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Background & Aims: Recurrent Clostridioides difficile infections (CDIs) remain common. While novel microbiome therapeutics gain approval, the efficacy of a full-spectrum, oral microbiome therapeutic is unknown. This study aimed to determine the safety and efficacy of CP101, an orally administered microbiome therapeutic, to restore a diverse microbiome and prevent recurrent CDI in a broad population., Methods: We conducted a multicenter, phase 2, double-blind, randomized, placebo-controlled trial in adults with recurrent CDI. Participants with one or more CDI recurrences and diagnosis by polymerase chain reaction or toxin enzyme immunoassay for the qualifying episode were included. Participants were randomized 1:1 to receive a single oral dose of either CP101 (∼6 × 10 11 colony-forming units of lyophilized microbial cells) or placebo after standard-of-care antibiotics. The primary efficacy endpoint was the proportion of participants without CDI recurrence through week 8. Safety, efficacy, and microbiome endpoints were evaluated through weeks 8 and 24., Results: A total of 198 participants were analyzed: CP101 (n = 102) and placebo (n = 96). Overall, 27.5% had a first recurrence, and 62.7% were diagnosed by polymerase chain reaction-based testing. The proportion without CDI recurrence through week 8 was significantly higher in the CP101 group compared to the placebo group (74.5% [76 of 102] vs 61.5% [59 of 96], respectively; P = .0488), with durable efficacy observed through week 24 (73.5% [75 of 102] vs 59.4% [57 of 96], respectively; P = .0347). Similar efficacy was observed regardless of diagnostic modality or number of CDI recurrences. Rapid and durable increase in microbiome diversity was observed in the CP101 group compared to the placebo group. The incidence of adverse events was similar between the 2 groups., Conclusions: CP101 was superior to placebo in reducing recurrent CDI with a safety profile similar to placebo. (ClinicalTrials.gov, Number NCT03110133)., (Copyright © 2025 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Background: Clostridioides difficile is a major cause of antimicrobial-associated colitis. While antimicrobial stewardship has reduced the incidence of C. difficile infection (CDI), managing CDI is challenging because knowledge about preventing it is limited among healthcare professionals. To address this, we examined associations between antimicrobial use and CDI development., Methods: This observational, nested case-controlled study was conducted using the Shizuoka Kokuho Database (SKDB). Individuals with no record of CDI or antimicrobial-associated enterocolitis within 1 year after SKDB enrolment, but who subsequently developed CDI, were included as the Case group. The Control group comprised individuals selected via 1:4 matching sampling without replacement for sex, age, and month of CDI onset. Conditional logistic regression analysis was performed to assess associations between antimicrobial use (number and combination) and CDI development, controlling for matched variables, background factors, and underlying conditions., Results: Of the 2,398,393 individuals, 4917 were assigned to the Case group and 19,668 to the Control group. The adjusted odds ratios (ORs) for CDI were 3.65 for one antimicrobial and 8.58, 17.3, and 38.9 for combinations of two, three, or four or more agents (all p < 0.001). Penicillins, fourth-generation cephems, and carbapenems exhibited high ORs. Similar results were observed in certain demographic and comorbidity-free subgroups. Several combinations (penicillins + carbapenems, penicillins + cephems + carbapenems, cephems + fluoroquinolones, and penicillins + cephems + carbapenems) were notably associated with CDI development., Conclusion: CDI prevalence increased with the number of antimicrobial classes used in combination. Certain types or combinations of antimicrobials may increase the OR for CDI., Competing Interests: Conflict of interest statement The authors declare no conflicts of interest., (Copyright © 2024 Japanese Society of Chemotherapy, Japanese Association for Infectious Diseases, and Japanese Society for Infection Prevention and Control. Published by Elsevier Ltd. All rights reserved.)

Purpose: Clostridioides difficile is the main cause of antibiotic related diarrhea and some ribotypes (RT), such as RT027, RT181 or RT078, are considered high risk clones. A fast and reliable approach for C. difficile ribotyping is needed for a correct clinical approach. This study analyses high-molecular-weight proteins for C. difficile ribotyping with MALDI-TOF MS., Methods: Sixty-nine isolates representative of the most common ribotypes in Europe were analyzed in the 17,000-65,000 m/z region and classified into 4 categories (RT027, RT181, RT078 and 'Other RTs'). Five supervised Machine Learning algorithms were tested for this purpose: K-Nearest Neighbors, Support Vector Machine, Partial Least Squares-Discriminant Analysis, Random Forest (RF) and Light-Gradient Boosting Machine (GBM)., Results: All algorithms yielded cross-validation results > 70%, being RF and Light-GBM the best performing, with 88% of agreement. Area under the ROC curve of these two algorithms was > 0.9. RT078 was correctly classified with 100% accuracy and isolates from the RT181 category could not be differentiated from RT027., Conclusions: This study shows the possibility of rapid discrimination of relevant C. difficile ribotypes by using MALDI-TOF MS. This methodology reduces the time, costs and laboriousness of current reference methods., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Clostridioides difficile ( C. difficile ) is a leading cause of healthcare-associated infections. Using opioids while infected with C. difficile may hypothetically lead to reduced clearance of the organism and higher risk of progressing to severe or fulminant infection. Objective: The objective of this study was to determine if opioid use leads to worsening of C. difficile infection. Methods: This was a single-center, retrospective cohort study of patients with C. difficile infection. The primary endpoint was progression to severe or fulminant disease, defined as serum creatinine greater than 1.5 mg/dL or over 50% of baseline, white blood cells above 15,000 cells/mm 3 , shock requiring vasopressors, ileus, toxic megacolon, or vancomycin dose increase. Secondary outcomes included hospital length of stay and time to resolution of diarrhea. The groups were stratified based on average morphine milligram equivalents received during the treatment. Results: A total of 73 patients were included in the non-opioid group and 93 patients in the opioid group. The composite outcome occurred in 16 patients (21.9%) without opioids vs 26 patients (28.0%) with opioids; ( P = 0.37). The average length of stay was 7.2 days without opioids and 9.3 days with opioids ( P = 0.11) and the average time to resolution of diarrhea was 3.5 days without opioids and 5.5 days with opioids ( P = 0.40). Conclusion: There was no significant difference in the rate of progression to severe or fulminant disease. There was a numerical trend towards increase in progression in patients who had opioids, primarily driven by those who had higher dosages of opioids used., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Clostridioides difficile is one of the leading causes of antibiotic-associated diarrhea. In this study, we characterized 76C. difficile isolates, obtained from three Mediterranean mussel (Mytilus galloprovincialis) farms in Slovenia from November 2014 to October 2015 (sampling period 1) and from January to December 2021 (sampling period 2). The overall isolation rate of C. difficile from all the examined mussels was 59.8 %. A statistically significant trend of seasonal variation was observed, with a higher isolation rate in the colder months of the year (87.9 %; sea temperature ≤ 15 °C) compared with the warmer months (31.8 %; sea temperature > 15 °C). Whole-genome sequencing (WGS) revealed that the isolates belonged to 31 different sequence types (STs), which were associated with three clades (1, 4, and 5) and two cryptic clades (C-II and C-III). Five isolates, which belonged to ST11 (clade 5), harbored all the main toxin genes (A + B + CDT + ) and chromosomal mutations conferring fluoroquinolone resistance. Core genome multilocus sequence typing (cgMLST) revealed four clusters of 2-3 isolates, three of which included isolates from different farms, suggesting that clonal C. difficile strains are circulating among the Slovenian mussel farms. The results highlight the presence of hypervirulent strains in mussels; therefore, at-risk population groups should be alerted to the risks associated with consuming shellfish., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Objectives: Diarrhea is a common disease that could threaten the welfare of newborn foals. While there are several forms of foal diarrhea, the etiologies can be considered known pathogenic or non-pathogenic in nature. Moreover, there are likely differences in the composition of microbial populations in the gastrointestinal tracts of foals depending upon the etiology of diarrhea. Our study aims to examine the microbial population in the feces of foals with both pathogenic and non-pathogenic diarrheas to discern differences in their microbial compositions., Results: Foal diarrhea samples tested positive or negative for common equine neonatal diarrhea pathogens by diagnostic polymerase chain reaction (PCR), which allowed for samples to be segregated as pathogenic or non-pathogenic. Pathogenic samples tested positive for combinations of Clostridium perfringens and/or Clostridioides difficile toxins. As a result, significantly higher alpha diversity was seen in the non-pathogenic samples than in pathogenic ones. Sequencing of the V4 domains of bacterial 16 S rRNA genes demonstrated that non-pathogenic samples had more alpha diversity. Furthermore, eight microbial families and eleven genera showed significant differences in their abundances between pathogenic and non-pathogenic diarrhea samples., Competing Interests: Declarations. Ethical animal research: Fecal samples were collected from university-owned horses and from non-university horses with permission of owners and, both with IACUC approval (#18998). Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Clostridioides difficile species lacking toxin genes (non-toxigenic C. difficile or NTCD) may confer protection against CDI. However, current diagnostic tests detect either toxin proteins or toxin genes and cannot detect NTCD. This study developed a molecular testing method that uniquely identified NTCD and assessed its prevalence in a clinical cohort. A quantitative PCR (qPCR) assay was developed and validated using reference strains. Analytic sensitivity was determined using DNA from reference NTCD strains, and qPCR efficiency was assessed using the slope of the standard curves of DNA dilutions. A random selection of 95 clinical stool samples, tested using the GDH enzyme and toxin enzyme immunoassay (EIA), was used to evaluate the prevalence of NTCD in hospitalized patients tested for CDI. The KB-1/KB-2 primers and probe designed were specific for NTCD strains and did not amplify with toxigenic C. difficile or other related strains. The NTCD qPCR assay analytical sensitivity was linear between 3 × 10 1 and 3 × 10 6 gDNA ( R 2 = 0.999; P < 0.0001). No NTCD was found in 25 GDH-EIA -/- samples compared to 5 of 25 (20%) GDH-EIA +/- samples and 2 of 23 (8.7%) GDH-EIA +/+ samples. Of samples detected with NTCD, median NTCD DNA was 33,039 (IQR: 22.449-45.688) in GDH-EIA +/- samples and 370 [IQR: 159-583] in GDH-EIA +/+ samples. The new qPCR NTCD assay identified NTCD colonization in 7% of hospitalized patients tested for CDI. This NTCD assay may have important implications for diagnostic and antimicrobial stewardship as colonization with NTCD strains may offer protection against CDI.IMPORTANCECurrent diagnostic strategies do not detect non-toxigenic Clostridioides difficile (NTCD) strains, which may provide protection against C. difficile infection (CDI). Detecting these strains is critical as it underscores the importance of avoiding unnecessary antibiotic treatment in patients colonized with NTCD. To better guide clinical decisions and enhance the understanding of NTCD epidemiology, molecular assays that specifically target non-coding regions unique to NTCD strains are needed. In this study, we developed and validated a qPCR assay capable of uniquely identifying NTCD strains. This innovative assay holds significant potential for applications in public health, infection control, diagnostic, and therapeutic strategies related to CDI., Competing Interests: The authors declare no conflict of interest.

