Skip to content
1887

Microbial Genomics logo Microbial Genomics

Volume 12, Issue 2

Comparative genomic analysis of isolates from symptomatic and asymptomatic paediatric patients Open Access

Abstract

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 , 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 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 and presence of , exhibited a high frequency of mutations. experiments demonstrated that these strains exhibited higher biofilm formation capacity and elevated transcriptional levels of both and . 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.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): asymptomatic carrier , children , Clostridioides difficile , symptomatic infection and whole-genome sequencing
Funding
This study was supported by the:
  • National Natural Science Foundation of China (Award 82470572)
    • Principal Award Recipient: TingZhang
© 2026 The Authors
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License.
Loading

Article metrics loading...

/content/journal/mgen/10.1099/mgen.0.001610
2026-02-02
2026-02-15

Metrics

Loading full text...

Full text loading...

/deliver/fulltext/mgen/12/2/mgen001610.html?itemId=/content/journal/mgen/10.1099/mgen.0.001610&mimeType=html&fmt=ahah

References

  1. Hall IC, O’Tooleo E. Intestinal flora in new-born infants: with a description of a new pathogenic anaerobe, bacillus difficilis. Am J Dis Child 1935; 49:390–402 [View Article]
     [Google Scholar]
  2. Yang J, Rui W, Zhong S, Li X, Liu W et al. Symbiotic biofilms formed by Clostridioides difficile and Bacteroides thetaiotaomicron in the presence of vancomycin. Gut Microbes 2024; 16:2390133 [View Article] [PubMed]
     [Google Scholar]
  3. Kordus SL, Thomas AK, Lacy DB. Clostridioides difficile toxins: mechanisms of action and antitoxin therapeutics. Nat Rev Microbiol 2022; 20:285–298 [View Article] [PubMed]
     [Google Scholar]
  4. Di Bella S, Sanson G, Monticelli J, Zerbato V, Principe L et al. Clostridioides difficile infection: history, epidemiology, risk factors, prevention, clinical manifestations, treatment, and future options. Clin Microbiol Rev 2024; 37:e0013523 [View Article] [PubMed]
     [Google Scholar]
  5. Wang L, Chen X, Pollock NR, Villafuerte Gálvez JA, Alonso CD et al. Metagenomic analysis reveals distinct patterns of gut microbiota features with diversified functions in C. difficile infection (CDI), asymptomatic carriage and non-CDI diarrhea. Gut Microbes 2025; 17:2505269 [View Article]
     [Google Scholar]
  6. Tougas SR, Lodha N, Vandermeer B, Lorenzetti DL, Tarr PI et al. Prevalence of detection of Clostridioides difficile among asymptomatic children: a systematic review and meta-analysis. JAMA Pediatr 2021; 175:e212328 [View Article] [PubMed]
     [Google Scholar]
  7. Jangi S, Lamont JT. Asymptomatic colonization by Clostridium difficile in infants: implications for disease in later life. J Pediatr Gastroenterol Nutr 2010; 51:2–7 [View Article] [PubMed]
     [Google Scholar]
  8. Enoch DA, Butler MJ, Pai S, Aliyu SH, Karas JA. Clostridium difficile in children: colonisation and disease. J Infect 2011; 63:105–113 [View Article] [PubMed]
     [Google Scholar]
  9. McFarland LV, Ozen M, Dinleyici EC, Goh S. Comparison of pediatric and adult antibiotic-associated diarrhea and Clostridium difficile infections. World J Gastroenterol 2016; 22:3078–3104 [View Article] [PubMed]
     [Google Scholar]
  10. van Dorp SM, Smajlović E, Knetsch CW, Notermans DW, de Greeff SC et al. Clinical and microbiological characteristics of Clostridium difficile infection among hospitalized children in the Netherlands. Clin Infect Dis 2017; 64:192–198 [View Article] [PubMed]
     [Google Scholar]
  11. Khanna S, Baddour LM, Huskins WC, Kammer PP, Faubion WA et al. The epidemiology of Clostridium difficile infection in children: a population-based study. Clin Infect Dis 2013; 56:1401–1406 [View Article] [PubMed]
     [Google Scholar]
  12. Knight DR, Elliott B, Chang BJ, Perkins TT, Riley TV. Diversity and evolution in the genome of Clostridium difficile. Clin Microbiol Rev 2015; 28:721–741 [View Article] [PubMed]
     [Google Scholar]
