Core genome multilocus sequence typing of Clostridioides difficile to investigate transmission in the hospital setting.

PURPOSE: Traditional epidemiological investigations of healthcare-associated Clostridioides difficile infection (HA-CDI) are often insufficient. This study aimed to evaluate a procedure that includes secondary isolation and genomic typing of single toxigenic colonies using core genome multilocus sequence typing (cgMLST) for the investigation of C. difficile transmission. METHODS: We analyzed retrospectively all toxigenic C. difficile-positive stool samples stored at the Lausanne University Hospital over 6 consecutive months. All isolates were initially typed and classified using a modified double-locus sequence typing (DLST) method. Genome comparison of isolates with the same DLST and clustering were subsequently performed using cgMLST. The electronic administrative records of patients with CDI were investigated for spatiotemporal epidemiological links supporting hospital transmission. A comparative descriptive analysis between genomic and epidemiological data was then performed. RESULTS: From January to June 2021, 86 C. difficile isolates were recovered from thawed samples of 71 patients. Thirteen different DLST types were shared by > 1 patient, and 13 were observed in single patients. A genomic cluster was defined as a set of isolates from different patients with ≤ 3 locus differences, determined by cgMLST. Seven genomic clusters were identified, among which plausible epidemiological links were identified in only 4/7 clusters. CONCLUSION: Among clusters determined by cgMLST analysis, roughly 40% included unexplained HA-CDI acquisitions, which may be explained by unidentified epidemiological links, asymptomatic colonization, and/or shared common community reservoirs. The use of DLST, followed by whole genome sequencing analysis, is a promising and cost-effective stepwise approach for the investigation of CDI transmission in the hospital setting.

Biogeographic distribution and molecular epidemiology of Clostridioides (Clostridium) difficile in Western Australian soils.

Clostridioides (Clostridium) difficile is a leading cause of infectious diarrhea in humans and production animals and can be found in a variety of environmental sources. The prevalence and diversity of multi-locus sequence type clade 5 strains of C. difficile in Australian production animals suggest Australia might be the ancestral home of this lineage of One Health importance. To better understand the role of the environment in the colonization of humans and animals in Australia, it is important to investigate these endemic sources. This study describes the prevalence, molecular epidemiology, and biogeographic distribution of C. difficile in soils of Western Australia. A total of 321 soil samples from remote geographical locations across the eight health regions of Western Australia were screened for C. difficile and isolates characterized by PCR ribotyping and toxin gene profiling. C. difficile was isolated from 31.15% of samples, with the highest prevalence in the Perth Metropolitan Health Region (49.25%, n = 33/67). Overall, 52 different strains [PCR ribotypes (RTs)] were identified, with 14 being novel, and 38% (38/100) of isolates being toxigenic, the most common of which was RT014/020. Five unique novel isolates showed characteristics similar to C. difficile clade 5. This is the first study of C. difficile isolated from soils in Australia. The high prevalence and heterogeneity of C. difficile strains recovered suggest that soils play a role in the survival and environmental dissemination of this organism, and potentially its transmission among native wildlife and production animals, and in community and hospital settings.IMPORTANCEClostridium difficile is a pathogen of One Health importance. To better understand the role of the environment in human and animal colonization/infection, it is critical that autochthonous reservoirs/sources of C. difficile be investigated. This is the first study of C. difficile isolated from soils of Western Australia (WA). Here, the ecology of C. difficile in WA is described by examining the geographic distribution, molecular epidemiology, and diversity of C. difficile isolated from soils across WA.

An epidemiological surveillance study (2021-2022): detection of a high diversity of Clostridioides difficile isolates in one tertiary hospital in Chongqing, Southwest China.