Current guidelines recommend a two-step algorithm rather than relying solely on a single test for diagnosing Clostridioides difficile infection. This algorithm starts with enzyme immunoassay (EIA) for detecting glutamate dehydrogenase (GDH) and toxins A/B, followed by nucleic acid amplification test (NAAT) for GDH-positive but toxin-negative cases. This study compared the performance of three commercial NAATs: the STANDARD M10 C. difficile , Xpert C. difficile , and BD MAX Cdiff assays, utilized as confirmatory testing of the two-step algorithm. Two hundred archived stool specimens, previously tested GDH-positive but toxin-negative by EIA, were analyzed in parallel with these NAATs and toxigenic culture, which served as the reference standard. Sensitivity, specificity, positive predictive value, and negative predictive value were 89.1%, 92.6%, 94.6%, and 85.2%, respectively, for the M10 assay; 95.8%, 86.4%, 91.2%, and 93.3%, respectively, for the Xpert assay; and 89.8%, 91.4%, 93.8%, and 86.0%, respectively, for the BD MAX assay. The rates of invalid results were 1.0%, 0.5%, and 1.0% for the M10, Xpert, and BD MAX assays, respectively. In conclusion, the M10 assay is a reliable diagnostic tool, performing comparably to the Xpert and BD MAX assays when used as confirmatory testing in the two-step algorithm.IMPORTANCEWhile numerous studies have assessed nucleic acid amplification tests (NAATs) as stand-alone tests for diagnosing Clostridioides difficile infection, limited research has compared their performance as confirmatory tests in a two-step algorithm. This study evaluated the performance of three commercial NAATs (M10, Xpert, and BD MAX assays) using 200 archived stool specimens initially tested as glutamate dehydrogenase (GDH)-positive but toxin-negative by GDH/toxin A/B enzyme immunoassay, the first step in the two-step algorithm. All three assays demonstrated high sensitivity (89.1% to 95.8%) and specificity (86.4% to 92.6%), with low rates of invalid results (≤1%). Our findings suggest that the M10 assay performs comparably to the Xpert and BD MAX assays when used as confirmatory testing in the two-step algorithm. Offering similar performance and turnaround time to these widely used assays at a slightly lower cost, the M10 assay serves as a practical alternative in this setting., Competing Interests: The authors declare no conflict of interest.

Background Clostridioides difficile infection (CDI) is a severe infection that needs to be monitored. This infection predominantly occurs in hospitalised patients after antimicrobial treatment, with high mortality in elderly patients.AimWe aimed at estimating the incidence of CDI in Italian hospitals over 4 months in 2022.MethodsWe estimated incidences of hospital-acquired CDI (HA-CDI), community or unknown CDI (CA/UA-CDI), recurrent CDI and overall CDI in 25 Italian hospitals, characterised C. difficile isolates using PCR ribotyping, analysed them for toxin genes and susceptibility to antimicrobials.Results Clostridioides difficile was detected in 9.7% (655/6,722) of samples from 550 patients, 18 patients died of CDI. The mean overall CDI incidence was 5.0 cases per 10,000 patient days (range: 0.7-11.9). For HA-CDI, mean incidence was 3.7 (range: 0.7-9.2), for CA/UA-CDI 0.8 (range: 0.0-3.2) and for recurrent CDI 0.5 (range: 0.0-3.4). Most patients were female (n = 295; 53.6%), aged ≥ 65 years (n = 422; 76.7%) and previously hospitalised (n = 275; 50.0%). Of the 270 culturable isolates, 267 (98.9%) had toxin A and B genes and 51 (18.9%) the binary toxin genes. Of the 55 PCR ribotypes (RTs) identified, RT 018 (n = 56; 20.7%) and RT 607 (n = 23; 8.5%) were the most common, RT 607 in the northern (p < 0.0001) and RT 018 in the central (p < 0.0001) regions of Italy. Most isolates (n = 158; 58.5%) were antimicrobial-resistant and 119 (44.1%) were multidrug-resistant (MDR).ConclusionHighly virulent and MDR C. difficile types are circulating in Italian hospitals which highlights the need of robust surveillance and stringent prevention and control measures.

Background: This study explores the infrastructural and organizational risk factors for health care-associated (HCA) Clostridioides difficile infections (CDIs) and methicillin-resistant Staphylococcus aureus (MRSA) in hospitals., Methods: This is a retrospective observational study involving all eligible inpatient units from 12 hospitals in British Columbia, Canada, from April 1, 2020 to September 16, 2021. The outcomes were the average HCA CDI or MRSA rates. Covariates included, but were not limited to, infection control factors (eg, hand hygiene rate), infrastructural factors (eg, unit age), and organizational factors (eg, hallway bed utilization). Multivariable regression was performed to identify statistically significant risk factors., Results: Older units were associated with higher HCA CDI rates (adjusted relative risk [aRR]: 0.012; 95% confidence interval (CI) [0.004, 0.020]). Higher HCA MRSA rates were associated with decreased hand hygiene rate (aRR: -0.035; 95% CI [-0.063, -0.008]), higher MRSA bioburden (aRR: 9.008; 95% CI [5.586, 12.429]), increased utilization of hallway beds (aRR: 0.680; 95% CI [0.094, 1.267]), increased nursing overtime rate (aRR: 5.018; 95% CI [1.210, 8.826]), and not keeping the clean supply room door closed (aRR: -0.283; 95% CI [-0.536, -0.03])., Conclusions: The study confirmed the multifaceted nature of infection prevention and emphasized the importance of interdepartmental collaboration to improve patient safety., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background and Purpose: Clostridioides difficile infection (CDI) is a leading cause of healthcare-associated infections worldwide, with hypervirulent strains linked to severe disease and higher mortality. This study aims to analyze the epidemiology of CDI at a tertiary-care hospital in Italy and compare clinical outcomes between patients infected with hypervirulent and non-hypervirulent strains., Methods: A retrospective comparative study was conducted on patients diagnosed with CDI at ASST Spedali Civili di Brescia, Italy, from January 2015 to June 2023. Hypervirulent strains were identified using the GeneXpert assay as positive for cytotoxin gene (tcdB), binary toxin genes (tcdA and tcdB) and a single nucleotide deletion at position 117 in the tcdC gene and compared to a randomized matched control group with non-hypervirulent CDI. Clinical data were collected and analyzed, with multivariate logistic regression employed to identify risk factors for hypervirulent CDI., Results: Of 1,059 positive C. difficile specimens, a statistically significant trend between January 2015 to June 2023 was found in the increasing incidence of CDI cases per 1,000 hospital admissions and 10,000 bed-days. Notably, a remarkable increase of hypervirulent strains was recorded in 2021 and 2022 when compared to previous years. A total of 130 patients were analyzed: 62 (47.7%) with hypervirulent CDI and 68 (52.3%) controls. Hypervirulent CDI was associated with higher 30-day mortality (18% vs. 5.8%, p = 0.03). Multivariate analysis showed that hypervirulent CDI significantly increased 30-day mortality risk (OR = 9.915, CI = 2.37-61.05, p = 0.005) and that prior antibiotic therapy was a significant risk factor (OR = 5.49, CI = 1.19-39.96, p = 0.047)., Discussion: Our epidemiological data, while suggesting a potential resurgence in CDI transmission during COVID-19 pandemic, are derived from a single-center experience with limited generalizability to the broader population. Nonetheless, they highlight the need for strengthened antimicrobial stewardship and national surveillance systems to effectively monitor and manage these strains., Competing Interests: Declarations. Ethical approval: The study protocol was approved by the ASST Spedali Civili Ethics Committee (protocol NP 6143). Competing interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

