Clostridioides difficile infection epidemiology during the COVID-19 pandemic in Greece.

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.

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Association of toxin-producing Clostridioides difficile with piglet diarrhea and potential transmission to humans.

The pathogenicity of Clostridioides difficile in piglets remains controversial. It is unknown whether C. difficile control helps protect piglet health. To clarify the association between C. difficile presence and piglet diarrhea, isolates were obtained from piglets with and without diarrhea. In addition, to determine the genetic relationship of C. difficile from pigs and humans, we performed whole-genome sequencing (WGS) of C. difficile isolates. Diarrheal and non-diarrheal stool samples were collected from neonatal piglets from five farms in Japan in 2021. To clarify the relationship between C. difficile derived from pigs and those from human clinical cases, WGS of C. difficile isolates was performed. Toxin-positive C. difficile were significantly more prevalent in piglets with diarrhea, although the overall frequency of C. difficile did not differ between piglets with and without diarrhea. This observation indicates an association between toxin-positive C. difficile and diarrhea in piglets. However, further studies are needed to establish a direct causal relationship and to explore other contributing factors to diarrhea in piglets. WGS results showed that C. difficile sequence type (ST) 11 including the hypervirulent PCR ribotype 078 isolates derived from Japanese pigs were closely related to ST11 of overseas strains (human clinical and animal-derived) and a Japanese human clinical strain. Toxin-positive C. difficile may cause diarrhea in piglets and hypervirulent C. difficile are spreading among pigs and human populations worldwide.

Clostridioides difficile in Pigs and Dairy Cattle in Northern Italy: Prevalence, Characterization and Comparison between Animal and Human Strains.

It has been observed that novel strains of Clostridioides difficile can rapidly emerge and move between animal and human hosts. The aim of this study was to investigate the prevalence of C. difficile in pigs and dairy cattle in northern Italy and to characterize and compare C. difficile animal strains with those from patients from the same geographical area. The C. difficile strains were isolated from animals from farms and slaughterhouses (cross-sectional studies) and from neonatal animals with enteric disorders in routine diagnostic investigations (passive surveillance). Samples positive for C. difficile were found in 87% of the pig farms and in 40% of the cattle farms involved in the cross-sectional studies, with a 20% prevalence among suckling piglets and 6.7% prevalence in neonatal calves, with no significant difference between animals with and without diarrheal symptoms. The prevalence of C. difficile in older animal categories was significantly lower. This result suggests that young age is an important risk factor for C. difficile colonization. In cross-sectional studies at slaughterhouses, in both the heavy pigs and dairy cows examined, only 2% of the intestinal content samples were positive for C. difficile and no contamination was found on the surface of the carcasses. Considering passive surveillance, the prevalence rates of positive samples were 29% in piglets and 1.4% in calves. Overall, 267 strains of animal origin and 97 from humans were collected. In total, 39 ribotypes (RTs) were identified, with RT 078 and RT 018 being predominant among animals and humans, respectively. Several RTs overlapped between animals and patients. In particular, RT 569 was identified as an emergent type in our country. Resistance to erythromycin and moxifloxacin was widely diffused among C. difficile strains, regardless of origin. This study supports C. difficile as a pathogen of one-health importance and highlights the need for a collaborative approach between physicians and veterinarians to control and prevent infections that are able to cross species and geographical barriers.

Predominance of Clostridioides difficile PCR ribotype 181 in northern Greece, 2016-2019.

