Liqing Hu ¹ Shan Lin ^2,3,4 Meng Zhang ^3,4 Mengting Cai ^3,4 Yuhang Shen ^3,4,5 Peng Zeng ^3,6 Xiaojun Song ⁷ Qiao Bian ⁸ Jina Gu ⁹ Yun Luo ¹⁰ Yu Chen ^3,4 Dazhi Jin ^11,3,7*

• ¹ Department of Laboratory Medicine, The First Affiliated Hospital of Ningbo University, Ningbo, China, Ningbo, China
• ² TEDA Institite of Biological Sciences and Biotechnology, Nankai University, Tianjin, China
• ³ Hangzhou Medical College, Hangzhou, China
• ⁴ Key Laboratory of Biomarkers and In Vitro Diagnosis Translation of Zhejiang province, Hangzhou, Jiangsu Province, China
• ⁵ Institute of Ageing Research, School of Basic Medical Sciences, Hangzhou Normal University, Hangzhou, China, Hangzhou, China
• ⁶ Department of Clinical Laboratory, Yuedong Hospital, The Third Affiliated Hospital of Sun Yat-sen University, Meizhou, China, Meizhou, China
• ⁷ Laboratory Medicine Center, Department of Clinical Laboratory, Zhejiang Provincial People’s Hospital, Hangzhou Medical College, Hangzhou, Jiangsu Province, China
• ⁸ Zhejiang Center for Disease Control and Prevention (Zhejiang CDC), Hangzhou, Zhejiang Province, China
• ⁹ Department of Infectious Diseases, Ningbo No.2 Hospital, Ningbo, China, Ningbo, China
• ¹⁰ School of Biotechnology and Biomolecular Sciences, Faculty of Science, University of New South Wales, Sydney, New South Wales, Australia
• ¹¹ School of Laboratory Medicine, Hangzhou Medical College, Hangzhou, Zhejiang Province, China

Background: Nosocomial transmission of Clostridioides difficile infection (CDI) has been documented in Ningbo, China. However, data on molecular characteristics, clonal transmission, and risk factors of CDI in this region remain limited. Methods: A cross-sectional study enrolled hospitalized patients with diarrhea during September to November 2021. Stool samples from all patients were tested for C. difficile, and isolated strains underwent toxin gene identification, genotyping, and antibiotic susceptibility testing. Whole-genome sequencing and epidemiological variables were analyzed in patients infected with C. difficile strains of the same sequence types (STs) to identify nosocomial transmission and risk factors for CDI. Results: Of the 907 investigated patients, 115 (12.7%) had microbiologically proven CDI, as their diarrhea was associated with toxigenic C. difficile strains, which comprised 106 A+B+CDT-, 3 A-B+CDT-, and 6 A+B+CDT+. Predominant genotypes (ST2, ST3, ST35, and ST54) exhibited distinct antibiotic resistance patterns. ST54 strains showed higher resistance to erythromycin (100%) but lower resistance to moxifloxacin (18.2%) and gatifloxacin (18.2%) (χ2 = 10.24 – 16.65, P < 0.05). ST35 strains exhibited higher resistance to ciprofloxacin (66.7%) and tetracycline (33.3%) than other STs (χ2 = 13.30 – 20.19, P < 0.05). Genomic and epidemiological analysis revealed two nosocomial clonal transmission events caused by 5 ST35 strains (with ≤ 2 single nucleotide polymorphism differences), elucidating clonal transmission among different floors and buildings within the hospital. Prolonged hospitalization (> 10 days) (odds ratio [95% confidence interval], 1.76 [1.05 – 2.93]) and penicillin-class antibiotics (1.69 [1.11 – 2.58]) were risk factors for CDI, with the latter being an independent risk factor (1.57 [1.02 – 2.42]). For C. difficile ST35 infection, intensive care unit (12.00 [2.77 – 52.05]) and neurology departments (8.08 [1.46 – 44.65]) admissions were risk factors, with the latter as an independent risk factor (1.56 [1.01 – 2.40]). Conclusions: Multiple C. difficile genotypes with varied antibiotic resistance patterns circulated in Ningbo, with ST35 causing nosocomial clonal transmission among different floors and buildings within the hospital. These findings and the identified risk factors necessitate enhanced surveillance and infection control in the region.