We present a case of tubo-ovarian abscess (TOA) caused by Clostridioides difficile (CD) in a 43-year-old female. Despite lacking a history of sexually transmitted diseases, the patient had undergone paraovarian cystectomy nine months before admission. Transvaginal ultrasonography performed eight months post-surgery revealed left ovarian enlargement, accompanied by subsequent lower abdominal pain and fever exceeding 38 °C. As oral antibiotic treatment was ineffective, the patient was admitted to our hospital. Computed tomography upon admission revealed a massive TOA. Surgical drainage of the abscess was performed, and CD was identified in the culture from the pus. The TOA was treated with a three-month course of metronidazole and oral amoxicillin/clavulanic acid. While CD is commonly associated with colitis, extraintestinal manifestations are exceptionally rare. This case represents the inaugural report of TOA resulting from CD. A literature review on abdominal and pelvic CD abscesses found that patients undergoing surgical drainage had a favorable prognosis. Therefore, surgical intervention plays an important role in the management of CD abscesses., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Japanese Society of Chemotherapy, Japanese Association for Infectious Diseases, and Japanese Society for Infection Prevention and Control. Published by Elsevier Ltd. All rights reserved.)

Background: The study of the epidemiology of Clostridioides difficile in populations is greatly facilitated by the ability to isolate and further characterize individual organisms, which requires effective culture protocols. In cattle, where little is known about the epidemiology of C. difficile, no studies have assessed or compared the performance of different assays for detecting C. difficile., Objectives: This study compared two culture protocols for detecting C. difficile in bovine faeces from 121 gestating cows and 70 of their neonatal calves, while situating results obtained with each protocol relative to those obtained with shotgun metagenomic sequencing., Methods: Protocol 1 involved direct plating enrichment onto taurocholine-cycloserine-cefoxitin-fructose agar (TCCFA), while Protocol 2 included an ethanol shock step before plating on CCFA/ChromID agar. For both protocols, one aliquot underwent broth enrichment prior to plating, while the other aliquot did not., Results: Clostridioides difficile was detected following broth enrichment in two of the same calf samples using both protocols, and an additional cow sample was found to be positive with Protocol 2, though the difference in detection rates was not statistically significant (p = 1.0)., Conclusions: The detection of C. difficile in a much high number of these samples by shotgun metagenomics, albeit at low levels of relative abundance, suggests that neither of these culture protocols is sensitive when levels of abundance are low., (© 2024 The Author(s). Veterinary Medicine and Science published by John Wiley & Sons Ltd.)

For Clostridioides difficile infections, highly sensitive rapid diagnostic test kits are necessary for prompt diagnose and infection control. In this study, we evaluated "Quick Chaser CD GDH/TOX" (evaluation kit), a rapid diagnostic kit for C. difficile , using 65 clinical stool specimens, comparing with GE test immunochromato-CD GDH/TOX "Nissui" (GE test) and TECHLAB C. DIFF QUIK CHEK COMPLETE (QUIK CHEK). The results of the evaluation kit showed a high concordance rate; 100% the positive concordance rate (31/31) and 97.1% the negative concordance rate (33/34) of GDH, 94.1% the positive concordance rate (16/17) and 97.9% the negative concordance rate (47/48) of toxin against GE test, respectively. Also 96.9% the positive concordance rate (31/32) and 97.0% the negative concordance rate (32/33) of GDH, 100% the positive concordance rate (12/12) and 90.6% the negative concordance rate (48/53) of toxin against QUIK CHEK, respectively. Additionally, comparison of discrepancy cases between kits based on Nucleic Acid Amplification test, one false negative each for GDH and toxin in the evaluation kit, two false negatives for GDH and one false negative for toxin in the GE test, one false negative for GDH and six false negatives for toxin in QUIK CHEK, and no false positive cases were observed. Therefore, the detection sensitivity of GDH and toxin of the evaluation kit was equivalent to that of two types of comparative kits.

Background: The most severe complications of antibiotic use are clostridial infection (CDI) and pseudomembranous colitis (PMC). There is a need for further study of these conditions and identification of their triggers., Aim: To identify risk factors for severe forms of antibiotic-associated diarrhea caused by Clostridioides difficile ., Materials and Methods: A clinical and laboratory examination of 440 patients with CDI who were treated at Botkin Clinical Infectious Diseases Hospital was conducted. CDI was confirmed by detection of toxins A and B of C. difficile in feces using enzyme-linked immunosorbent assay and immunochromatography. Metronidazole (2 g/day, in patients without comorbid pathology) and vancomycin (0.5-2 g/day) were used as a starting drug for up to 14 days. Statistical processing of the obtained data was performed using the Statistica for Windows, v.10 (StatSoft, USA) program., Results: CDI was confirmed by enzyme-linked immunosorbent assay in 202 (45.91%) patients, immunochromatography - in 149 (33.86%), endoscopically - in 203 (46.14%). CDI was most frequently recorded in 2 age groups of patients: 18-30 years old - in 137 (31.14%); over 60 years old - in 205 (46.59%). CDI was characterized by a combination of colitic and intoxication syndromes. In 43 (9.77%) patients the disease resulted in death. The diagnosis of PMС was established in 61 (13.86%) people. The risks of developing PMC when prescribing antibacterial drugs decreased in the following order: fluoroquinolones, cephalosporins, macrolides, penicillins, nitrofurans and chloramphenicol. Probiotic drugs reduced the risk of developing PMC (relative risk 0.5 [0.3; 0.7]; p =0.002)., Conclusion: PMC was characterized by a combination of diarrhea, intoxication and abdominal pain. The formation of PMC was preceded by courses of antibacterial drugs of the fluoroquinolone and cephalosporin groups, eradication therapy for Helicobacter infection, the use of high doses of glucocorticosteroids, as well as intestinal superinfection in patients with previous episodes of CDI.

Clostridioides difficile ( C. difficile ) was a gram-positive anaerobic bacterium in the gut, exhibiting clinical manifestations ranging from mild diarrhoea to fatal pseudomembranous colitis. C. difficile infection (CDI) remains a serious public health problem and accounted for an estimated 360,075 cases in the United States in 2021. It has attracted the utmost attention of the world health organization (WHO). Since publication of a review of the diagnosis of CDI in adults, new clinical diagnostic assays have become available and clinical practice guidelines were updated. This paper presents a comprehensive review of contemporary laboratory diagnostic approaches for CDI in adult patients, with a focus on the utilisation and potential advancements of five sophisticated methodologies, CRISPR in conjunction with nucleic acid amplification tests (NAATs), gene sequencing technology, ultra-high performance liquid chromatography-mass spectrometry, Raman spectroscopy, and real-time cell analysis (RTCA). It can provide new perspectives and ideas for the early diagnosis of CDI in clinical settings., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Liu, Chen, Hu, Qiu, Chen, Xu, Yuan, Zhang and Li.)

Diagnosis of Clostridioides difficile infection (CDI) remains challenging as it involves in the first instance recognition (clinical awareness) of the patients' symptoms for clinical suspicion of CDI to warrant testing, and secondly, different laboratory tests have been described for CDI. Due to the overwhelming amount of information in the literature on CDI tests and their performance, with separately published guidelines, this review aims to provide a comprehensive but concise summary of the current state of CDI diagnostic testing. Current knowledge and the impact of using different laboratory diagnostic procedures for CDI, including the most recommended approach as a two-step algorithm and the concept of diagnostic stewardship, are being discussed. This review provides an updated overview and valuable take-home messages in the field of CDI laboratory testing and highlights that timely diagnosis is important for the clinical management of CDI and that the recommended testing procedures are increasingly becoming more widely accepted.

Clostridioides difficile is a One Health pathogen found in humans, animals, and the environment, with food representing a potential transmission route. One Health studies are often limited to a single country or selected reservoirs and ribotypes. This study provides a varied and accessible collection of C. difficile isolates and sequencing data derived from human, animal, and food sources across 13 European countries. A total of 441 strains (human hospital- and community-associated cases n  = 280, animal n  = 96, food n  = 65) were analysed by ribotyping, toxinotyping and whole-genome sequencing (WGS). We detected 83 sequence types (STs), with ST11 ( n  = 80 isolates) and ST1 ( n  = 54 isolates) being the most represented. Several STs included strains originating from all source combinations. Further genomic analysis confirmed close genetic relatedness in some of the STs. Additionally, the genomic analysis identified 10 strains from cryptic clades (C-I to C-III) and 4 of them were mono-toxigenic possessing only a variant form of tcd A gene. Amongst 106 ribotypes, 10 were shared between all 3 sources and 68 were source-specific. Some ribotypes were only found at the intersection of human and food source (RT023, RT027), or between human and animal source (RT009, RT045, RT046). C. difficile ribotypes and STs in Europe were diverse. In this collection, some ribotypes showed potential association with food or animal transmission routes. C. difficile strains from divergent clades CI-III, currently emerging in the human population, were rare and mostly food-associated. Trial registration: ClinicalTrials.gov identifier: NCT03503474.