OBJECTIVES: The epidemiology of Clostridioides difficile infection (CDI) has undergone many changes since the beginning of this century and continues to evolve based on recent studies. Here, we performed a molecular analysis of C. difficile isolates in northern Greece across 10 health-care facilities, spanning from 2016 to 2019. METHODS: 221 C. difficile isolates were cultured from stool samples of hospitalized patients with diarrhea and screened by PCR for the presence of the toxin A (tcdA), toxin B (tcdB), the binary toxin (cdtA and cdtB) genes and the regulating gene of tcdC. PCR ribotyping of the cultured isolates was performed by a standardized protocol for capillary gel-based PCR ribotyping and an international database with well-documented reference strains. RESULTS: Thirty-five different PCR ribotypes were identified. The most common RTs identified were: 181 (36%, 80/221), 017 (10%, 21/221), 126 (9%, 19/221), 078 (4%, 9/221) and 012 (4%, 8/221). Notably, the predominant RT181, with toxin profile tcdA+tcdB+cdtA+cdtB+, was identified in seven out of ten participating hospitals. CONCLUSIONS: Multiple C. difficile ribotypes have been circulating in the northern Greece region with RTs 181 (closely related to 027), 017, 126 and 078 being predominant.

Implementation of a Clostridioides difficile sentinel surveillance system in Germany: First insights for 2019-2021.

OBJECTIVES: Clostridioides difficile is a major cause of nosocomial diarrhea. Several "hypervirulent" lineages such as ribotype 027 (RT027) and RT078 are of high epidemiological importance, leading to outbreaks and more severe courses of disease. An active surveillance system targeting molecular epidemiology and corresponding antimicrobial resistance has not been established in Germany. METHODS: Since October 2019, University Hospitals throughout Germany collected by two dates every year (1st April and October, respectively) their first ten unselected samples being tested positive for C. difficile. RESULTS: Out of 1026 samples received from 29 sites, 876 toxigenic C. difficile strains could be cultivated. PCR ribotyping of these strains revealed a large strain diversity with RT014 (17.5%) dominating, followed by isolates of the major nosocomial lineage RT001 (7.1%) and the "hypervirulent" lineage RT078 (5.9%). Notably, prevalence of RT027 was low with ∼3.5% at all time points analyzed, while the abundance of RT001 isolates significantly declined from 12.3% to 3.7% during the sampling period (P < 0.001). Antimicrobial resistance against clarithromycin, moxifloxacin, and rifampicin was detected in 18%, 15%, and 4% of the tested isolates, respectively. Highest resistance rates were found among RT027 isolates (83%, 87% and 63% for clarithromycin, moxifloxacin, and rifampicin, respectively). Vancomycin resistance was not detected, and metronidazole resistance was observed only for a single RT027 isolate. CONCLUSIONS: This Germany-wide continuing surveillance effort with a standardized mode of isolate acquisition indicates that isolates of RT027 were only sporadically detected under these strain acquisition conditions, and RT001 seems to become less important in the hospital setting, being replaced by other RTs.

Molecular Epidemiology, Clinical Course, and Implementation of Specific Hygiene Measures in Hospitalised Patients with Clostridioides difficile Infection in Brandenburg, Germany.

(1) Background: Clostridioides difficile infections (CDI) have increased worldwide, and the disease is one of the most common healthcare-associated infections (HAI). This study aimed to evaluate the molecular epidemiology of C. difficile, the clinical outcome, and the time of initiation of specific hygiene measures in patients with CDI in a large tertiary-care hospital in Brandenburg. (2) Methods: Faecal samples and data from hospitalised patients diagnosed with CDI were analysed from October 2016 to October 2017. The pathogens were isolated, identified as toxigenic C. difficile, and subsequently subtyped using PCR ribotyping and whole genome sequencing (WGS). Data regarding specific hygiene measures for handling CDI patients were collected. (3) Results: 92.1% of cases could be classified as healthcare-associated (HA)-CDI. The recurrence rate within 30 and 90 days after CDI diagnosis was 15.7% and 18.6%, and the mortality rate was 21.4% and 41.4%, respectively. The most frequent ribotypes (RT) were RT027 (31.3%), RT014 (18.2%), and RT005 (14.1%). Analysis of WGS data using cgMLST showed that all RT027 isolates were closely related; they were assigned to two subclusters. Single-room isolation or barrier measures were implemented in 95.7% patients. (4) Conclusions: These data show that RT027 is regionally predominant, thus highlighting the importance of specific hygiene measures to prevent and control CDI and the need to improve molecular surveillance of C. difficile at the local and national level.

