Comparative genomic analysis of Clostridioides difficile isolates from symptomatic and asymptomatic paediatric patients

<i>Clostridioides difficile</i> infection (CDI) imposes a substantial clinical burden in paediatric populations. However, the high prevalence of asymptomatic colonization, especially in children under 2 years of age, complicates the distinction between true infection and non-pathogenic carriage. This diagnostic uncertainty hinders appropriate treatment decisions and complicates infection prevention efforts. A 6-year retrospective cohort study was performed at Shanghai Children's Hospital to characterize the epidemiology and clinical features of CDI and asymptomatic colonization in paediatric patients. Stool specimens were cultured for <i>C. difficile</i>, and isolates underwent whole-genome sequencing to perform multilocus sequence typing, identify SNPs and characterize functional gene content via Clusters of Orthologous Groups (COG) analysis. Mutations in genes associated with toxin production were analysed to assess genetic differences between clinical isolates from infected patients and asymptomatic carriers. In addition, comparative genomic analysis was performed to assess variations in virulence-associated genes, antimicrobial resistance (AMR) genes and genes involved in quorum sensing (QS). Colonization factors (CFs) were also characterized to elucidate potential mechanisms differentiating asymptomatic colonization from symptomatic infection. And we conducted <i>in vitro</i> experiments on toxin B-variant strains. A total of 23 sequence types (STs) were identified among isolates from 39 asymptomatic carriers and 61 symptomatic patients, with greater ST diversity observed in the infection group compared to the colonization group. COG analysis demonstrated an increased representation of uncharacterized functional categories in the infection group, suggesting a potential role for novel genes in pathogenesis. Patterns of virulence factor presence, AMR genes and QS gene distribution were comparable between the two groups, as were mutations in toxin regulation genes. Notably, six isolates belonging to ST37 and ST81, characterized by the absence of <i>tcdA</i> and presence of <i>tcdB</i>, exhibited a high frequency of mutations. <i>In vitro</i> experiments demonstrated that these strains exhibited higher biofilm formation capacity and elevated transcriptional levels of both <i>tcdB</i> and <i>spoA</i>. Additionally, no significant differences were detected in the distribution of CFs. Our findings contribute to the growing understanding of the genomic determinants and their functional roles underlying paediatric CDI severity, providing more evidence for improved diagnostic and therapeutic strategies.