Introduction: In the past decade, the incidence of community-acquired Clostridioides difficile infection (CA-CDI) has increased, suggesting a role for community reservoirs such as animals in its spread., Objective: This study aimed to isolate and characterize C. difficile strains from domestic dogs at veterinary clinics to enhance our understanding of C. difficile epidemiology in Rio de Janeiro., Material and Methods: For this study 90 stool samples from dogs were collected and cultured in a selective medium (Clostridioides difficile Brucella agar - CDBA) for isolation. Species were identified by MALDI-TOF MS, with confirmation provided by PCR targeting the tpi gene. The antibiotic susceptibility test of the strains was performed using five antibiotics: vancomycin, metronidazole, moxifloxacin, rifampicin, and erythromycin. Strains resistant to metronidazole were further analyzed for the presence of the plasmid pCD-METRO using PCR. The presence of toxin genes (tcdA, tcdB, and cdtB) was investigated, alongside ribotyping and tcdC sequencing analyses. The strains were also tested for biofilm formation and motility., Results: C. difficile was isolated in 15.5 % (14/90) of the samples. Among the strains analyzed, 87.71 % (12/14) tested positive for both toxin genes tcdA and tcdB and belonged to ribotypes 106 (10/14) and 014/020 (2/14). The remaining 14.3 % (2/14) were non-toxigenic and were identified as ribotype 010. Regarding the antibiotic profile, 42.85 % (6/14) of the strains exhibited resistance to at least one antibiotic, including vancomycin (1/14) and metronidazole (1/14). The metronidazole-resistant strain was also positive for the plasmid pCD-METRO. All strains exhibited both biofilm formation and motility. Among the 12 toxigenic strains sequenced for the tcdC gene, two exhibited a deletion in the same region as the epidemic strain, NAP1 (RT027)., Conclusion: Our study found some overlap between C. difficile ribotypes isolated from dogs and from cases of CDI in humans, and the C. difficile prevalence was higher in dogs with diarrhea (p = 0.034)., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Clostridioides difficile sequence type (ST) 35 has been found in humans and animals worldwide. However, its genomic epidemiology and clonal transmission have not been explored in detail. In this study, 176 C. difficile ST35 isolates from six countries were sequenced. Genomic diversity, clonal transmission and epidemiological data were analyzed. Sporulation and virulence capacities were measured. Four ribotypes (RT) were identified including RT046 (97.2%), RT656 (1.1%), RT427 (0.6%), and RT AI-78 (1.1%). Phylogenetic analysis of 176 ST35 genomes, along with 50 publicly available genomes, revealed two distinctive lineages without time-, region-, or source-dependent distribution. However, the distribution of antimicrobial resistance genes differed significantly between the two lineages. Nosocomial and communal transmission occurred in humans with the isolates differed by ≤ two core-genome single-nucleotide polymorphism (cgSNPs) and clonal circulation was found in pigs with the isolates differed by ≤ four cgSNPs. Notably, interspecies clonal transmission was identified among three patients with community acquired C. difficile infection and pigs with epidemiological links, differed by ≤ nine cgSNPs. Toxin B (TcdB) concentrations were significantly higher in human isolates compared to pig isolates, and ST35 isolates exhibited stronger sporulation capacities than other STs. Our study provided new genomic insights and epidemiological evidence of C. difficile ST35 intraspecies and interspecies clonal transmission, which can also be facilitated by its strong sporulation capacity.

Hospital surfaces are known to contribute to the spread of healthcare-associated antimicrobial pathogens. Environmental sampling can help locate reservoirs and determine intervention strategies, although sampling and detection can be labor intensive. Composite approaches may help reduce time and costs associated with sampling and detection. We investigated optimum surface areas for sampling antimicrobial-resistant organisms (AROs) with a single side of cellulose sponge, created theoretical composites (TC) by adding recovery results from multiple optimum areas, then compared the TC to the standard Centers for Disease Control and Prevention sampling method (one sponge using all sides, whole tool; (WT)). Five AROs were evaluated: carbapenemase-producing KPC+ Klebsiella pneumoniae (KPC), Acinetobacter baumannii (AB), methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecalis (VRE) and Clostridioides difficile spores (CD). Steel coupons comprising four surface areas (323; 645; 1,290 and 2,258 cm2) were inoculated, dried, and sampled with one sampling pass using the larger side (face) or the smaller side (edge) of a pre-moistened cellulose sponge tool. Based on the optimum areas determined for each organism, composite areas were 1,290 cm2 for MRSA and VRE, 1,936 cm2 for AB, 2,580 cm2 for CD spores and 3,870 cm2 for KPC. Total colony forming units (CFU) recovered using a composite approach was greater or comparable than using multiple WT samplings (over the same area as the composite) for MRSA, VRE and AB (130%; 144% and 95%) yet less than if using multiple WT samplings for KP and CD (47% and 66%). We propose a conservative composite sampling strategy if the target organism is unknown; 323 cm2 sampling area for each of the four sides of the sponge, (1290 cm2 total). The conservative composite sampling strategy improved the recovery of KP (from 47% to 85% of multiple WT samplings), while MRSA, VRE, AB and CD (131%; 144%; 97% and 66%) remained within 5% to that of the optimum area TC., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Background: Clostridiodes difficile bacteremia (CDB) develops in less than 1% of all the cases, nonetheless, with a significant increase in mortality. The objective of the present article is to report the first case of CDB in the Mexican population, empathizing with the treatment difficulty derived from the lack of consensus., Clinical Case: A 42-year-old female with previous history of chronic thromboembolic pathologies and grade III obesity, develops community acquired atypical pneumonia requiring hospitalization, having parenteral antibiotics. During hospitalization, non severe C. difficile infection was concluded and started treatment with Vancomycin for 10 days. One week later the patient presented to the emergency department at HES CMN SXXI with gastrointestinal symptomatology, mostly epigastric pain, fever and diarrhea. After proper approach, C. difficile was isolated in blood cultures through mass spectrophotometry analysis, for which a 10-day treatment of metronidazole was prescribed with resolution of the infection being discharged afterwards., Conclusions: CDB is an exceptional and lethal clinical condition, mainly seen in patients with previous gastrointestinal diseases. IV Metronidazole demonstrated therapeutic efficacy in this case report and could be a successful and available therapeutic option in Mexican health care units., (Licencia CC 4.0 (BY-NC-ND) © 2024 Revista Médica del Instituto Mexicano del Seguro Social.)

Background: The increasing incidence of antibiotic-associated diarrhoea (AAD) is a serious health care problem. Dysbiosis of the gut microbiota is suspected to play a role in the pathogenesis of AAD, but its impact on the clinical outcomes of patients remains unclear., Methods: Between May and October 2022, 210 patients with AAD admitted to a university hospital and 100 healthy controls were recruited. DNA extraction from stool specimens and shotgun sequencing were performed. Machine learning was conducted to assess profiling at different taxonomic levels and to select variables for multivariable analyses., Results: Patients were classified into two groups: Clostridioides difficile infection (CDI, n = 39) and non-CDI AAD (n = 171). The in-hospital mortality rate for the patients was 20.0%, but the presence of C. difficile in the gut microbiota was not associated with mortality. Machine learning showed that taxonomic profiling at the genus level best reflected patient prognosis. The in-hospital mortality of patients was associated with the relative abundance of specific gut microbial genera rather than alpha-diversity: each of the five genera correlated either positively (Enterococcus, Klebsiella, Corynebacterium, Pseudomonas, and Anaerofustis) or negatively (Bifidobacterium, Bacteroides, Streptococcus, Faecalibacterium, and Dorea). Genes for vancomycin resistance were significantly associated with in-hospital mortality in patients with AAD (adjusted hazard ratios, 2.45; 95% CI, 1.20-4.99)., Conclusion: This study demonstrates the potential utility of metagenomic studies of the gut microbial community as a biomarker for prognosis prediction in AAD patients., (Copyright © 2024 Elsevier Ltd and International Society of Antimicrobial Chemotherapy. All rights reserved.)