Rapid Classification of Clostridioides difficile Strains Using MALDI-TOF MS Peak-Based Assay in Comparison with PCR-Ribotyping.

Typing methods are needed for epidemiological tracking of new emerging and hypervirulent strains because of the growing incidence, severity and mortality of Clostridioides difficile infections (CDI). The aim of this study was the evaluation of a typing Matrix-Assisted Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS (T-MALDI)) method for the rapid classification of the circulating C. difficile strains in comparison with polymerase chain reaction (PCR)-ribotyping results. Among 95 C. difficile strains, 10 ribotypes (PR1-PR10) were identified by PCR-ribotyping. In particular, 93.7% of the isolates (89/95) were grouped in five ribotypes (PR1-PR5). For T-MALDI, two classifying algorithm models (CAM) were tested: the first CAM involved all 10 ribotypes whereas the second one only the PR1-PR5 ribotypes. Better performance was obtained using the second CAM: recognition capability of 100%, cross-validation of 96.6% and agreement of 98.4% (60 correctly typed strains, limited to PR1-PR5 classification, out of 61 examined strains) with PCR-ribotyping results. T-MALDI seems to represent an alternative to PCR-ribotyping in terms of reproducibility, set up time and costs, as well as a useful tool in epidemiological investigation for the detection of C. difficile clusters (either among CAM included ribotypes or out-of-CAM ribotypes) involved in outbreaks.

Comparison of Clostridioides difficile strains from animals and humans: First results after introduction of C. difficile molecular typing and characterization at the Istituto Zooprofilattico Sperimentale of Piemonte, Liguria e Valle d'Aosta, Italy.

PCR ribotypes (RTs027 and 078) are known causes of Clostridioides difficile infection (CDI) in humans. Molecular typing and characterization of 39 C. difficile strains isolated from samples from humas and animals in 2016-2018 indicated an overlap of RTs between community-acquired patients (CA-CDI) and domestic animals from the same geographical area; 14 RTs were identified: 12 RTs were positive for toxins A/B; RT078, RT080 and RT126 were also positive for binary toxin (CDT). Most of the RTs from the animals (RTs020, 078, 106, 126) were also detected in the samples from humans. Strains grouped into three clusters: cluster I included prevalently human strains, mainly RT 018; clusters II and III included strains from humans and animals, mainly RT078 and RT020. The CA-CDI strains suggested animals as a reservoir of C. difficile isolated together with other microorganisms from animals, highlighting the association of enteric pathogens as a cause of infection and death.

Simultaneous detection and ribotyping of Clostridioides difficile, and toxin gene detection directly on fecal samples.

BACKGROUND: Clostridioides difficile is the most common cause of nosocomial diarrhea. Ribotyping of cultured strains by a PCR-based test is used to study potential transmission between patients. We aimed to develop a rapid test that can be applied directly on fecal samples for simultaneous detection and ribotyping of C. difficile, as well as detection of toxin genes. METHODS: We developed a highly specific and sensitive primer set for simultaneous detection and ribotyping of C. difficile directly on total fecal DNA. Toxin genes were detected with primers adapted from Persson et al. (Clin Microbiol Infect 14(11):1057-1064). Our study set comprised 130 fecal samples: 65 samples with positive qPCR for C. difficile toxin A/B genes and 65 C. difficile qPCR negative samples. PCR products were analyzed by capillary gel electrophoresis. RESULTS: Ribosomal DNA fragment peak profiles and toxin genes were detected in all 65 C. difficile positive fecal samples and in none of the 65 C. difficile negative samples. The 65 samples were assigned to 27 ribotypes by the Dutch reference laboratory. Our peak profiles corresponded to these ribotypes, except for two samples. During a C. difficile outbreak, patients were correctly allocated to the outbreak-cluster based on the results of direct fecal ribotyping, before C. difficile isolates were cultured and conventionally typed. CONCLUSION: C. difficile ribotyping directly on fecal DNA is feasible, with sensitivity and specificity comparable to that of diagnostic toxin gene qPCR and with ribotype assignment similar to that obtained by conventional typing on DNA from cultured isolates. This supports simultaneous diagnosis and typing to recognize an outbreak.

