Evaluation of cobas® Liat® Cdiff, STANDARD™ M10 C. difficile and Xpert® C. difficile BT assays for rapid detection of toxigenic Clostridioides difficile.

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.

Development and evaluation of a rapid visual loop-mediated isothermal amplification assay for the tcdA gene in Clostridioides difficile detection.

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 (X2 = 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.

Virulence factors, antibiotic susceptibility and sequence type distribution of hospital-associated Clostridioides difficile isolates in Israel, 2020-2022.

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.

A molecular epidemiological and transmission analysis of Clostridioides difficile using draft whole-genome sequencing in a single hospital.

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.

[Molecular characteristics and antibiotic resistance of Clostridioides difficile isolated from children in China].

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.

Regional and temporal genotype profiling of Clostridioides difficile in a multi-institutional study in Japan.

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.

Genomic epidemiology of Clostridioides difficile sequence type 35 reveals intraspecies and interspecies clonal transmission.

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.

Diversity of Clostridioides difficile PCR ribotypes isolated from freshwater sediments depends on the isolation method.

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.IMPORTANCEClostridioides 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.

The impact of 30-day antecedent antibiotic exposure on Clostridioidesdifficile ribotype patterns and the relationship with clinical outcomes: A single center study.

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.

Impact of terminal cleaning in rooms previously occupied by patients with healthcare-associated infections.

Healthcare associated infections (HAIs) are costly but preventable. A limited understanding of the effects of environmental cleaning on the riskiest HAI associated pathogens is a current challenge in HAI prevention. This project aimed to quantify the effects of terminal hospital cleaning practices on HAI pathogens via environmental sampling in three hospitals located throughout the United States. Surfaces were swabbed from 36 occupied patient rooms with a laboratory-confirmed, hospital- or community-acquired infection of at least one of the four pathogens of interest (i.e., Acinetobacter baumannii (A. baumannii), methicillin resistant Staphylococcus aureus (MRSA), vancomycin resistant Enterococcus faecalis/faecium (VRE), and Clostridioides difficile (C. difficile)). Six nonporous, high touch surfaces (i.e., chair handrail, bed handrail, nurse call button, desk surface, bathroom counter near the sink, and a grab bar near the toilet) were sampled in each room for Adenosine Triphosphate (ATP) and the four pathogens of interest before and after terminal cleaning. The four pathogens of interest were detected on surfaces before and after terminal cleaning, but their levels were generally reduced. Overall, C. difficile was confirmed on the desk (n = 2), while MRSA (n = 24) and VRE (n = 25) were confirmed on all surface types before terminal cleaning. After cleaning, only MRSA (n = 6) on bed handrail, chair handrail, and nurse call button and VRE (n = 5) on bathroom sink, bed handrail, nurse call button, toilet grab bar, and C. difficile (n = 1) were confirmed. At 2 of the 3 hospitals, pathogens were generally reduced by >99% during terminal cleaning. One hospital showed that VRE increased after terminal cleaning, MRSA was reduced by 73% on the nurse call button, and VRE was reduced by only 50% on the bathroom sink. ATP detections did not correlate with any pathogen concentration. This study highlights the importance of terminal cleaning and indicates room for improvement in cleaning practices to reduce surface contamination throughout hospital rooms.

Clinical characteristics of community-onset Clostridioides difficile infections at a tertiary hospital in mainland China: A fourteen-year (2010-2023) retrospective study.

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.

Epidemiology of Clostridioides difficile infection at one hospital 10 years after an outbreak of the epidemic C. difficile strain BI/027: changing strain prevalence, antimicrobial susceptibilities, and patient antibiotic exposures.

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.

Antimicrobial susceptibilities, resistance mechanisms and molecular characteristics of toxigenic Clostridioides difficile isolates in a large teaching hospital in Chongqing, China.

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.

More than a new name: Updates in the management of Clostridioides difficile infection.

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.

A case of community-acquired Clostridioides difficile infection causing intussusception, severe pneumonia, and severe hypokalemia.

BACKGROUND: Clostridioides difficile infection is associated with antibiotic use and manifests as diarrhea; however, emerging cases of fulminant diarrhea caused by binary toxin-producing C. difficile unrelated to prior antibiotic exposure have been reported. Although fulminant colitis caused by C. difficile has been documented, instances of intussusception remain scarce. Here, we present a case of adult intussusception with severe hypokalemia and pneumonia resulting from a community-acquired C. difficile infection in Japan. CASE PRESENTATION: An 82-year-old male presented with dizziness, progressive weakness, and diarrhea. Initial vital signs indicated severe respiratory and circulatory distress, and laboratory findings revealed hypokalemia, pneumonia, and septic shock. Imaging confirmed intussusception of the ascending colon. Although colonoscopy suggested a potential tumor, no malignancy was found. The C. difficile rapid test result was positive, indicating community-acquired C. difficile infection. Treatment with vancomycin was initiated; however, intussusception relapsed. Surgical intervention was successful and led to clinical improvement. The patient's complex pathophysiology involved community-acquired C. difficile-induced severe diarrhea, hypokalemia, hypermetabolic alkalosis, and subsequent intussusception. Although adult intussusception is uncommon, this case was uniquely linked to binary toxin-producing C. difficile. The identified strain, SUH1, belonged to a novel sequence type (ST1105) and clade 3, suggesting a highly virulent clone. Resistome analysis aligned with phenotypic susceptibility to metronidazole and vancomycin, confirming their treatment efficacy. CONCLUSION: This case report highlights a binary toxin-producing C. difficile that caused intussusception. The consideration of community-acquired C. difficile in the differential diagnosis of severe enteritis is necessary, even in Japan.