We evaluated the analytical performance of three commercial molecular assays for rapid detection of Clostridioides difficile toxin B in stool samples. The results were compared with results from the BD MAX™ Cdiff assay. We analyzed forty negative and thirty-two positive stool samples with three rapid assays: Roche cobas® Liat® Cdiff, SD Biosensor STANDARD™ M10 C. difficile and Cepheid Xpert® C. difficile BT. The assays demonstrated a sensitivity of 96.9 %, 96.9 % and 100.0 % and a specificity of 100 %, 97.5 % and 97.5 %, respectively. There is limited data available on the analytical performance of the newly introduced STANDARD™ M10 C. difficile assay. In this study, all three rapid assays demonstrated similarly high analytical performance and can be used for detection of toxigenic C. difficile., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Clostridioides difficile infection (CDI) is one of the most significant causes of diarrhea in hospitalized patients. The aim of this study was to investigate the incidence and epidemiology of CDI after the COVID-19 pandemic in hospitalized patients in a rehabilitation center in Thessaloniki, Greece. Α retrospective observational cohort study was performed in inpatients diagnosed with diarrhea of all ages (January 2023 - December 2023) who were initially screened for CDI. From the total cohort of patients with proven CDI, some patients were randomly selected based on their monthly isolation incidence throughout the study period, to investigate their epidemiological data and clinical characteristics. Laboratory diagnosis of CDI was performed by enzyme immunoassay, followed by specific anaerobic culture and molecular testing for detection of toxigenic C. difficile. The isolated C. difficile strains were further characterized by PCR ribotyping. The annual incidence of CDI during the study period was 27.1% (130/480). The linear trend of CDI incidence decreased from 32.5% to 18.2% (P = 0.024). The all-cause mortality rate was 5.0% (3/60). A positive correlation was observed between the length of hospital stay and the number of recurrences (r = 0.546, P < 0.001), while 28 patients (46.7%) experienced recurrence of the infection. Seven different PCR ribotypes were identified in this study. C. difficile tcdA+, tcdB+, cdtA+, cdtB+ PCR ribotype 181 (RT181) was the predominant (76.6%, 46/60), followed by toxin A-negative PCR RT017 (11.6%, 7/60). The annual incidence of CDI decreased after the COVID-19 pandemic. Our study demonstrates predominance of C. difficile RT181 with tcdA+, tcdB+, cdtA+, cdtB+ toxin gene profile after COVID-19 pandemic in Northern Greece.

Background: Inflammatory bowel disease (IBD) is a chronic, relapsing condition wherein biologics have improved disease prognosis but introduced elevated infection susceptibility. Vedolizumab (VDZ) demonstrates unique safety advantages; however, a comprehensive systematic comparison regarding the risk of Clostridioides difficile infection (CDI) between vedolizumab and alternative medications remains absent., Method: Medline, Embase, Cochrane, and clinicaltrials.gov registry were comprehensively searched. Pooled estimates of CDI proportion, incidence, pooled risk ratio between ulcerative colitis (UC) and Crohn's disease (CD), vedolizumab and other medications were calculated. Data synthesis was completed in R using the package "meta"., Results: Of the 338 studies initially identified, 30 met the inclusion/exclusion criteria. For CDI risk, the pooled proportion was 0.013 (95% CI 0.010-0.017), as well as the pooled proportion of serious CDI was 0.004 (95% CI 0.002-0.008). The comparative pooled risk ratios revealed: UC versus CD at 2.25 (95% CI 1.73-2.92), vedolizumab versus anti-TNF agents at 0.15 (95% CI 0.04-0.63) for UC and 1.29 (95% CI 0.41-4.04) for CD., Conclusion: The overall CDI risk in IBD patients exposed to vedolizumab was estimated to be 0.013. An increased risk of CDI was noted in UC patients receiving vedolizumab compared to those with CD. Vedolizumab potentially offers an advantage over anti-TNF agents for UC regarding CDI risk, but not for CD., Trial Registration: The study was registered on the PROSPERO registry (CRD42023465986)., (© 2024. The Author(s).)

Clostridioides difficile is an intestinal pathogen of humans and animals. In community-associated infections, the environment is suggested to play a significant role in overall transmission routes. Although the prevalence of C. difficile in freshwater and soil has been widely studied, little is known about its presence in sediments. In this study, we tested 15 sediment samples collected from various freshwater sources. C. difficile was isolated from all sampled sites, yielding a total of 171 strains grouped into 26 ribotypes, with 001/072 and 014/020 being the most prevalent. Genome sequencing of 37 isolates from 17 PCR ribotypes confirmed the presence of highly related strains in the geographically distant and unlinked water samples. Eight divergent PCR ribotypes from clades C-II and C-III were found in six samples. In each sample, the unbound fraction (supernatant after sediment wash) and bound fraction (sonicated sediment sample) were subjected to enrichment. Sonication was only slightly better than washing in terms of sample positivity (14 positive samples with sonication and 11 with washing). However, sonication substantially increased the diversity of the PCR ribotypes obtained (23 in sonicated samples vs nine in washed samples). In conclusion, sediments are a rich source for investigating the diversity of environmental C. difficile, including isolates from divergent lineages. Selection of the isolation method can significantly impact the diversity of captured PCR ribotypes.IMPORTANCE Clostridioides difficile , a pathogenic bacterium that can cause intestinal infections in humans and animals, thrives in the gut but also disperses widely through spores found in the environment. Clinical and environmental strains often overlap with common PCR ribotypes, which are consistently isolated worldwide. Environmental studies have mostly focused on water and soil, but sediments have been very poorly studied. In this study, we investigated the presence of C. difficile in various freshwater sediments and evaluated the effectiveness of two different isolation approaches on positivity rates and strain diversity. C. difficile was found to be highly prevalent in sediments, with an isolation rate of 100%. Sonication proved to be more effective than simple washing for capturing a greater diversity of PCR ribotypes. Overall, this study underscores the widespread presence of C. difficile in freshwater sediments and emphasizes the importance of continued surveillance and monitoring to understand its ecology and transmission dynamics., Competing Interests: The authors declare no conflict of interest.

Clostridioides difficile strains of sequence type (ST) 37, primarily including PCR ribotype (RT) 017, are prevalent in mainland China. Our study aimed to compare the major virulence factors of an epidemic C. difficile isolate of ST37 type (Xy06) from China with the well-characterized C. difficile reference strains R20291 (RT027) and CD630E (ST54), as well as a Chinese ST54 strain (Xy07) isolated from the same hospital. The Xy06 genome was predicted to harbor two complete prophages and several transposon-like elements. Comparative analysis of PaLoc revealed a truncated tcdA gene, a functional tcdB gene, a functional tcdC gene, and well-conserved tcdR and tcdE genes. Phenotypic comparisons showed that Xy06 was a robust producer of TcdB, readily sporulated and germinated, and strongly bound to human gut epithelial cells. In a mouse model of C. difficile infection, Xy06 was more virulent than strains CD630E and Xy07 and was comparable to strain R20291 in virulence. Our data suggest the potential threat of the epidemic ST37 strains in China., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Li, Heuler, Zhu, Meng, Chakraborty, Harmanus, Wang, Peng, Smits, Wu and Sun.)

Background: Outcomes of inflammatory bowel disease (IBD) following flare complicated by enteric infection (EI) are limited by follow-up duration and insufficient assessment of the role of non-Clostridioides difficile pathogens. We compared 2-year IBD outcomes following flare with and without EI., Methods: We performed a retrospective cohort study of adults evaluated with stool PCR testing for IBD flare. Subjects were stratified by presence of EI at flare and were matched for age, sex, and date to those without EI. The primary outcome was a composite of steroid-dependent IBD, colectomy, and/or IBD therapy class change/dose escalation at 2 years. Additional analyses were performed by dividing the EI group into C. difficile infection (CDI) and non-CDI EI, and further subdividing non-CDI EI into E. coli subtypes and other non-CDI EI., Results: We identified 137 matched subjects, of whom 62 (45%) had EI (40 [29%] CDI; 17 [12%] E. coli). Enteric infection at flare was independently associated with the primary outcome (adjusted odds ratio, 4.14; 95% confidence interval [CI], 1.62-11.5). After dividing EI into CDI and non-CDI EI, only CDI at flare was independently associated with the primary outcome (adjusted odds ratio, 4.04; 95% CI, 1.46-12.6). After separating E. coli subtypes from non-CDI EI, E. coli infection and CDI at flare were both independently associated with the primary outcome; other EI was not., Conclusions: Enteric infection at flare-specifically with CDI-is associated with worse IBD outcomes at 2 years. The relationship between E. coli subtypes at flare and subsequent IBD outcomes requires further investigation., (© The Author(s) 2023. Published by Oxford University Press on behalf of Crohn’s & Colitis Foundation. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Background: Antibiotic exposure is a known risk factor for Clostridioides difficile infection (CDI) and recurrence and can lead to infection with specific C. difficile strains. In this study, we sought to explore the relationship between antecedent antibiotic exposure and C. difficile antimicrobial resistance, and the impact of resistance on clinical outcomes., Methods: This was a single center retrospective study evaluating patients with CDI between 2011 and 2021. A logistic regression model was used to evaluate the relationship between antecedent antibiotics in the 30 days prior to CDI and resistance among isolates. In addition, an exploratory analysis using a cause-specific Cox proportional hazards model evaluated the association between resistance and a composite outcome of clinical failure, relapse at 30 days or CDI-related death., Results: we analyzed one isolate from 510 patients; resistance was noted in 339 (66.5 %) of the isolates. Exposure to fluoroquinolones and macrolides was associated with 2.4 (95 % CI 1.4-4.4) and 4.7 (95 % CI 1.1-20.5) increased odds of having resistance compared to other antibiotic class exposure, respectively. There were 58 (17.0 %) patients in the resistance group who developed the composite outcome and 24 (14.2 %) patients who lacked resistance who developed the composite outcome (HR 1.32, 95 % CI 0.81-2.14)., Conclusion: These findings suggest that fluoroquinolone and macrolide exposure were significantly associated with isolating a resistant strain, but we did not find significant differences in clinical outcomes based on the presence of antimicrobial resistance., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: DSR has research contracts at Tufts Medical Center from Prolacta, Summit Therapeutics, Seres Health and Merck. He is also a consultant to Prolacta and Merck. MA has grant funding from CARB-X. JC has research contracts at Tufts Medical Center from Merck and Kamada. All other authors do not have any conflicts of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: The nosocomial transmission of toxin-producing Clostridioides difficile is a significant concern in infection control. C. difficile, which resides in human intestines, poses a risk of transmission, especially when patients are in close contact with medical staff., Methods: To investigate the nosocomial transmission of C. difficile in a single center, we analyzed the genetic relationships of the bacteria. This was done using draft whole-genome sequencing (WGS) and examining single nucleotide polymorphisms (SNPs) in core-genome, alongside data regarding the patient's hospital wards and room changes. Our retrospective analysis covered 38 strains, each isolated from a different patient, between April 2014 and January 2015., Results: We identified 38 strains that were divided into 11 sequence types (STs). ST81 was the most prevalent (n = 11), followed by ST183 (n = 10) and ST17 (n = 7). A cluster of strains that indicated suspected nosocomial transmission (SNT) was identified through SNP analysis. The draft WGS identified five clusters, with 16 of 38 strains belonging to these clusters. There were two clusters for ST81 (ST81-SNT-1 and ST81-SNT-2), two for ST183 (ST183-SNT-1 and ST183-SNT-2), and one for ST17 (ST17-SNT-1). ST183-SNT-1 was the largest SNT cluster, encompassing five patients who were associated with Wards A, B, and K. The most frequent room changer was a patient labeled Pt08, who changed rooms seven times in Ward B. Patients Pt36 and Pt10, who were also in Ward B, had multiple admissions and discharges during the study period., Conclusions: Additional culture tests and SNP analysis of C. difficile using draft WGS revealed silent transmission within the wards, particularly in cases involving frequent room changes and repeated admissions and discharges. Monitoring C. difficile transmission using WGS-based analysis could serve as a valuable marker in infection control management., (© 2024. The Author(s).)