  13. McDonald LC, Gerding DN, Johnson S, Bakken JS, Carroll KC et al. Clinical practice guidelines for Clostridium difficile infection in adults and children: 2017 update by the Infectious Diseases Society of America (IDSA) and Society for Healthcare Epidemiology of America (SHEA). Clin Infect Dis 2018; 66:e1–e48 [View Article] [PubMed]
     [Google Scholar]
  14. Li X, Wang Y, Cao R, Xiao F, Wang X et al. Antimicrobial resistance of Clostridioides difficile in children from a tertiary pediatric hospital in Shanghai, China. Infect Drug Resist 2024; 17:329–339
     [Google Scholar]
  15. Jolley KA, Bray JE, Maiden MCJ. Open-access bacterial population genomics: BIGSdb software, the PubMLST.org website and their applications. Wellcome Open Res 2018; 3:124 [View Article] [PubMed]
     [Google Scholar]
  16. Gardner SN, Slezak T, Hall BG. kSNP3.0: SNP detection and phylogenetic analysis of genomes without genome alignment or reference genome. Bioinformatics 2015; 31:2877–2878 [View Article] [PubMed]
     [Google Scholar]
  17. Zhou Y, Zhou W, Xiao T, Chen Y, Lv T et al. Comparative genomic and transmission analysis of Clostridioides difficile between environmental, animal, and clinical sources in China. Emerg Microbes Infect 2021; 10:2244–2255 [View Article] [PubMed]
     [Google Scholar]
  18. Emms DM, Kelly S. OrthoFinder: phylogenetic orthology inference for comparative genomics. Genome Biol 2019; 20:238 [View Article] [PubMed]
     [Google Scholar]
  19. Sullivan MJ, Petty NK, Beatson SA. Easyfig: a genome comparison visualizer. Bioinformatics 2011; 27:1009–1010 [View Article] [PubMed]
     [Google Scholar]
  20. Ahmed UKB, Ballard JD. Autoinducing peptide-based quorum signaling systems in Clostridioides difficile. Curr Opin Microbiol 2022; 65:81–86 [View Article] [PubMed]
     [Google Scholar]
  21. Okada Y, Okugawa S, Ikeda M, Kobayashi T, Saito R et al. Genetic diversity and epidemiology of accessory gene regulator loci in Clostridioides difficile. Access Microbiol 2020; 2:acmi000134 [View Article] [PubMed]
     [Google Scholar]
  22. Chen L, Yang J, Yu J, Yao Z, Sun L et al. VFDB: a reference database for bacterial virulence factors. Nucleic Acid Res 2005; 33:D325–8 [View Article] [PubMed]
     [Google Scholar]
  23. Alcock BP, Raphenya AR, Lau TTY, Tsang KK, Bouchard M et al. CARD 2020: antibiotic resistome surveillance with the comprehensive antibiotic resistance database. Nucleic Acid Res 2020; 48:D517–D525 [View Article] [PubMed]
     [Google Scholar]
  24. Buchfink B, Xie C, Huson DH. Fast and sensitive protein alignment using DIAMOND. Nat Methods 2015; 12:59–60 [View Article] [PubMed]
     [Google Scholar]
  25. Liu M, Blattman SB, Takahashi M, Mandayam N, Jiang W et al. Conserved genetic basis for microbial colonization of the gut. Cell 2025; 188:2505–2520 [View Article] [PubMed]
     [Google Scholar]
  26. Ouyang Z, Yang J, Zhang H, Zhao M, Yang H et al. Differences in virulence and drug resistance between Clostridioides difficile ST37 and ST1 isolates. Virulence 2025; 16:2502554 [View Article] [PubMed]
     [Google Scholar]
  27. Xu X, Luo Y, Chen H, Song X, Bian Q et al. Genomic evolution and virulence association of Clostridioides difficile sequence type 37 (ribotype 017) in China. Emerg Microbes Infect 2021; 10:1331–1345 [View Article] [PubMed]
     [Google Scholar]
  28. Gu W, Wang W, Li W, Li N, Wang Y et al. New ribotype Clostridioides difficile from ST11 group revealed higher pathogenic ability than RT078. Emerg Microbes Infect 2021; 10:687–699 [View Article] [PubMed]
     [Google Scholar]
  29. Ahmed UKB, Shadid TM, Larabee JL, Ballard JD. Combined and distinct roles of agr proteins in Clostridioides difficile 630 sporulation, motility, and toxin production. mBio 2020; 11:e03190-20 [View Article] [PubMed]
     [Google Scholar]
  30. Shirley DA, Tornel W, Warren CA, Moonah S. Clostridioides difficile infection in children: recent updates on epidemiology, diagnosis, therapy. Pediatrics 2023; 152:e2023062307 [View Article] [PubMed]
     [Google Scholar]
  31. Shuai H, Bian Q, Luo Y, Zhou X, Song X et al. Molecular characteristics of Clostridium difficile in children with acute gastroenteritis from Zhejiang. BMC Infect Dis 2020; 20:343 [View Article] [PubMed]
     [Google Scholar]