BACKGROUND: Clostridioides difficile is a bacterium that causes antibiotic-associated infectious diarrhea and pseudomembranous enterocolitis. The impact of C. difficile infection (CDI) in China has gained significant attention in recent years. However, little epidemiological data are available from Chongqing, a city located in Southwest China. This study aimed to investigate the epidemiological pattern of CDI and explore the drug resistance of C. difficile isolates in Chongqing. METHODS: A case-control study was conducted to investigate the clinical infection characteristics and susceptibility factors of C. difficile. The features of the C. difficile isolates were evaluated by testing for toxin genes and using multi-locus sequence typing (MLST). The susceptibility of strains to nine antibiotics was determined using agar dilution technique. RESULTS: Out of 2084 diarrhea patients, 90 were tested positive for the isolation of toxigenic C. difficile strains, resulting in a CDI prevalence rate of 4.32%. Tetracycline, cephalosporins, hepatobiliary disease, and gastrointestinal disorders were identified as independent risk factors for CDI incidence. The 90 strains were classified into 21 sequence types (ST), with ST3 being the most frequent (n = 25, 27.78%), followed by ST2 (n = 10, 11.11%) and ST37 (n = 9, 10%). Three different toxin types were identified: 69 (76.67%) were A+B+CDT-, 12 (13.33%) were A-B+CDT-, and 9 (10%) were A+B+CDT+. Although substantial resistance to erythromycin (73.33%), moxifloxacin (62.22%), and clindamycin (82.22%), none of the isolates exhibited resistance to vancomycin, tigecycline, or metronidazole. Furthermore, different toxin types displayed varying anti-microbial characteristics. CONCLUSIONS: The strains identified in Chongqing, Southwest China, exhibited high genetic diversity. Enhance full awareness of high-risk patients with HA-CDI infection, particularly those with gastrointestinal and hepatocellular diseases, and emphasize caution in the use of tetracycline and capecitabine. These findings suggest that a potential epidemic of CDI may occur in the future, emphasizing the need for timely monitoring.

Development and clinical application of a rapid, visually interpretable polymerase spiral reaction for tcdB gene of Clostridioides difficile in fecal cultures.

In the surveillance of outbreaks of Clostridioides difficile infection, the rapid detection and diagnosis of C. difficile remain a major challenge. Polymerase spiral reaction (PSR) is a nucleic acid amplification technique that uses mixed primers and the strand displacement activity of Bst DNA polymerase to achieve a pair of primers and a single enzyme in an isothermal environment. The primer design is simple, the reaction is efficient, and a color indicator can be used to visualize the result. In this study, we developed a rapid and visually interpretable PSR to detect C. difficile by analyzing artificially contaminated feces samples and clinical isolates from patient feces samples. We designed two pairs of primers for a PSR that specifically targeted the conserved tcdB gene of C. difficile. The amplification results were visualized with the chromogenic dye hydroxynaphthol blue. The entire process was accomplished in 50 min at 64°C, with high specificity. The limit of detection of C. difficile with PSR was 150 fg/µl genomic DNA or 2 × 10 CFU/ml in artificially contaminated feces samples. With this method, we analyzed four clinical isolates and also compared the PSR with an isolation-and-culture detection method, polymerase chain reaction, and the Sanger sequencing. The four clinical isolates were found positive for tcdB, which confirmed the high specificity of the primers. The positive rates of tcdB in toxigenic C. difficile detected with PSR, PCR, and Sanger sequencing were 100%. The proportions of toxin types in these clinical C. difficile strains were 50% tcdA+tcdB+CDT- and 50% tcdA+tcdB+CDT+. The assay described should extend our understanding of the incidence of C. difficile. This may allow the rapid diagnosis and screening of C. difficile-related disease outbreaks in the field.

Prevalence of Clostridioides difficile in dogs (Canis familiaris) with gastrointestinal disorders in Rio de Janeiro.