Clostridioides difficile, a cause of healthcare-associated infections, poses a significant global health threat. This multi-institutional retrospective study focuses on epidemic dynamics, emphasizing minor and toxin-negative clinical isolates through high-resolution genotyping. The genotype of the C. difficile clinical isolates during 2005 to 2022 was gathered from 14 hospitals across Japan (N = 982). The total number of unique genotypes was 294. Some genotypes were identified in every hospital (cross-regional genotypes), while others were unique to a specific hospital or those in close geographic proximity (region-specific genotypes). Notably, a hospital located in a sparsely populated prefecture exhibited the highest prevalence of region-specific genotypes. The isolation rate of cross-regional genotypes positively correlated with the human mobility flow. A 6-month interval analysis at a university hospital from 2019 to 2021 revealed a temporal transition of the genotype dominance. The frequent isolation of identical genotypes over a brief timeframe did not always align with the current criteria for defining nosocomial outbreaks. This study highlights the presence of diverse indigenous C. difficile strains in regional environments. The cross-regional strains may have a higher competency to spread in the human community. The longitudinal analysis underscores the need for further investigation into potential nosocomial spread., (© 2024. The Author(s).)

Objective: To understand molecular characteristics and antibiotic resistance of Clostridioides ( C. ) difficile isolated from children in China, and provide data support the development of disease risk assessment and burden studies. Methods: A total of 155 strains of C. difficile isolated from children aged <12 years in 14 provinces (autonomous regions, municipalities) in China from 2010 to 2023 were used for the analyses on molecular characteristics and antibiotic resistance of C. difficile by PCR and drug susceptibility test. Results: A total of 26 sequence types (STs) and 18 ribotypes (RTs) were identified in the 155 C. difficile isolates, in which ST3 (20.65%), ST54 (16.13%), ST35 (12.90%), and RT012/ICDC007 (14.84%), RT001/ICDC001 (11.61%), RT046/ICDC018 (8.39%) were the most common. One highly virulent strain with RT078 and 27 non-toxin-producing strains were also found; the predominant toxin gene was tcdA + tcdB + cdt -. All the strains were sensitive to metronidazole and vancomycin, and there were 29 multidrug-resistant strains, in which 1 strain was resistant to all the seven antibiotics except for vancomycin and metronidazole. Conclusions: Molecular characteristics and antibiotic resistance of C. difficile in children were similar to those in whole population in China, but there were regional distribution differences. It is necessary to strengthen the routine drug-resistance surveillance for C. difficile infection in children in China.

Biofilm formation and toxin production are some of the virulence factors of Clostridioides difficile (C. difficile), which causes hospital-acquired C. difficile infection (HA-CDI). This work investigated the prevalence and distribution of different strains recovered from HA-CDI patients hospitalized in 4 medical centres across Israel, and characterized strains' virulence factors and antibiotic susceptibility. One-hundred and eighty-eight faecal samples were collected. C. difficile 's toxins were detected by the CerTest Clostridium difficile GDH + Toxin A + B combo card test kit. Toxin loci PaLoc and PaCdt were detected by whole-genome sequencing (WGS). Multi-locus sequence typing (MLST) was performed to classify strains. Biofilm production was assessed by crystal violet. Antibiotic susceptibility was determined using Etest. Fidaxomicin susceptibility was tested via agar dilution. Sequence type (ST) 42 was the most (13.8%) common strain. All strains harboured the 2 toxins genes; 6.9% had the binary toxin. Most isolates were susceptible to metronidazole (98.9%) and vancomycin (99.5%). Eleven (5.85%) isolates were fidaxomicin-resistant. Biofilm production capacity was associated with ST (p < 0.001). In conclusion, a broad variety of C. difficile strains circulate in Israel's medical centres. Further studies are needed to explore the differences and their contribution to HA-CDI epidemiology., (© 2024. The Author(s).)

Objectives: Clostridioides difficile ranks among the primary sources of healthcare-related infections and diarrhoea in numerous nations. We evaluated the drug susceptibility and resistance mechanisms of C. difficile isolates from a hospital in Chongqing, China, and identified resistance rates and resistance mechanisms that differed from previous findings., Methods: The toxin genes and drug resistance genes of clinical strains were detected using Polymerase Chain Reaction (PCR), and these strains were subjected to Multilocus Sequence Typing (MLST). The agar dilution technique was employed for assessing susceptibility of antibiotics. Clinical data collection was completed through a review of electronic medical records., Results: A total of 67 strains of toxin-producing C. difficile were detected. All C. difficile isolates demonstrated susceptibility to both metronidazole and vancomycin. However, resistance was observed in 8.95%, 16.42%, 56.72%, 56.72%, 31.34% and 5.97% of the isolates for tigecycline, tetracycline, clindamycin, erythromycin, moxifloxacin and rifampin, respectively. Among the strains with toxin genotypes A + B + CDT - and belonging to the ST3, six strains exhibited reduced susceptibility to tigecycline (MIC=0.5mg/L) and tetracycline (MIC=8mg/L). The tetA(P) and tetB(P) genes were present in these six strains, but were absent in tetracycline-resistant strains. Resistance genes (ermB, tetM, tetA(P) and tetB(P)) and mutations (in gyrA, gyrB, and rpoB) were identified in resistant strains., Conclusions: In contrast to prior studies, we found higher proportions of ST3 isolates with decreased tigecycline sensitivity, sharing similar resistance patterns and resistance genes. In the resistance process of tigecycline and tetracycline, the tetA(P) and tetB(P) genes may play a weak role., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Aim: The aim was to highlight the incidence and epidemiology of C. difficile infections (CDI) in a tertiary Greek hospital during the COVID-19 pandemic. Methods: A single-center prospective observational cohort study was conducted (October 2021 until April 2022). 125 C. difficile isolates were cultured from hospitalized patients stool samples and screened by PCR for toxin A ( tcdA ), toxin B ( tcdB ), binary toxin ( cdtA and cdtB ) genes and the regulating gene of tcdC . Results: The incidence of CDI increased to 13.1 infections per 10,000 bed days. The most common PCR ribotypes identified included hypervirulent RT027-related RT181 (73.6%), presumably hypervirulent RT126 (8.0%) and toxin A negative RT017 (7.2%). Conclusion: Although the incidence of CDI increased significantly, the CDI epidemiology remained stable.

