Objective Clostridioides difficile infection（CDI）is a leading cause of healthcare-associated diarrhea. However, the research on CDI in key areas of Gansu province remains limited. This study aims to investigate the epidemiological and molecular characteristics of C. difficile isolated from diarrhea patients in this province, and provide scientific basis for development of targeted prevention and control strategies.

Methods From January to December 2024, a total of 1109 stool samples or anal swabs were collected from diarrhea patients in 6 prefectures (municipality) in Gansu: i.e. Linxia Hui Autonomous Prefecture（Linxia）, Lanzhou, Baiyin, Dingxi, Jinchang, and Jiuquan. The samples were tested for C. difficile by nucleic acid detection, followed by anaerobic culture for strain isolation. All the isolates underwent whole-genome sequencing. Sequence types (STs) were determined by using PubMLST database. Phylogenetic relationships based on core genome multilocus sequence typing (cgMLST) and the carriages of drug resistance genes were analyzed.

Results The overall nucleic acid positive rate was 9.20% (102/1109). Linxia had the highest positive rate (16.81%, 38/226), followed by Lanzhou (11.48%, 14/122). Anaerobic culture yielded 28 C. difficile isolates, with an isolation rate of 27.45% (28/102). Sequencing revealed that 82.14% (23/28) of the isolates were toxigenic, with three strains carrying binary toxin genes (cdtA⁺/cdtB⁺). A total of 15 STs were identified, predominated by ST3 (n=6), ST5 (n=3), ST42 (n=3), and ST54 (n=3). the strains from Jiuquan showed the highest ST diversity. The drug resistance gene analysis showed that 50.00% (14/28) of the strains carried genes conferring resistance to three or more classes of antibiotics, indicating a multidrug resistance potential. The phylogenetic analysis based on cgMLST revealed that some strains isolated in Gansu had close genetic relationships with strains from other provinces in China, suggesting possible cross-provincial transmission.

Conclusion The incidence of C. difficile infection varied in Gansu. It is necessary to pay close attention to the high infection incidence in Linxia and the transmission risk associated with the predominant genotypes in Jiuquan. The genomic data obtained from these 28 isolates established a foundation for a provincial MLST database. Future studies should integrate the data from drug susceptibility testing and clinical practice to further improve local prevention and control of C. difficile infection.