Clostridioides difficile infections (CDI) have a high morbidity and mortality rate and have always been considered a nosocomial disease. Nonetheless, the number of cases of community-acquired CDI is increasing, and new evidence suggests additional C. difficile reservoirs exist. Pathogenic C. difficile strains have been found in livestock, domestic animals, and meat, so a zoonotic transmission has been proposed. OBJECTIVE: The goal of this study was to isolate C. difficile strains in dogs at a veterinary clinic in Rio de Janeiro, Brazil, and characterize clinical and pathological findings associated with lower gastrointestinal tract disorders. METHODS: Fifty stool samples and biopsy fragments from dogs were obtained and cultured in the CDBA selective medium. All suggestive C. difficile colonies were confirmed by MALDI-TOF MS and PCR (tpi gene). Vancomycin, metronidazole, moxifloxacin, erythromycin, and rifampicin were tested for antibiotic susceptibility. Biofilm, motility assays, and a PCR for the toxins (tcdA, tcdB, and cdtB), as well as ribotyping, were also performed. RESULTS: Blood samples and colonic biopsy fragments were examined in C. difficile positive dogs. Ten animals (20%) tested positive for C. difficile by using stool samples, but not from biopsy fragments. Most C. difficile strains were toxigenic: six were A+B+ belonging to RT106; two were A+B+ belonging to RT014/020; and two were A-B- belonging to RT010. All strains were biofilm producers. In the motility test, 40% of strains were as motile as the positive control, CD630 (RT012). In the disc diffusion test, two strains (RT010) were resistant to erythromycin and metronidazole; and another to metronidazole (RT014/020). In terms of C. difficile clinicopathological correlations, no statistically significant morphological changes, such as pseudomembranous and "volcano" lesions, were observed. Regarding hematological data, dogs positive for C. difficile had leucopenia (p = 0.02) and lymphopenia (p = 0.03). There was a significant correlation between senility and the presence of C. difficile in the dogs studied (p = 0,02). CONCLUSIONS: Although C. difficile has not been linked to canine diarrheal disorders, it appears to be more common in dogs with intestinal dysfunctions. The isolation of ribotypes frequently involved in human CDI outbreaks around the world supports the theory of C. difficile zoonotic transmission.

Transcriptomic analysis of biofilm formation in strains of Clostridioides difficile associated with recurrent and non-recurrent infection reveals potential candidate markers for recurrence.

The transcriptomic profile in a biofilm model of ribotypes (RT) 001 and 027 associated with recurrent Clostridioides difficile infection (R-CDI) and not associated with recurrent (NR)-CDI was analyzed to identify genes that may favor the recurrence. Twenty strains were selected, 10 RT001 and 10 RT027. From each ribotype, 5 were R-CDI and 5 NR-CDI. Biofilm and nonadherent cells were prepared from each clinical isolate, and the RNA was extracted. RNA samples were pooled in 8 combinations implying ribotype, recurrence, and biofilm formation. Each pool was separately labeled with Cy3 dye and hybridized on a microarray designed for this study. Slides were scanned, analyzed, and gene expression was compared between unique and grouped pools using the Student's t-test with Benjamini-Hochberg correction when appropriate. Validation was carried out by qRT-PCR for selected genes. Results: After comparisons of differentially expressed genes from both ribotypes of R-CDI strains (nonadherent cells vs. biofilm) and both ribotypes in biofilm (R-CDI vs. NR-CDI), we found 3 genes over-expressed and 1 under-expressed in common (adj. p ≤ 0.05). Overexpressed genes were CAJ70148 (a putative dehydrogenase), CAJ68100 (a secretion type II system protein from the GspH (pseudopilins) family), and CAJ69725 (a putative membrane protein); under-expressed was CAJ68151 (a segregation and condensation protein A). Because CAJ70148, CAJ68100, CAJ69725 and CAJ68151 were differentially expressed in biofilm in strains associated with R-CDI, they may support the biofilm favoring the recurrence of CDI. However, further studies will be needed for poorly studied genes.

Clostridioides difficile PCR Tcdb Cycle Threshold predicts toxin EIA positivity but not severity of infection.

BACKGROUND: Diagnosis of Clostridioides difficile Infection (CDI) entails compatible clinical presentation and laboratory findings. We evaluated real-time polymerase chain reaction (qPCR) cycle threshold (CT) as a predictor for disease severity and TcdB enzyme immunoassay (EIA) results. METHODS: Inpatients or emergency department patients who tested positive for tcdB gene by PCR were evaluated. Patients' stools underwent testing for GDH and TcdA/B by EIA. Medical health records were reviewed for demographic, clinical presentation, laboratory, treatment and outcome data. Severity of CDI was calculated using various severity score indexes. RESULTS: The median CT of cases was 32.05 ± 5.45. The optimal cut-off for predicting toxin EIA positivity and severe CDI based on chart review was 32.6 and 29.8, respectively, with the area under the receiver operator characteristics curve (AUC) of 0.74 and 0.60 respectively. CONCLUSION: CT value was an acceptable predictor for EIA toxin but less so for clinical severity. Our study potentially supports a diagnostic algorithm including CT value to reduce the number of EIA toxin assays performed.