Impact of the introduction of nucleic acid amplification testing on Clostridioides difficile detection and ribotype distribution in Wales.

OBJECTIVES: To determine the impact of the 2018 introduction of nucleic acid amplification tests (NAATs) for C. difficile detection on the laboratory diagnosis of C. difficile infection (CDI), and the distribution of C. difficile ribotypes. METHODS: A retrospective analysis of five years (2015-2019) of C. difficile diagnostic laboratory and PCR ribotyping test results. RESULTS: A total of 255,104 diagnostic results, from 136,353 patients were analysed: 199,794 samples where glutamate dehydrogenase (GDH) was used as the primary screen; and 55,310 where NAATs were employed. An overall decrease in frontline positivity from 2015 to 2019 (10.3% [n = 5017] to 6% [n = 3190] - p < 0.0001) was observed, despite an increase in the number of samples tested (48,778 to 52,839). NAAT positivity was lower than GDH (p < 0.0001) for the two years where it was implemented. The variance was accounted for by increased overall C. difficile isolation and reduced toxin negative strain culture from NAAT positive samples (p < 0.0001). Ribotype distribution (6546 samples) remained stable with decreasing RT27 isolation in each year except 2017 (p < 0.0001). RT78 was associated with toxin A/B EIA positivity (p < 0.0001). CONCLUSIONS: Use of NAAT for the detection of C. difficile, as part of a 2-step algorithm, has not led to an increase in CDI laboratory diagnostic test positivity. In spite of ribotype distribution being comparable for screening in toxin A/B positive samples, there is a significantly greater correlation between NAAT positivity and culture of toxigenic strains compared to GDH.

Ribotypes in isolates and testing algorithm of C. difficile infections in the studied sample.

BACKGROUND: Clostridium (Clostridioides) difficile is the most common pathogen of nosocomial and antibiotic-related diarrhea in health-care facilities. The aim of the analysis was to show the testing algorithm and to identify hypervirulent strains (suspected RT 027). METHODS: The retrospective analysis of patient samples suspected on CDI was carried out by a two-step algorithm. Biological specimens were analysed by GDH or culture, immunoenzymatic assay on toxins A/B and selected samples also by a real-time PCR. RESULTS: In 1006 specimen suspected on CDI, 202 specimens were evaluated as positive in the two-step algorithm. Conflicting results (64 C. difficile isolates) were tested in a three-step algorithm by a real-time PCR and revealed 59 toxigenic and non RT 027 ribotypes. Statistically significant dependence among the independent variables, such as: diagnostic parameters and length of hospitalization (p = 0.175) and C. difficile (suspected RT027) ribotypes was not found. CONCLUSION: The results of PCR ribotyping showed a high prevalence of hypervirulent and toxigenic ribotypes in the studied sample. A resistance to vancomycin was found in one isolate. The PCR method contributed to the rapid laboratory diagnosis and thus treatment of high risk patients or was used as a third step in in the case of unclear results of standard diagnostic methods(Tab. 1, Fig. 4, Ref. 18). Text in PDF www.elis.sk.

Microbiological characteristics of human and animal isolates of Clostridioides difficile in Italy: Results of the Istituto Superiore di Sanità in the years 2006-2016.

