Little is known about zoonotic pathogens and their antimicrobial resistance in South American camelids (SAC) in Germany including Clostridioides (C.) difficile. The aim of this study was to investigate prevalence, molecular characteristics and antimicrobial resistance of C. difficile in SAC. Composite SAC faecal samples were collected in 43 husbandries in Central Germany and cultured for C. difficile. Toxinotyping and ribotyping was done by PCR. Whole genome sequencing was performed with Illumina^® Miseq™. The genomes were screened for antimicrobial resistance determinants. Genetic relatedness of the isolates was investigated using core genome multi locus sequence typing (cgMLST) and single nucleotide polymorphism analysis. Antimicrobial susceptibility testing was done using the Etest^® method. Eight C. difficile isolates were recovered from seven farms. The isolates belonged to different PCR ribotypes. All isolates were toxinogenic. cgMLST revealed a cluster containing isolates recovered from different farms. Seven isolates showed similar resistance gene patterns. Different phenotypic resistance patterns were found. Agreement between phenotypic and genotypic resistance was identified only in some cases. Consequently, SAC may act as a reservoir for C. difficile. Thus, SAC may pose a risk regarding zoonotic transmission of toxinogenic, potentially human-pathogenic and resistant C. difficile isolates.

Clostridioides difficile has significant clinical importance as a leading cause of healthcare-associated infections, with symptoms ranging from mild diarrhoea to severe colitis, and possible life-threatening complications. C. difficile ribotype (RT) 002, mainly associated with MLST sequence type (ST) 8, is one of the most common RTs found in humans. This study aimed at investigating the genetic characteristics of 537 C. difficile genomes of ST8/RT002. To this end, we sequenced 298 C. difficile strains representing a new European genome collection, with strains from Germany, Denmark, France and Portugal. These sequences were analysed against a global dataset consisting of 1,437 ST8 genomes available through Enterobase. Our results showed close genetic relatedness among the studied ST8 genomes, a diverse array of antimicrobial resistance (AMR) genes and the presence of multiple mobile elements. Notably, the pangenome analysis revealed an open genomic structure. ST8 shows relatively low overall variation. Thus, clonal isolates were found across different One Health sectors (humans, animals, environment and food), time periods, and geographical locations, suggesting the lineage’s stability and a universal environmental source. Importantly, this stability did not hinder the acquisition of AMR genes, emphasizing the adaptability of this bacterium to different selective pressures. Although only 2.4 % (41/1,735) of the studied genomes originated from non-human sources, such as animals, food, or the environment, we identified 9 cross-sectoral core genome multilocus sequence typing (cgMLST) clusters. Our study highlights the importance of ST8 as a prominent lineage of C. difficile with critical implications in the context of One Health. In addition, these findings strongly support the need for continued surveillance and investigation of non-human samples to gain a more comprehensive understanding of the epidemiology of C. difficile.

Background/Objectives: Antimicrobial agents play an important role in the pathogenesis and treatment of Clostridioides (C.) difficile infections. C. difficile isolates have shown different genotypic and phenotypic resistance patterns and could serve as antimicrobial resistance reservoirs. Methods: To gain insight into accordance and potential disagreements between genotypic and phenotypic antimicrobial resistances in C. difficile, we compared the genotypic and phenotypic resistance patterns of 108 bovine C. difficile isolates collected in Germany between 2010 and 2012. These isolates represent a collection of different ribotypes (RT) and originated from different husbandries in Germany. Whole genome sequencing of all isolates was performed with Illumina^® Miseq™, and sequences were screened for antimicrobial resistance determinants. For phenotypic antimicrobial susceptibility testing, the agar dilution procedure according to the CLSI document M11 was used. Minimal inhibitory concentration values were determined for penicillin, meropenem, tetracycline, moxifloxacin, vancomycin, metronidazole, erythromycin and clindamycin. Results: Various phenotypic and genotypic antimicrobial resistances were found in the isolates examined that belonged to different ribotype/sequence type (ST) lineages, even if these originated from the same source and geographical region (bovine isolates from Germany). Agreement between phenotypic and genotypic resistance was seen for most antimicrobial agents tested. A total of 92% (83/90) of the investigated ST11 isolates showed phenotypic resistance or were classified as non-wild type to at least one of the antimicrobials tetracycline, moxifloxacin, erythromycin and clindamycin. Conclusions: The results of this comparison contribute to a better understanding of antimicrobial resistance in C. difficile by relating phenotypic susceptibility patterns to genomic resistance determinants.