Molecular epidemiology, antimicrobial susceptibility, and toxin production of clinical Clostridioides difficile isolates from a teaching hospital in Northern China.

To grasp the epidemiological trend and drug resistance mechanisms of Clostridioides difficile (C. diff) in Beijing, 302 C. diff isolates were obtained from patients with diarrhea. The sequence types (STs) from mainstream strains were all susceptible to metronidazole, vancomycin, piperacillin/tazobactam, meropenem, and tigecycline but almost resistant to ciprofloxacin and clindamycin. The missense mutation of GyrA/GyrB and RpoB resulted in fluoroquinolone and rifamycin resistance, respectively. Toxigenic strains from clade IV were likely to be missed due to the deficiency of tcdA gene. Four tcdC genotypes were first detected in strains from clade III and IV. The truncating mutation of TcdC disabled its function working as a toxin suppressor. In conclusion, the molecular epidemiology of C. diff in Beijing is different from other regions of China. The antimicrobial resistance patterns and toxin-producing abilities of strains with different STs varied greatly, which suggests that continuous surveillance and control are meaningful and urgent.

A network of small RNAs regulates sporulation initiation in Clostridioides difficile.

The obligate anaerobic, enteric pathogen Clostridioides difficile persists in the intestinal tract by forming antibiotic-resistant endospores that contribute to relapsing and recurrent infections. Despite the importance of sporulation for C. difficile pathogenesis, environmental cues and molecular mechanisms that regulate sporulation initiation remain ill-defined. Here, by using RIL-seq to globally capture the Hfq-dependent RNA-RNA interactome, we discovered a network of small RNAs that bind to mRNAs encoding sporulation-related genes. We show that two of these small RNAs, SpoX and SpoY, regulate translation of the master regulator of sporulation, Spo0A, in an opposing manner, which ultimately leads to altered sporulation rates. Infection of antibiotic-treated mice with SpoX and SpoY deletion mutants revealed a global effect on gut colonization and intestinal sporulation. Our work uncovers an elaborate RNA-RNA interactome controlling the physiology and virulence of C. difficile and identifies a complex post-transcriptional layer in the regulation of spore formation in this important human pathogen.

Clostridioides (Clostridium) difficile in adults with diarrhoea in Vietnam.

BACKGROUND: Clostridioides (Clostridium) difficile causes antimicrobial-associated diarrhoea, however, presentations may range from asymptomatic carriage to severe diarrhoea, life-threatening toxic megacolon and even death. Reports on C. difficile infection (CDI) in Vietnam remain limited. The objectives of this study were to evaluate the epidemiology, molecular characteristics, and antimicrobial susceptibility of C. difficile isolated from adults with diarrhoea in Vietnam. METHODS: Diarrhoeal stool samples from adult patients aged ≥17 years old were collected at Thai Binh General Hospital in northern Vietnam between March 1, 2021 and February 28, 2022. All samples were transported to The University of Western Australia, Perth, Western Australia for C. difficile culture, toxin gene profiling, PCR ribotyping and antimicrobial susceptibility testing. RESULTS: A total of 205 stool samples were collected from patients aged from 17 to 101 years old. The overall prevalence of C. difficile was 15.1% (31/205) with the recovery of toxigenic and non-toxigenic isolates 9.8% (20/205) and 6.3% (13/205), respectively. Thus 33 isolates were recovered comprising 18 known ribotypes (RTs) and one novel RT (two samples contained two different RTs in each sample). The most prevalent strains were RT 012 (five strains) and RTs 014/020, 017 and QX 070 three strains each. All C. difficile were susceptible to amoxicillin/clavulanate, fidaxomicin, metronidazole, moxifloxacin and vancomycin, while resistance to varying degrees was seen to clindamycin, erythromycin, tetracycline and rifaximin, 78.8% (26/33), 51.5% (17/33), 27.3% (9/33) and 6.1% (2/33), respectively. The prevalence of multidrug resistance was 27.3% (9/33) and multidrug resistance was most common in toxigenic RT 012 and non-toxigenic RT 038 strains. CONCLUSION: The prevalence of C. difficile in adults with diarrhoea and multidrug resistance in C. difficile isolates was relatively high. A clinical assessment to differentiate between CDI/disease and colonisation is required.