The increased incidence of Clostridioides difficile infection (CDI) and the emergence of highly virulent types highlight the need of microbiological characterization to gain insight CDI epidemiological changes. This paper, reporting data obtained by the Istituto Superiore di Sanità Central Laboratory Service for C. difficile (ISS-CLSCD) in 2006-2016, provides a first long-term microbiological analysis of human and animal C. difficile strains circulating in Italy. The number of human isolates analyzed by ISS-CLSCD significantly increased over the time (170 in 2006-2011 vs 661 in 2012-2016). Independently from the year of isolation, 42% of the clinical isolates belonged to the PCR-ribotype (RT) 018-lineage (RT 018, RT 607, RT 541, PR07661 and PR14328), with RT 018 and RT 607 grouping the majority of isolates. This lineage was significantly associated to CDIs occurred in the General Medicine Units, Clinic Units or Long-Term Care Facilities, while it was rarely found in pediatric patients. Although the percentage of isolates positive for the binary toxin (CDT) was stable during the study (20%), several CDT-positive RTs emerged in 2012-2016, including RT 027. In total, 32 RTs overlapped between animals and humans and six of these RTs were non-toxigenic. The two lineages prevalent in animals, the RT 078-lineage and the RT 569-lineage (RT 569, RT 049, RT 056 and RT 727), were also found in humans, while the RT 018-lineage was rarely detected in animals, suggesting that it is prevalently associated to human infections. Sixty-two percent of clinical isolates showed a multidrug-resistance (MDR) phenotype, with resistance to rifampicin characterizing successful RTs. A MDR phenotype was also observed in 18% of animal isolates, in particular from dogs, supporting animals as potential reservoirs of resistant C. difficile strains. Interestingly, multiple resistances were observed in both human and animal non-toxigenic isolates suggesting their contribution to antibiotic resistance spread among C. difficile population. All these data indicate that CDI is an issue of growing concern in Italy, highlighting the need for a standardized surveillance in our Country and an interdisciplinary approach to deal successfully with this infection.

Microevolution within ST11 group Clostridioides difficile isolates through mobile genetic elements based on complete genome sequencing.

BACKGROUND: Clade 5 Clostridioides difficile diverges significantly from the other clades and is therefore, attracting increasing attention due its great heterogeneity. In this study, we used third-generation sequencing techniques to sequence the complete whole genomes of three ST11 C. difficile isolates, RT078 and another two new ribotypes (RTs), obtained from three independent hospitalized elderly patients undergoing antibiotics treatment. Mobile genetic elements (MGEs), antibiotic-resistance, drug resistance genes, and virulent-related genes were analyzed and compared within these three isolates. RESULTS: Isolates 10,010 and 12,038 carried a distinct deletion in tcdA compared with isolate 21,062. Furthermore, all three isolates had identical deletions and point-mutations in tcdC, which was once thought to be a unique characteristic of RT078. Isolate 21,062 (RT078) had a unique plasmid, different numbers of transposons and genetic organization, and harboring special CRISPR spacers. All three isolates retained high-level sensitivity to 11 drugs and isolate 21,062 (RT078) carried distinct drug-resistance genes and loss of numerous flagellum-related genes. CONCLUSIONS: We concluded that capillary electrophoresis based PCR-ribotyping is important for confirming RT078. Furthermore, RT078 isolates displayed specific MGEs, indicating an independent evolutionary process. In the further study, we could testify these findings with more RT078 isolates of divergent origins.

Local outbreak of Clostridioides difficile PCR-Ribotype 018 investigated by multi locus variable number tandem repeat analysis, whole genome multi locus sequence typing and core genome single nucleotide polymorphism typing.

The prevalence of Clostridioides difficile PCR-ribotype (RT) 018 is low in Europe but variations are observed across countries. We report here the first RT 018-related outbreak in France that took place in 4 geriatric units (GU) in Strasbourg, France. From January to December 2017, 38 patients were diagnosed with C. difficile infection (CDI). Strains were first characterized by PCR ribotyping: 19 out of 38 (50%) strains belonged to RT 018. These strains as well as 12 RT 018 isolated in other French healthcare facilities and 2 strains of RT 018 isolated in the GU in 2015 were characterized by multi locus variable-number tandem repeat (VNTR) analysis (MLVA), whole genome multi locus sequence typing (wgMLST) and core genome single nucleotide polymorphism typing (cgSNP). The MLVA indicated that 15 out of 19 epidemic strains of RT 018 were included in 2 Clonal Complexes (CC). Four RT 018 strains from the outbreak did not belong to the CC. The wgMLST and cgSNP typing analysis revealed a single CC that included 19 strains from the geriatric unit (17 from GU in 2017 and 2 from GU in 2015) and 4 strains (33%) from other healthcare facilities (HCF). Our results suggest that a specific RT 018 clone has spread in the geriatric unit and has evolved slowly over time. MLVA, wgMLST and cgSNP typing results provided fairly consistent information but wgMLST and cgSNP typing better separated epidemic strains from non-epidemic strains. Compared to wgMLST, the cgSNP typing did not provide additional information.