Molecular epidemiology and antimicrobial resistance in Clostridioides difficile strains isolated from children and adolescents in a tertiary referral pediatric hospital in Fortaleza, Brazil.

BACKGROUND: C. difficile has been increasingly reported as a cause of gastrointestinal disease in children, ranging from mild self-limiting diarrhea to severe conditions such as pseudomembranous colitis and toxic megacolon. Only two pediatric research groups reported the presence of C. difficile infection in Brazilian children, but no previous research has examined C. difficile infection among children in northeastern Brazil. This prospective cross-sectional study investigated the molecular epidemiology and antimicrobial resistance of C. difficile strains isolated from children and adolescents with diarrhea referred to a tertiary pediatric hospital in Brazil while exploring the associated risk factors. RESULTS: Toxin positivity or C. difficile isolation was found in 30.4 % (17/56) samples. C. difficile was isolated from 35 % (6/17) samples. Four toxigenic strains were identified (tpi+, tcdA+, tcdB+, cdtB-, without tcdC deletions) belonging to PCR ribotypes and PFGE-pulsotypes: 046 (new pulsotype 1174), 106 (NAP11), 002 (new pulsotype 1274), 012 (new pulsotype NML-1235). Two of the six isolates belonging to ribotypes 143 and 133 were non-toxigenic. All toxigenic strains were sensitive to metronidazole and vancomycin. Regarding the clinical manifestation, diarrhea lasted an average of 11 days, ranging from 3 to 50 days and was often associated with mucus and/or blood. All six patients from whom the C. difficile was isolated had a chronic disease diagnosis, with these comorbidities as the main risk factors. CONCLUSION: Our study enhances our understanding of the present epidemiological landscape of C. difficile-associated diarrhea (CDI) among children in northeastern Brazil, reveling a substantial CDI frequency of 30.4 %, with toxigenic strains detected in 76.4 % of cases, highlighting a higher prevalence compared to earlier Brazilian studies. In the globalized world, an understanding of disease-generating strains, the associated risk factors, clinical manifestation, and antimicrobial sensitivity has fundamental epidemiological importance and draws attention to preventive measures, allowing for more decisive action.

Genomic and phenotypic studies among Clostridioides difficile isolates show a high prevalence of clade 2 and great diversity in clinical isolates from Mexican adults and children with healthcare-associated diarrhea.

Clostridioides difficile (C. difficile) is widely distributed in the intestinal tract of humans, animals, and in the environment. It is the most common cause of diarrhea associated with the use of antimicrobials in humans and among the most common healthcare-associated infections worldwide. Its pathogenesis is mainly due to the production of toxin A (TcdA), toxin B (TcdB), and a binary toxin (CDT), whose genetic variants may be associated with disease severity. We studied genetic diversity in 39 C. difficile isolates from adults and children attended at two Mexican hospitals, using different gene and genome typing methods and investigated their association with in vitro expression of toxins. Whole-genome sequencing in 39 toxigenic C. difficile isolates were used for multilocus sequence typing, tcdA, and tcdB typing sequence type, and phylogenetic analysis. Strains were grown in broth media, and expression of toxin genes was measured by real-time PCR and cytotoxicity in cell-culture assays. Clustering of strains by genome-wide phylogeny matched clade classification, forming different subclusters within each clade. The toxin profile tcdA+/tcdB+/cdt+ and clade 2/ST1 were the most prevalent among isolates from children and adults. Isolates presented two TcdA and three TcdB subtypes, of which TcdA2 and TcdB2 were more prevalent. Prevalent clades and toxin subtypes in strains from children differed from those in adult strains. Toxin gene expression or cytotoxicity was not associated with genotyping or toxin subtypes. In conclusion, genomic and phenotypic analysis shows high diversity among C. difficile isolates from patients with healthcare-associated diarrhea. IMPORTANCE: Clostridioides difficile is a toxin-producing bacterial pathogen recognized as the most common cause of diarrhea acquired primarily in healthcare settings. This bacterial species is diverse; its global population has been divided into five different clades using multilocus sequence typing, and strains may express different toxin subtypes that may be related to the clades and, importantly, to the severity and progression of disease. Genotyping of children strains differed from adults suggesting toxins might present a reduced toxicity. We studied extensively cytotoxicity, expression of toxins, whole genome phylogeny, and toxin typing in clinical C. difficile isolates. Most isolates presented a tcdA+/ tcdB+/cdt+ pattern, with high diversity in cytotoxicity and clade 2/ST1 was the most prevalent. However, they all had the same TcdA2/TcdB2 toxin subtype. Advances in genomics and bioinformatics tools offer the opportunity to understand the virulence of C. difficile better and find markers for better clinical use.

Differences in enteric pathogens and intestinal microbiota between diarrheic weaned piglets and healthy penmates.

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.