Clostridioides difficile infections were predominantly driven by fluoroquinolone-resistant Clostridioides difficile ribotypes 176 and 001 in Slovakia in 2018-2019.

AIM: To investigate the epidemiology of Clostridioides difficile infection (CDI) in Slovakian hospitals after the emergence of ribotype 176 (027-like) in 2016. METHODS: Between 2018 and 2019, European Centre for Disease Control and Prevention CDI surveillance protocol v2.3 was applied to 14 hospitals, with additional data collected on recent antimicrobial use and the characterization of C. difficile isolates. RESULTS: The mean hospital incidence of CDI was 4.1 cases per 10,000 patient bed-days. One hundred and five (27.6%) in-hospital deaths were reported among the 381 cases. Antimicrobial treatment within the previous 4 weeks was recorded in 90.5% (333/368) of cases. Ribotype (RT)176 was detected in 50% (n=185/370, 14 hospitals) and RT001 was detected in 34.6% (n=128/370,13/14 hospitals) of cases with RT data. Overall, 86% (n=318/370) of isolates were resistant to moxifloxacin by Thr82Ile in GyrA (99.7%). Multi-locus variable tandem repeat analysis showed clonal relatedness of predominant RTs within and between hospitals. Seven of 14 sequenced RT176 isolates and five of 13 RT001 isolates showed between zero and three allelic differences by whole-genome multi-locus sequence typing. The majority of sequenced isolates (24/27) carried the erm(B) gene and 16/27 also carried the aac(6')-aph(2'') gene with the corresponding antimicrobial susceptibility phenotypes. Nine RT176 strains carried the cfr(E)gene and one RT001 strain carried the cfr(C) gene, but without linezolid resistance. CONCLUSIONS: The newly-predominant RT176 and endemic RT001 are driving the epidemiology of CDI in Slovakia. In addition to fluoroquinolones, the use of macrolide-lincosamide-streptogramin B antibiotics can represent another driving force for the spread of these epidemic lineages. In C. difficile, linezolid resistance should be confirmed phenotypically in strains with detected cfr gene(s).

Screening for Clostridioides difficile colonization at admission to the hospital: a multi-centre study.

OBJECTIVES: To assess the value of screening for Clostridioides difficile colonization (CDC) at hospital admission in an endemic setting. METHODS: A multi-centre study was conducted at four hospitals located across the Netherlands. Newly admitted patients were screened for CDC. The risk of development of Clostridioides difficile infection (CDI) during admission and 1-year follow-up was assessed in patients with and without colonization. C. difficile isolates from patients with colonization were compared with isolates from incident CDI cases using core genome multi-locus sequence typing to determine whether onwards transmission had occurred. RESULTS: CDC was present in 108 of 2211 admissions (4.9%), whereas colonization with a toxigenic strain (toxigenic Clostridoides difficile colonization [tCDC]) was present in 68 of 2211 admissions (3.1%). Among these 108 patients with colonization, diverse PCR ribotypes were found and no 'hypervirulent' PCR ribotype 027 (RT027) was detected (95% CI, 0-0.028). None of the patients with colonization developed CDI during admission (0/49; 95% CI, 0-0.073) or 1-year follow-up (0/38; 95% CI, 0-0.93). Core genome multi-locus sequence typing identified six clusters with genetically related isolates from patients with tCDC and CDI; however, in these clusters, only one possible transmission event from a patient with tCDC to a patient with CDI was identified based on epidemiological data. CONCLUSION: In this endemic setting with a low prevalence of 'hypervirulent' strains, screening for CDC at admission did not detect any patients with CDC who progressed to symptomatic CDI and detected only one possible transmission event from a patient with colonization to a patient with CDI. Thus, screening for CDC at admission is not useful in this setting.

Infrequent intrahousehold transmission of Clostridioides difficile between pet owners and their pets.