Introduction: The performance of MALDI-TOF MS combined with analysis platform for identification of toxin-producing Clostridiodes difficile is yet to be known., Methods: Between August 2018 and September 2020, 61 isolates from stool specimens of patients with C. difficile-associated diarrhea were analyzed using the MALDI Biotyper system. A C. difficile toxin-producer detection model was developed using ClinProTools. The model was validated using 28 known strains that differed from the isolates used to develop the model., Results: The sensitivity and specificity of the Genetic Algorithm (GA) model using isolates grown on Brucella with hemin and vitamin K (BHK) agar plates were 91.7% and 44.4%, respectively. When isolates grown on cycloserine-cefoxitin mannitol agar were analyzed by the model, sensitivity and specificity were 6.3% and 100%, respectively. The GA model using BHK medium showed the highest discriminatory performance in detection of toxin-producing C. difficile. However, a discrepancy in detection of toxin-producing C. difficile was observed in the results generated when the model was being developed and when the model was validated which suggests that incubation conditions may have affected the results., Conclusion: MALDI-TOF analysis using ClinProTools has a potential to be a cost-effective tool for rapid diagnosis and contribute to antimicrobial stewardship by differentiating toxin-producing C. difficile from non-producers., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Japanese Society of Chemotherapy, Japanese Association for Infectious Diseases, and Japanese Society for Infection Prevention and Control. Published by Elsevier Ltd. All rights reserved.)

Goals: To assess fecal microbiota, live-jslm (REBYOTA, abbreviated as RBL, formerly RBX2660) efficacy and safety in participants grouped by recurrent Clostridioides difficile infection (rCDI) risk factors and treatment-related variables., Background: RBL is the first microbiota-based live biotherapeutic approved by the US Food and Drug Administration for the prevention of rCDI in adults after antibiotic treatment for rCDI., Study: Treatment success rates across subgroups for PUNCH CD3 (NCT03244644) were estimated using a Bayesian hierarchical model, borrowing data from PUNCH CD2 (NCT02299570). Treatment-emergent adverse events were summarized for the double-blind treatment period within 8 weeks., Results: Treatment differences between RBL and placebo at 8 weeks were similar to the total population for most subgroups. Treatment effect sizes were similar between CDI tests, higher for oral vancomycin courses >14 days versus ≤14 days and higher for antibiotic washout periods of 3 days versus ≤2 days. The largest reductions in the rate of rCDI with RBL versus placebo were observed for participants with a 3-day CDI antibiotic washout period and participants with ≥4 previous CDI episodes. Most RBL-treated participants experienced TEAEs that were mild or moderate in severity and related to preexisting conditions., Conclusion: This analysis provides further evidence of RBL efficacy and safety across subgroups, including those at high risk for rCDI., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc.)

Background: Clostridioides difficile infection (CDI) is an increasingly common disease in healthcare facilities and community settings. However, there are limited reports of community-onset CDI (CO-CDI) in China., Methods: We collected diarrheal stool samples from 3885 patients who went to outpatient department or emergency department in a tertiary hospital in China during 2010-2023, analyzed the correlation between patients' basic information and the detection rate of CDI. Besides, all stool samples from 3885 outpatients included were tested by culturing. Moreover, we randomly selected 89 patients' stools during the 14 years and isolated 126 C. difficile strains from them. The presence of toxin genes (tcdA, tcdB, cdtA, and cdtB) were confirmed by PCR. Toxigenic strains were typed using multilocus sequence typing (MLST). Susceptibility to 9 antimicrobials was evaluated using the E-test., Results: 528 of 3885 patients (13.6 %) with diarrhea were finally diagnosed as CDI. The median age of patients included was 51 years (6 months-95 years), while the median of patients with CDI was older than patients with negative results [55.5 years (6 months-93 years) vs. 50 years (9 months -95 years), p < 0.001]. In winter, patients with diarrhea might be more likely to have CDI. The detection rate of CDI of patients in emergency department was much higher than those in other outpatients (20.7 % vs. 12.4 %, p < 0.001), and did differ from each outpatient departments (p < 0.05). There were 95 isolated strains detected as toxigenic C. difficile. Among these strains, 82 (86.3 %) had the tcdA and tcdB genes (A+B+) and 5 of these 82 strains were positive for the binary toxin genes (cdtA and cdtB) (A+B+CDT+). There were 15 different sequence types (STs) by multilocus sequence typing (MLST), while the most ST was ST-54 (23.2 %). ST types composition was relatively stable over the time span of this study. Some strains had high resistance to ciprofloxacin, clindamycin, and erythromycin. Twenty-three isolates (24.2 %) were multidrug-resistant., Conclusions: Outpatients with CDI were common among patients having diarrhea during this period in our hospital. Elderly patients and patients went to emergency department may be susceptible to CDI. Based on MLST, the result revealed that the C. difficile isolates had high genetic diversity and maintained stability in this period. All isolates were susceptible to metronidazole and vancomycin, and nearly one quarter of all isolates had multidrug resistance., Competing Interests: Conflict of Interest The authors declare that they have no competing interests., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Background: The tcdA gene codes for an important toxin produced by Clostridioides difficile ( C. difficile ), but there is currently no simple and cost-effective method of detecting it. This article establishes and validates a rapid and visual loop-mediated isothermal amplification (LAMP) assay for the detection of the tcdA gene., Methods: Three sets of primers were designed and optimized to amplify the tcdA gene in C. difficile using a LAMP assay. To evaluate the specificity of the LAMP assay, C. difficile VPI10463 was used as a positive control, while 26 pathogenic bacterial strains lacking the tcdA gene and distilled water were utilized as negative controls. For sensitivity analysis, the LAMP assay was compared to PCR using ten-fold serial dilutions of DNA from C. difficile VPI10463, ranging from 207 ng/µl to 0.000207 pg/µl. The tcdA gene of C.difficile was detected in 164 stool specimens using both LAMP and polymerase chain reaction (PCR). Positive and negative results were distinguished using real-time monitoring of turbidity and chromogenic reaction., Results: At a temperature of 66 °C, the target DNA was successfully amplified with a set of primers designated, and visualized within 60 min. Under the same conditions, the target DNA was not amplified with the tcdA12 primers for 26 pathogenic bacterial strains that do not carry the tcdA gene. The detection limit of LAMP was 20.700 pg/µl, which was 10 times more sensitive than that of conventional PCR. The detection rate of tcdA in 164 stool specimens using the LAMP method was 17% (28/164), significantly higher than the 10% (16/164) detection rate of the PCR method (X 2 = 47, p < 0.01)., Conclusion: LAMP method is an effective technique for the rapid and visual detection of the tcdA gene of C. difficile , and shows potential advantages over PCR in terms of speed, simplicity, and sensitivity. The tcdA -LAMP assay is particularly suitable for medical diagnostic environments with limited resources and is a promising diagnostic strategy for the screening and detection of C. difficile infection in populations at high risk., Competing Interests: The authors declare that they have no competing interests., (© 2024 Lin et al.)

In contrast to the epidemiology 10 years earlier at our hospital when the epidemic restriction endonuclease analysis (REA) group strain BI accounted for 72% of Clostridioides difficile isolates recovered from first-episode C. difficile infection (CDI) cases, BI represented 19% of first-episode CDI isolates in 2013-2015. Two additional REA group strains accounted for 31% of isolates (Y, 16%; DH, 12%). High-level resistance to fluoroquinolones and azithromycin was more common among BI isolates than among DH, Y, and non-BI/DH/Y isolates. Multivariable analysis revealed that BI cases were 2.47 times more likely to be associated with fluoroquinolone exposure compared to non-BI cases (95% confidence interval [CI]: 1.12-5.46). In addition, the odds of developing a CDI after third- or fourth-generation cephalosporin exposure was 2.83 times for DH cases than for non-DH cases (95% CI: 1.06-7.54). Fluoroquinolone use in the hospital decreased from 2005 to 2015 from a peak of 113 to a low of 56 antimicrobial days/1,000 patient days. In contrast, cephalosporin use increased from 42 to 81 antimicrobial days/1,000 patient days. These changes correlated with a decrease in geometric mean MIC for ciprofloxacin (61.03 to 42.65 mg/L, P = 0.02) and an increase in geometric mean MIC for ceftriaxone (40.87 to 86.14 mg/L, P < 0.01) among BI isolates. The BI strain remained resistant to fluoroquinolones, but an overall decrease in fluoroquinolone use and increase in cephalosporin use were associated with a decrease in the prevalence of BI, an increased diversity of C. difficile strain types, and the emergence of strains DH and Y., Competing Interests: A.M.S. has served as a consultant for Bio-K+, Recursion Pharmaceuticals, and Ferring Pharmaceuticals and has received research funding from Paratek Pharmaceuticals (paid to institution). D.N.G. licensed technology the treatment of CDI to Destiny Pharma plc and is a consultant for Destiny, Sebela, and AstraZeneca. S.J. has served as a consultant for Acurx Pharmaceuticals, Inc, Bio-K+ International, and Ferring Pharmaceuticals. S.J. has received research funding from Paratek Pharmaceuticals (paid to institution). J.T.W., A.C., L.A.P., V.W.S., and L.M.W. have no disclosures.

