Impact of Phage CDHS-1 on the Transcription, Physiology and Pathogenicity of a Clostridioides difficile Ribotype 027 Strain, R20291

All known Clostridioides difficile phages encode integrases rendering them potentially able to lyse or lysogenise bacterial strains. Here, we observed the infection of the siphovirus, CDHS-1 on a ribotype 027 strain, R20291 and determined the phage and bacterial gene expression profiles, and impacts of phage infection on bacterial physiology and pathogenicity. Using RNA-seq and RT-qPCR we analysed transcriptomic changes during early, mid-log and late phases of phage replication at an MOI of 10. The phage has a 20 min latent period, takes 80 min to lyse cells and a burst size of ~37. All phage genes are highly expressed during at least one time point. The Cro/C1-transcriptional regulator, ssDNA binding protein and helicase are expressed early, the holin is expressed during the mid-log phase and structural proteins are expressed from mid-log to late phase. Most bacterial genes, particularly the metabolism and toxin production/regulatory genes, were downregulated from early phage replication. Phage-resistant strains and lysogens showed reduced virulence during Galleria mellonella colonization as ascertained by the larval survival and expression of growth (10), reproduction (2) and infection (2) marker genes. These data suggest that phage infection both reduces colonization and negatively impacts bacterial pathogenicity, providing encouraging data to support the development of this phage for therapy to treat C. difficile infection.

Community-acquired fulminant Clostridioides (Clostridium) difficile infection by ribotype 027 isolate in Japan: a case report

Background Clostridioides (Clostridium) difficile infection (CDI) has become an increasingly significant disease not only as healthcare-associated infection, but also as community-acquired (CA) infection worldwide. CDI caused by the NAP1/BI/027 strain is reported to be more severe, difficult to cure, and frequently associated with recurrences in North America and Europe. Case presentation A 68-year-old woman was referred to our hospital for continuous lower abdominal pain 4 weeks after eradication therapy against Helicobacter pylori. While she was treated with fasting on the suspicion of ischemic colitis, she experienced septic shock. Emergent subtotal proctocolectomy revealed fulminant pseudomembranous C. difficile colitis. The C. difficile isolate recovered from the patient was identified as ribotype 027, which has been reported to be uncommon in Japan. Conclusion We report a rare case of CA fulminant pseudomembranous colitis caused by ribotype 027 C. difficile after H. pylori eradication therapy.

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

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

675. Evaluation of PLST for Clostridioides difficile Sequence-based Strain Typing

Background Clostridioides difficile is a leading cause of healthcare-associated infection (HAI), most often following antibiotic therapy. The source of these infections may be endogenous or nosocomial; effective intervention requires distinquishing between these, which in turn requires strain typing. Numerous methods have been developed for C. difficile typing, ranging from length-based ribotyping and MLVA to whole genome sequencing. However, none are routinely used in clinical settings due to low resolution, high cost, technical complexity, or requirement for cultured isolates. The application of polymorphic locus sequence typing (PLST) to epidemiological analysis of HAI and foodborne infections has recently been described; here this approach is extended to C. difficile. Methods Tandem repeats were bioinformatically identified in the genome sequence of ribotype 027 strain R20291. These were screened by BLASTN of GenBank databases for the most polymorphic locus, which identified CdMT1 (Mbp 3.149). DNA was purified from colonies or environmental (Banana Broth) cultures; bead-beating and PCR inhibitor removal steps were required for consistent results. Results CdMT1 encompassed MLVA repeat C6cd which, based on length alone, yielded the highest diversity index (DI) of 0.96. In contrast, CdMT1 sequence analysis yielded DI of >0.99. Comparison to ribotype further illustrated high level resolution; e.g., 9 ribotype 027 strains were resolved into 8 CdMT1 alleles. For initial laboratory evalulation, veterinary C. difficile isolates (44 canine, 4 bovine) were CdMT1 typed. Bioinformatic analysis of the 48 sequences resolved 24 CdMT1 alleles, including 8 clusters of 2 to 6 canine strains. Six of these clusters represented isolates from individual puppies in the same litter, or from different litters but the same household, while the bovine isolates formed a phylogenetically distinct group. Using the same DNA purification protocol, CdMT1 typing demonstrated compatibility with C. difficile-spiked stool samples and Banana Broth environmental cultures. Conclusion CdMT1 typing represents a potentially useful tool for outbreak detection and investigation in healthcare facilities, particularly in light of its compatibility with both stool and environmental samples. Disclosures Tom Edlind, PhD, MicrobiType LLC (Employee, Scientific Research Study Investigator)

Characterisation and antibiotic susceptibility profile of Clostridioides (Clostridium) difficile isolated from chicken carcasses

IntroductionClostridioides (Clostridium) difficile is a Gram+, anaerobic, spore-forming, rod-shaped bacterium that can produce toxins, and it is mainly because its virulence is attributed. The objective of this study was to evaluate the presence of C. difficile and hyper virulent ribotypes in chicken carcasses and the antibiotic susceptibility of isolated strains.Material and MethodsC. difficile was isolated from chicken carcasses by microbiological methods, its ribotypes were identified by means of PCR, the toxin production ability was defined by ELISA, and the susceptibility of the isolates to selected antibiotics was determined by minimum inhibitory concentration evaluator strips.ResultsThe bacterium was isolated from 69 out of 185 (37.3%) examined chicken carcass samples, and six out of the 69 (8.7%) isolates were identified as ribotype 027. All isolates were susceptible to amoxicillin-clavulanic acid (100.0%), vancomycin (97.1%), metronidazole (88.4%), and tetracycline (95.7%), whereas they were resistant to cefotaxime (97.1%) and imipenem (89.9%).ConclusionThe results of this study demonstrate the presence of toxigenic C. difficile isolates such as ribotype 027 (one of the most common causes of C. difficile infection in humans) in chicken carcasses. Although there is no case for stating that C. difficile is a food-borne pathogen, the presence of C. difficile in chicken may be considered to be a potential risk to consumers.