Companion animals have been shown to carry Clostridioides difficile strains that are similar or identical to strains found in people, and a small number of studies have shown that pets carry genetically identical C. difficile isolates as their owners, suggesting inter-species transmission. However, the directionality of transmission is ultimately unknown, and the frequency with which animals acquire C. difficile following their owners' infection is unclear. The goal of this study was to assess how often pets belonging to people with C. difficile infection carry genetically related C. difficile isolates. We enrolled pet owners from two medical institutions (University of Pennsylvania Health System (UPHS) and The Ohio State University Wexner Medical Center (OSUWMC)) who had diarrhoea with or without positive C. difficile assays and tested their faeces and their pets' faeces for C. difficile using both anaerobic culture and PCR assays. When microorganisms were obtained from both the owner and pet and had the same toxin profile or ribotype, isolates underwent genomic sequencing. Faecal samples were obtained from a total of 59 humans, 72 dogs and 9 cats, representing 47 complete households (i.e. where a sample was available from the owner and at least one pet). Of these, C. difficile was detected in 30 humans, 10 dogs and 0 cats. There were only two households where C. difficile was detected in both the owner and pet. In one of these households, the C. difficile isolates were of different toxin profiles/ribotypes (A+/B+ / RT 499 from the owner, A-/B- / RT PR22386 from the dog). In the other household, the isolates were genetically identical (one SNP difference). Interestingly, the dog from this household had recently received a course of antibiotics (cefpodoxime and metronidazole). Our findings suggest that inter-species transmission of C. difficile occurs infrequently in households with human C. difficile infections.

Molecular Epidemiology and Genetic Relatedness of Clostridioides difficile Isolates in Pediatric Oncology and Transplant Patients Using Whole Genome Sequencing.

BACKGROUND: The incidence of Clostridioides difficile infection (CDI) has been rising among hospitalized children, with poor understanding of genomic variability of C. difficile isolates in this population. METHODS: This was a retrospective cohort study of CDI in inpatient and outpatient pediatric oncology and cell transplant patients (POTPs) in 2016 and 2017. CDI cases were identified by positive C. difficile toxin polymerase chain reaction tests. Retrieved residual stool specimens were cultured anaerobically and toxin-producing C. difficile isolates underwent whole genome sequencing (WGS) followed by core genome multilocus sequence typing. Plausible time and location epidemiologic links among the closely related strains were evaluated to identify potential transmission events. RESULTS: Among 226 CDI episodes in 157 patients, 202 stool samples were cultured and had positive cytotoxicity tests. Sequencing identified 33 different strain types in 162 (80%) isolates. Thirty-nine (28%) patients had multiple episodes of CDI, and 31 clusters of related isolates were identified, 15 (47%) of which involved exclusively multiple specimens from the same patient. For the 16 clusters involving multiple patients, epidemiologic investigation revealed only 2 (12.5%) clusters with potential transmission events. CONCLUSIONS: WGS identified a highly diverse group of C. difficile isolates among POTPs with CDI. Although WGS identified clusters of closely related isolates in multiple patients, epidemiologic investigation of shared inpatient exposures identified potential transmission in only 2 clusters. Clostridioides difficile transmission was uncommon in this population. More than 70% of new CDI reinfections in POTPs are actually recurrences caused by a previous CDI strain.

Reference Susceptibility Testing and Genomic Surveillance of Clostridioides difficile, United States, 2012-17.

BACKGROUND: Antimicrobial susceptibility testing (AST) is not routinely performed for Clostridioides difficile and data evaluating minimum inhibitory concentrations (MICs) are limited. We performed AST and whole genome sequencing (WGS) for 593 C. difficile isolates collected between 2012 and 2017 through the Centers for Disease Control and Prevention's Emerging Infections Program. METHODS: MICs to 6 antimicrobial agents (ceftriaxone, clindamycin, meropenem, metronidazole, moxifloxacin, and vancomycin) were determined using the reference agar dilution method according to Clinical and Laboratory Standards Institute guidelines. Whole genome sequencing was performed on all isolates to detect the presence of genes or mutations previously associated with resistance. RESULTS: Among all isolates, 98.5% displayed a vancomycin MIC ≤2 µg/mL and 97.3% displayed a metronidazole MIC ≤2 µg/mL. Ribotype 027 (RT027) isolates displayed higher vancomycin MICs (MIC50: 2 µg/mL; MIC90: 2 µg/mL) than non-RT027 isolates (MIC50: 0.5 µg/mL; MIC90: 1 µg/mL) (P < .01). No vanA/B genes were detected. RT027 isolates also showed higher MICs to clindamycin and moxifloxacin and were more likely to harbor associated resistance genes or mutations. CONCLUSIONS: Elevated MICs to antibiotics used for treatment of C. difficile infection were rare, and there was no increase in MICs over time. The lack of vanA/B genes or mutations consistently associated with elevated vancomycin MICs suggests there are multifactorial mechanisms of resistance. Ongoing surveillance of C. difficile using reference AST and WGS to monitor MIC trends and the presence of antibiotic resistance mechanisms is essential.