  32. Leibowitz J, Soma VL, Rosen L, Ginocchio CC, Rubin LG. Similar proportions of stool specimens from hospitalized children with and without diarrhea test positive for Clostridium difficile. Pediatr Infect Dis J 2015; 34:261–266 [View Article] [PubMed]
     [Google Scholar]
  33. Al-Rawahi GN, Al-Najjar A, McDonald R, Deyell RJ, Golding GR et al. Pediatric oncology and stem cell transplant patients with healthcare-associated Clostridium difficile infection were already colonized on admission. Pediatr Blood Cancer 2019; 66:e27604 [View Article] [PubMed]
     [Google Scholar]
  34. De-la-Rosa-Martínez D, Villaseñor-Echavarri R, Vilar-Compte D, Mosqueda-Larrauri V, Zinser-Peniche P et al. Heterogeneity of Clostridioides difficile asymptomatic colonization prevalence: a systematic review and meta-analysis. Gut Pathog 2025; 17:6 [View Article] [PubMed]
     [Google Scholar]
  35. Baron SW, Ostrowsky BE, Nori P, Drory DY, Levi MH et al. Screening of Clostridioides difficile carriers in an urban academic medical center: understanding implications of disease. Infect Control Hosp Epidemiol 2020; 41:149–153 [View Article] [PubMed]
     [Google Scholar]
  36. Miles-Jay A, Snitkin ES, Lin MY, Shimasaki T, Schoeny M et al. Longitudinal genomic surveillance of carriage and transmission of Clostridioides difficile in an intensive care unit. Nat Med 2023; 29:2526–2534 [View Article] [PubMed]
     [Google Scholar]
  37. Reasoner SA, Zhang LS, Bernard R, Edwards KM, Nicholson MR. Prevalence and sequelae of asymptomatic Clostridioides difficile colonization in children with inflammatory bowel disease. J Pediatr Gastroenterol Nutr 2025; 80:446–449 [View Article] [PubMed]
     [Google Scholar]
  38. Mani J, Levy S, Angelova A, Hazrati S, Fassnacht R et al. Epidemiological and microbiome associations of Clostridioides difficile carriage in infancy and early childhood. Gut Microbes 2023; 15:2203969 [View Article] [PubMed]
     [Google Scholar]
  39. Liao F, Li W, Gu W, Zhang W, Liu X et al. A retrospective study of community-acquired Clostridium difficile infection in southwest China. Sci Rep 2018; 8:3992 [View Article] [PubMed]
     [Google Scholar]
  40. Su T, Chen W, Wang D, Cui Y, Ni Q et al. Complete genome sequencing and comparative phenotypic analysis reveal the discrepancy between Clostridioides difficile ST81 and ST37 isolates. Front Microbiol 2021; 12:776892 [View Article] [PubMed]
     [Google Scholar]
  41. Maslanka JR, Gu CH, Zarin I, Denny JE, Broadaway S et al. Detection and elimination of a novel non-toxigenic Clostridioides difficile strain from the microbiota of a mouse colony. Gut Microbes 2020; 12:1–15 [View Article] [PubMed]
     [Google Scholar]
  42. Wilson KH, Sheagren JN. Antagonism of toxigenic Clostridium difficile by nontoxigenic C. difficile. J Infect Dis 1983; 147:733–736 [View Article] [PubMed]
     [Google Scholar]
  43. Slater RT, Frost LR, Jossi SE, Millard AD, Unnikrishnan M. Clostridioides difficile LuxS mediates inter-bacterial interactions within biofilms. Sci Rep 2019; 9:9903 [View Article] [PubMed]
     [Google Scholar]
  44. Goorhuis A, Debast SB, Dutilh JC, van Kinschot CM, Harmanus C et al. Type-specific risk factors and outcome in an outbreak with 2 different Clostridium difficile types simultaneously in 1 hospital. Clin Infect Dis 2011; 53:860–869 [View Article] [PubMed]
     [Google Scholar]
  45. Marshall A, McGrath JW, Graham R, McMullan G. Food for thought-The link between Clostridioides difficile metabolism and pathogenesis. PLoS Pathog 2023; 19:e1011034 [View Article] [PubMed]
     [Google Scholar]
  46. Kumar N, Browne HP, Viciani E, Forster SC, Clare S et al. Adaptation of host transmission cycle during Clostridium difficile speciation. Nat Genet 2019; 51:1315–1320 [View Article] [PubMed]
     [Google Scholar]
/content/journal/mgen/10.1099/mgen.0.001610
Loading
Comparative genomic analysis of Clostridioides difficile isolates from symptomatic and asymptomatic paediatric patients
M Gen 12, 001610 (2026); https://doi.org/10.1099/mgen.0.001610
/content/journal/mgen/10.1099/mgen.0.001610
/content/journal/mgen/10.1099/mgen.0.001610
Loading

Data & Media loading...

Supplements

Supplementary material 1

EXCEL

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An error occurred
Approval was partially successful, following selected items could not be processed due to error