Objective: This study aimed to evaluate the fecal shedding of C. difficile in calves on farms in Sao Paulo State, Brazil., Materials and Methods: Fecal samples (n = 300) were collected from diarrheic (n = 78) and nondiarrheic (n = 222) calves less than 60 days of age from 20 farms. Fecal samples were inoculated into enrichment broth supplemented with taurocholate and cultured under anaerobic conditions. Colonies suspected to be C. difficile were harvested for DNA extraction and then multiplex PCR for the detection of genes encoding toxins A and B and binary toxins. All toxigenic isolates were ribotyped and tested for antimicrobial susceptibility, and five selected strains were subjected to whole-genome sequencing to determine their sequence type., Results and Discussion: C. difficile was isolated from 29.3 % (88/300) of the samples. All toxigenic isolates (17/88, 19.3 %) were classified as ribotypes RT046 (13/17-79.47 %, A + B + CDT - ) and RT126 (4/17 = 20.53 %, A + B + CDT + ). The sequenced strains from RT046 were classified as ST35 (Clade 1), while those from RT126 were classified as ST11 (Clade 5). No associations between the epidemiological factors in any of the groups and C. difficile isolation were observed. Most of the toxigenic isolates (16/17 = 94.41 %) were classified as multidrug-resistant. Calves can be an important source of toxigenic C. difficile strains, including multidrug-resistant isolates from ribotypes commonly observed in humans., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Objectives: Infection with Clostridioides difficile usually occurs after antibiotic treatment for other infections and can cause gastro-intestinal disorders of variable severity. C. difficile can be resistant to a wide spectrum of antimicrobials. Detection of antimicrobial resistance (AMR) is important to direct optimal treatment and surveillance of AMR patterns in the overall population. Correlation between genotypic markers and phenotypic AMR is not yet well defined. The aim for this study is to assess whether and to what extent genotypic determinants of AMR correlate with phenotypic resistance., Methods: C. difficile isolates (n = 99) were phenotypically characterized for resistance to eight antibiotics using Sensititre plates or E-tests. Their genomes were screened for genetic markers of resistance. Accuracy, sensitivity, specificity, positive and negative predictive values were calculated., Results: We found high rates of resistance (>50 %) to cefoxitin and clindamycin, intermediate rates of resistance (10 %-50 %) to moxifloxacin and tetracycline and low rates of resistance (<10 %) to imipenem, metronidazole, vancomycin, and rifampicin. For moxifloxacin, tetracycline, and clindamycin, we found a good correlation between genotypic and phenotypic AMR, with an overall accuracy of 98 % (95 % CI 93%-100 %), 78 % (95 % CI 68%-86 %) and 86 % (95 % CI 77%-92 %) respectively. For the other five antibiotics, accurate estimates on the correlation could not be made., Conclusion: Our results suggest that for moxifloxacin, tetracycline and clindamycin, phenotypic resistance in C. difficile can be predicted by genetic indicators and used for public health purposes. However, for the other five antibiotics, the model is not accurate and further development is necessary., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Inappropriate testing for Clostridiodes difficile infection (CDI) increases health care onset cases and contributes to overdiagnosis and overtreatment of patients in a community health care system., Methods: An electronic smart order set for the testing of CDI was created and implemented to improve the appropriateness of testing. A retrospective review of patients who were tested for CDI, pre and post, was conducted to determine if inappropriate stool testing for CDI decreased post-implementation of the order set., Results: 224 patients were tested for CDI during the study period with the post-implementation period having a higher proportion of patients who met appropriate testing criteria defined by presence of diarrhea (80.5% vs 61.3%; P = .002). The rate of inappropriate CDI stool testing decreased from 31.1% to 11.0% after implementation (P < .001). A higher proportion of CDI patients were readmitted within 30 days of discharge (54.2% vs 33.0%; P = 0.001) during the post-implementation period., Conclusions: There was a significant reduction in inappropriate CDI testing following the implementation of the order set. There was an observed increase in the proportion of patients who underwent recent gastrointestinal surgery which may have contributed to the increase in 30-day readmission rates during the post-implementation period., (Copyright © 2024. Published by Elsevier Inc.)

Clostridioides difficile (C. difficile) is the most common agent of antibiotic-associated diarrhea, leading to intestinal infection through the secretion of two major toxins. Not all strains of this bacterium are toxigenic, but some of them cause infection via their accessory virulence factors, such as surface layer protein (SlpA). SlpA is conserved in both toxigenic and non-toxigenic strains of C. difficile. In the present work, an amplification-free electrochemical genosensor was designed for the detection of the slpA gene. A glassy carbon electrode coated with gold nanoparticle-reduced graphene oxide nanocomposite was used as the working electrode, and its surface was modified using a simple thiolated linear oligonucleotide as the bioreceptor. Moreover, the hexaferrocenium tri[hexa(isothiocyanato) iron(III)] trihydroxonium (HxFc) complex was used as an intercalator, and its redox signal was recorded using differential pulse voltammetry. Scan rate studies indicated a quasi-reversible adsorption-controlled process for the HxFc complex. This genosensor showed high sensitivity with a limit of detection of 0.2 fM, a linear response range of 0.46-1900 fM, and a satisfactory specificity toward the synthetic slpA target gene. Also, the genosensor indicated responses in the mentioned linear range toward the genome extracted from either toxigenic or non-toxigenic strains of C. difficile., Competing Interests: Declaration of competing interest The authors have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Abstract: Infections from Clostridioides difficile (often called C. diff) have long presented challenges for both patients and clinicians. Traditionally, C. diff has been considered a nosocomial infection, but in recent years, a noticeable spike in community-acquired cases has occurred. C. diff infection (CDI) testing is often complicated, as various testing options with differing sensitivity and specificity for active infection are available. Also, recent guideline changes have altered the recommended treatment of infection. This article discusses recent changes to both the diagnosis and management of CDI and how they can be applied to everyday NP practice., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Multiplexed gastrointestinal PCR panels (MGPPs) are frequently used to aid the diagnosis and management of diarrhea in hematopoietic stem cell transplantation (HCT) recipients. Many issues related to the optimal use of MGPPs in HCT patients remain to be clarified. We aimed to better define MGPP diagnostic and therapeutic stewardship in HCT recipients, including indications for and benefits of testing, optimal timing of tests, and interpretation of results. We retrieved 463 consecutive MGPPs ordered on 651 consecutive first HCT (312 allogeneic, 339 autologous) performed at our institution between June 2015 and June 2023. One hundred and sixteen of the 463 MGPPs (25%) identified at least 1 diarrheagenic pathogen, and 12 (3%) identified more than 1 diarrheagenic pathogen. A positive result was more likely if the test was ordered within 48 hours of a hospital admission (41%; 32 of 78) or as an outpatient (41%; 46 of 111) compared with evaluation of hospital-onset diarrhea (14%; 38 of 274). Among the positive results, the most frequent pathogens identified included Clostridioides difficile (64%), diarrheagenic Escherichia coli (20%), norovirus (9%), and adenovirus 40/41 (5%). Thirty-eight percent of the positive C. difficile MGPP determinations were associated with a positive test for toxin. In our allogeneic HCT cohort, 3% of MGPPs for hospital-onset diarrhea yielded an organism other than C. difficile. Fifty-six percent of positive and 14% of all submitted tests resulted in a change in treatment. For organisms other than C. difficile, only 1% of all tests and 5% of positive tests resulted in initiation of therapy. For patients at risk for acute graft-versus-host disease (aGVHD), a positive or negative MGPP result was not predictive of a new diagnosis of aGVHD in proximity to diarrhea onset. These results suggest that MGPP testing is most useful when performed at hospital admission or on an outpatient basis. Because MGPPs are sensitive and do not distinguish between colonization and causes of diarrhea, caution is needed when interpreting results, especially for toxin-negative C. difficile and diarrheagenic gram-negative organisms., (Copyright © 2024 The American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Postweaning diarrhea (PWD) is a multifactorial disease caused by different aetiological agents, like viruses or bacteria and where the role of the microbiota remains unclear. The aim of this study was to assess differences between healthy and diarrheic weaned pigs concerning the prevalence of pathogens and changes in the intestinal microbiota. Eighteen farms with PWD were selected and 277 fecal samples were collected (152 diarrheic vs 125 healthy). Presence of Rotavirus A (RVA), B (RVB), C (RVC) and Porcine Epidemic Diarrhea Virus (PEDV), virulence factors of Escherichia coli and Clostridioides difficile were analyzed by PCR. Finally, the microbiota composition was also study by 16 S rRNA sequencing on 148 samples (102 diarrheic vs 46 healthy). RVA (53.95 % vs 36 %, p=0.04) and RVB (49.67 % vs 28.8 %, p<0.001) were more frequent in diarrheic animals. Furthermore, RVA viral load was higher in diseased animals. VT2 toxin was significantly associated with diarrhea, whereas other virulence factors were not. Presence of C. difficile and PEDV was almost negligible. Regarding microbiota changes, Fusobacteriota phylum was more frequent in diarrheic samples and Ruminococcaceae family in healthy penmates. During the first week postweaning, Enterobacteriace and Campylobacteria were enriched in animals presenting diarrhea. Furthermore, Lactobacillus was detected in those individuals with no RVA infection. In conclusion, RVA seems to play a primary role in PWD. Classic E. coli virulence factors were not associated with diarrhea, indicating the need for revising their implication in disease. Moreover, Lactobacillus was found frequently in animals negative for RVA, suggesting some protective effect., Competing Interests: Declaration of Competing Interest The authors declare to not have competing interests., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)