Carriage of three plasmids in a single human clinical isolate of Clostridioides difficile.

A subset of clinical isolates of Clostridioides difficile contains one or more plasmids and these plasmids can harbor virulence and antimicrobial resistance determinants. Despite their potential importance, C. difficile plasmids remain poorly characterized. Here, we provide the complete genome sequence of a human clinical isolate that carries three high-copy number plasmids from three different plasmid families that are therefore compatible. For two of these, we identify a region capable of sustaining plasmid replication in C. difficile that is also compatible with the plasmid pCD630 that is found in many laboratory strains. Together, our data advance our understanding of C. difficile plasmid biology.

Clostridioides difficile as a Potential Pathogen of Importance to One Health: A Review.

Clostridioides difficile (basonym Clostridium) is a bacterial enteropathogen associated with cases of C. difficile infection that can result in pseudomembranous colitis, rapid fluid loss, and death. For decades following its isolation, C. difficile was thought to be a solely nosocomial pathogen, being isolated from individuals undergoing antimicrobial therapy and largely affecting elderly populations. More recently, C. difficile spores have been identified in the broader environment, including in food-producing animals, soil, and food matrices, in both ready-to-eat foods and meat products. Furthermore, evidence has emerged of hypervirulent ribotypes (RTs), such as RT078, similar to those cultured in asymptomatic carriers, also being identified in these environments. This finding may reflect on adaptations arising in these bacteria following selection pressures encountered in these niches, and which occurs due to an increase in antimicrobial usage in both clinical and veterinary settings. As C. difficile continues to adapt to new ecological niches, the taxonomy of this genus has also been evolving. To help understand the transmission and virulence potential of these bacteria of importance to veterinary public health, strategies applying multi-omics-based technologies may prove useful. These approaches may extend our current understanding of this recognized nosocomial pathogen, perhaps redefining it as a zoonotic bacterium. In this review, a brief background on the epidemiological presentation of C. difficile will be highlighted, followed by a review of C. difficile in food-producing animals and food products. The current state of C. difficile taxonomy will provide evidence of Clade 5 (ST11/RT078) delineation, as well as background on the genomic elements linked to C. difficile virulence and ongoing speciation. Recent studies applying second- and third-generation sequencing technologies will be highlighted, and which will further strengthen the argument made by many throughout the world regarding this pathogen and its consideration within a One Health dimension.

Make It Less difficile: Understanding Genetic Evolution and Global Spread of Clostridioides difficile.

Clostridioides difficile is an obligate anaerobic pathogen among the most common causes of healthcare-associated infections. It poses a global threat due to the clinical outcomes of infection and resistance to antibiotics recommended by international guidelines for its eradication. In particular, C. difficile infection can lead to fulminant colitis associated with shock, hypotension, megacolon, and, in severe cases, death. It is therefore of the utmost urgency to fully characterize this pathogen and better understand its spread, in order to reduce infection rates and improve therapy success. This review aims to provide a state-of-the-art overview of the genetic variation of C. difficile, with particular regard to pathogenic genes and the correlation with clinical issues of its infection. We also summarize the current typing techniques and, based on them, the global distribution of the most common ribotypes. Finally, we discuss genomic surveillance actions and new genetic engineering strategies as future perspectives to make it less difficile.

Identification of novel, cryptic Clostridioides species isolates from environmental samples collected from diverse geographical locations.