Clostridium difficile isolated from faecal samples in patients with ulcerative colitis.

BACKGROUND: Ulcerative colitis (UC) is an inflammatory bowel disease (IBD) that is widely identified worldwide. This study aimed to investigate the phenotypic characterization and molecular typing of Clostridium difficile isolates among patients with UC at an inflammatory bowel disease clinic in Iran. METHODS: In this cross-sectional study, conducted from April 2015 to December 2015, 85 UC patients were assessed for C.difficile infection (CDI). C. difficile isolates were characterized based on their toxin profile and antimicrobial resistance pattern. Multi-locus sequence typing analysis (MLST) and PCR ribotyping were performed to define the genetic relationships between different lineages of toxigenic strains. RESULTS: The prevalence of C. difficile isolates was 31.8% (27/85) in patients, of those 15 patients (17.6%) had CDI. Three different sequence types (STs) identified based on MLST among the toxigenic isolates, that is ST54 (33.3%), ST2 (53.3%), and ST37 (13.6%). C. difficile strains were divided into four different PCR-ribotypes (012, 014, 017 and IR1). The most common ribotype was 014 accounting for 48.3% (7/15) of all strains. The strains isolated during the first episode and recurrence of CDI usually belonged to PCR ribotype 014 (ST2). A high rate of CDI recurrence (14.1%, 12/85) experienced in UC patients. Colonization of the gastrointestinal tract with non-toxigenic C. difficile strains was frequent among patients with mild disease. All C. difficile isolates were susceptible to metronidazole, and vancomycin, 86 and 67% of isolates were resistant to clindamycin and erythromycin respectively. There was no correlation between the toxin type and antibiotic resistance (p > 0.05). CONCLUSION: Overall CDI is rather prevalent in UC patients. All patients with CDI experienced moderate to severe disease and exposed to different antimicrobial and anti-inflammatory agents. Close monitoring and appropriate management including early detection and fast treatment of CDI will improve UC outcomes.

Direct detection and characterization of Clostridium difficile from a novel collection device to improve laboratory workflow.

Emergence of Clostridium difficile strains with increased virulence emphasizes the importance of early diagnosis and surveillance of C. difficile infection (CDI). In this study, the new FecalSwab™ collection and transport system was evaluated to improve C. difficile diagnosis. The FecalSwab™ was used for direct C. difficile molecular detection, C. difficile culture/toxigenic culture (TC) and bacterial genomic DNA (bgDNA) extraction. Our results demonstrated that the FecalSwab™ medium could be successfully used as template for Xpert C. difficile binary toxin (BT), regardless of the bacterial load of samples, and for C. difficile culture also after a long storage (30 days) of FecalSwab™ tubes at 4 °C. Furthermore, good-quality bgDNA was extracted from the FecalSwab™ medium for the majority (75%) of the samples analyzed. Typing was performed to fully characterize C. difficile strains isolated during this study and 17 different PCR-ribotypes (RTs) were identified. The results obtained indicate that the FecalSwab™ can be successfully used not only in daily diagnostic routine of C. difficile but also in surveillance and retrospective studies.

Epidemiological investigation of Clostridioides difficile colonization in Chinese community infants.

