization and 5-fold cross validation. The area under the receiver operator characteristic curve (AuROC) was computed.ResultsWe included 225 subjects, with 124 females (55.1%), average age 58.5 (±17), and more PCR+ than toxin EIA+ (170 vs. 55, respectively). IDSA severity, death, and DRCs occurred in 79 (35.1%), 14 (6.2%), and 12 (5.3%) subjects, respectively. PCA and PERMANOVA showed IDSA severity (P = 0.009) but not death or DRCs associated with the panel (figure). Several inflammatory mediators associated with IDSA severity and death (table). Machine learning models had AuROCs of 0.77 (IDSA severity), 0.84 (death), and 0.7 (DRCs).ConclusionWe found that specific inflammatory mediators from the stool of patients with CDI associate with severity and complications. These results are promising, but need replication in a larger dataset and should be incorporated into models that include clinical covariates prior to deployment. Disclosures All authors: No reported disclosures.