Clostridioides difficile is a pathogen often associated with hospital-acquired infection or antimicrobial-induced disease; however, increasing evidence indicates infections can result from community or environmental sources. Most genomic sequencing of C. difficile has focused on clinical strains, although evidence is growing that C. difficile spores are widespread in soil and water in the environment. In this study, we sequenced 38 genomes collected from soil and water isolates in Flagstaff (AZ, USA) and Slovenia in an effort targeted towards environmental surveillance of C. difficile. At the average nucleotide identity (ANI) level, the genomes were divergent to C. difficile at a threshold consistent with different species. A phylogenetic analysis of these divergent genomes together with Clostridioides genomes available in public repositories confirmed the presence of three previously described, cryptic Clostridioides species and added two additional clades. One of the cryptic species (C-III) was almost entirely composed of Arizona and Slovenia genomes, and contained distinct sub-groups from each region (evidenced by SNP and gene-content differences). A comparative genomics analysis identified multiple unique coding sequences per clade, which can serve as markers for subsequent environmental surveys of these cryptic species. Homologues to the C. difficile toxin genes, tcdA and tcdB, were found in cryptic species genomes, although they were not part of the typical pathogenicity locus observed in C. difficile, and in silico PCR suggested that some would not amplify with widely used PCR diagnostic tests. We also identified gene homologues in the binary toxin cluster, including some present on phage and, for what is believed to be the first time, on a plasmid. All isolates were obtained from environmental samples, so the function and disease potential of these toxin homologues is currently unknown. Enzymatic profiles of a subset of cryptic isolates (n=5) demonstrated differences, suggesting that these isolates contain substantial metabolic diversity. Antimicrobial resistance (AMR) was observed across a subset of isolates (n=4), suggesting that AMR mechanisms are intrinsic to the genus, perhaps originating from a shared environmental origin. This study greatly expands our understanding of the genomic diversity of Clostridioides. These results have implications for C. difficile One Health research, for more sensitive C. difficile diagnostics, as well as for understanding the evolutionary history of C. difficile and the development of pathogenesis.

Molecular epidemiology of Clostridioides difficile in companion animals: Genetic overlap with human strains and public health concerns.

Introduction: The changing epidemiology of Clostridioides difficile reflects a well-established and intricate community transmission network. With rising numbers of reported community-acquired infections, recent studies tried to identify the role played by non-human reservoirs in the pathogen's transmission chain. This study aimed at describing the C. difficile strains circulating in canine and feline populations, and to evaluate their genetic overlap with human strains to assess the possibility of interspecies transmission. Methods: Fecal samples from dogs (n = 335) and cats (n = 140) were collected from two populations (group A and group B) in Portugal. C. difficile isolates were characterized for toxigenic profile and PCR-ribotyping. The presence of genetic determinants of antimicrobial resistance was assessed in all phenotypically resistant isolates. To evaluate the genetic overlap between companion animals and human isolates from Portugal, RT106 (n = 42) and RT014/020 (n = 41) strains from both sources were subjected to whole genome sequencing and integrated with previously sequenced RT106 (n = 43) and RT014/020 (n = 142) genomes from different countries. The genetic overlap was assessed based on core-single nucleotide polymorphism (SNP) using a threshold of 2 SNP. Results: The overall positivity rate for C. difficile was 26% (76/292) in group A and 18.6% (34/183) in group B. Toxigenic strains accounted for 50% (38/76) and 52.9% (18/34) of animal carriage rates, respectively. The most prevalent ribotypes (RT) were the toxigenic RT106 and RT014/020, and the non-toxigenic RT010 and RT009. Antimicrobial resistance was found for clindamycin (27.9%), metronidazole (17.1%) and moxifloxacin (12.4%), associated with the presence of the ermB gene, the pCD-METRO plasmid and point mutations in the gyrA gene, respectively. Both RT106 and RT014/020 genetic analysis revealed several clusters integrating isolates from animal and human sources, supporting the possibility of clonal interspecies transmission or a shared environmental contamination source. Discussion: This study shows that companion animals may constitute a source of infection of toxigenic and antimicrobial resistant human associated C. difficile isolates. Additionally, it contributes with important data on the genetic proximity between C. difficile isolates from both sources, adding new information to guide future work on the role of animal reservoirs in the establishment of community associated transmission networks and alerting for potential public health risk.