Clostridioides difficile is a colonizer of the human gut; asymptomatic colonization has been reported to be more common in infants and is highly variable across regions even with no symptoms of diarrhea or death. Antibiotic treatment strategies might increase the antibiotic resistance of C. difficile. We performed a one-point study involving 1098 healthy infants (0-36 months) to address the deficiency of reports on C. difficile colonization in Chinese community infants. The C. difficile colonization rate was 22.8% (250/1098), and more than half of the strains (55.2%) were toxigenic isolates. Among the 138 toxigenic isolates, 111 were of the A+B+CDT- genotype, 26 strains were A-B+CDT-, and one strain was A+B+CDT+. Fifteen different PCR ribotypes were found among the 250 isolates, and PCR-ribotype HB03 appeared to be dominant type, accounting for 19.6% (49/250). High levels of resistance to antimicrobial agents were observed. Our study showed that age and hospitalization before stool collection were positively correlated with the C. difficile colonization rate, whereas the delivery term was negatively related to the colonization rate. Particular attention should be paid to the increasing resistance of C. difficile to rifamycin.

Survey, characterization and antimicrobial susceptibility of Clostridium difficile from marine bivalve shellfish of North Adriatic Sea.

Clostridium difficile is a major cause of infectious diarrhea associated to healthcare settings. Community-acquired infections are increasingly reported in the last decade and exposure other than to symptomatic patients rather to contaminated foods or animals is feasible. Occurrence of C. difficile in shellfish raises concern because spores can survive the cooking temperatures given that shellfish is often consumed poorly cooked or raw. Aim of our study was to investigate whether shellfish represents a reservoir of C. difficile human PCR-ribotypes (RTs). 702 shellfish samples of farmed and wild bivalve mollusc species were collected over the 2015-2017 period in North Adriatic Italian Sea to investigate contamination with C. difficile and characterize the isolates in terms of genotypic variability and antimicrobial resistance profile. C. difficile was detected in 16.9% (CI: 14.1%-19.8%) samples: 11.6% mussels and 23.2% clams. Compared to mussels, clams were significantly associated with detection of C. difficile (OR = 2.4, P < 0.01). Overall 113 C. difficile isolates were genotyped and 75 (66.4%) were toxigenic. Fifty-three different RTs were identified. 40.7% C. difficile isolates were among the RTs most commonly involved in human infection in Europe. The profile of antimicrobial susceptibility was determined by E-test; microbiological resistance was frequent against clindamycin (17%), erythromycin (23%), rifampicin (8.8%) and moxifloxacin (10.6%). All isolates were susceptible to metronidazole and one showed MIC > ECOFF for vancomycin. C. difficile strains showed high variety in RTs, most of them already detected in other animals or known as highly virulent and epidemic in humans. These results prompt towards investigating on specific risk mitigation measures against C. difficile and are preliminary for any source attribution and risk assessment study.

Molecular typing of Clostridioides difficile isolates from clinical and non-clinical samples in Iran.

Clostridioides difficile is a major cause of nosocomial infectious diarrhea in hospitalized patients throughout the world. We aimed to characterize C. difficile isolates among hospitalized patients, hospital staffs, and hospital environment samples obtained in three tertiary care hospitals of Iran with regard to their molecular types between June 2016 and November 2017. The toxigenicity of C. difficile isolates was determined by toxigenic culture and multiplex-PCR. Toxigenic C. difficile isolates collected were ribotyped using capillary gel electrophoresis-based PCR and the database of WEBRIBO (http://webribo.ages.at). Of 500 clinical and non-clinical samples, toxigenic C. difficile were identified in 35 of 250 stool samples (14%) and in 3 of 250 swabs (1.2%). The most frequently found ribotypes (RTs) were 039, AI-12, and AI-21 (15.8, 10.52, and 10.52% of all isolates, respectively). Further RTs were: 017, 001, AI-3, AI-15, AI-18, AI-10, AI-4, and PR21195 (as new ribotype). The epidemic RTs (027 and 078) seen in the Europe, North America, and Asia were completely absent in this study.

Characterization of Clostridium difficile isolates collected during a phase 2b clinical study with SYN-004 (ribaxamase) for the prevention of C. difficile infection.

During a Phase 2b study with SYN-004 (ribaxamase) for prevention of Clostridium difficile infection (CDI) conducted in North America and Eastern Europe, 45 C. difficile isolates from subjects with laboratory-confirmed CDI and or colonized with C. difficile were collected and characterized. Several C. difficile PCR ribotypes, including 027 and 198, were identified.