Skip to content
1887

Journal of Medical Microbiology logo Journal of Medical Microbiology

Volume 74, Issue 1

Modified multiplex PCR for serotyping and pathotyping of No Access

Abstract

is a zoonotic pathogen that causes invasive infections in humans who have been in close contact with infected pigs or contaminated pork-derived products. There is currently no consensus on the universal virulence factors or markers that can differentiate pathogenic from non-pathogenic or commensal isolates.

A diagnostic tool for serotyping and pathotyping of is required for active public health surveillance and the One-Health approach.

To improve the former multiplex PCR to serotyping all 29 recognized ‘true’ serotypes and distinguish pathogenic pathotypes using primers targeting the capsule and pathogenic marker genes.

Four sets of multiplex PCRs were modified and improved to detect all 29 recognized serotypes of and distinguish their pathogenic pathotypes using the gene.

This multiplex PCR allowed for the simultaneous amplification of -specific, serotype-specific and pathogenic pathotypes from the DNA of each serotype in each reaction. The accuracy, sensitivity, specificity, positive predictive value and negative predictive value of the pathogenic marker genes were 84.7% (625/738), 96.4% (423/439), 67.6% (202/299), 81.4% (423/520) and 92.7% (202/218), respectively. There was a significant (-value <0.001), high positive likelihood ratio [2.9 with 2.5–3.5 of 95% confidence interval (CI)] and a significant odds ratio (55.1 with 31.6–95.9 of 95 % CI), which indicated that the gene could be used as the pathogenic pathotype marker. No cross-reactions were observed with other bacterial species.

This modified multiplex PCR was able to distinguish 29 well-known serotypes and predicted the pathogenic pathotypes of isolates from humans and pigs in a single assay. It is useful for One-Health surveillance of human and pig isolates of .

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): pathotype , PCR , serotype and Streptococcus suis
Funding
This study was supported by the:
  • Agricultural Research Development Agency (Public Organization) (Award PRP6705030040)
    • Principle Award Recipient: SuphachaiNuanualsuwan
  • Kasetsart University Research and Development Institute (Award FF(KU)26.67)
    • Principle Award Recipient: AnusakKerdsin
© 2025 The Authors
Loading

Article metrics loading...

/content/journal/jmm/10.1099/jmm.0.001950
2025-01-07
2025-01-13
Loading full text...

Full text loading...

References

  1. Gottschalk M, Xu J, Calzas C, Segura M. Streptococcus suis: a new emerging or an old neglected zoonotic pathogen?. Future Microbiol 2010; 5:371–391 [View Article] [PubMed]
     [Google Scholar]
  2. Kerdsin A, Segura M, Fittipaldi N, Gottschalk M. Sociocultural factors influencing human Streptococcus suis disease in Southeast Asia. Foods 2022; 11:1190 [View Article] [PubMed]
     [Google Scholar]
  3. Lunha K, Chumpol W, Jiemsup S, Yongkiettrakul S, Li J et al. Serotype distribution and pathotypic characteristics of Streptococcus suis isolates from slaughtered pigs in a high‐density pig farming Area in Thailand. Trans Emerg Dis 20243186518 [View Article]
     [Google Scholar]
  4. King SJ, Leigh JA, Heath PJ, Luque I, Tarradas C et al. Development of a multilocus sequence typing scheme for the pig pathogen Streptococcus suis: identification of virulent clones and potential capsular serotype exchange. J Clin Microbiol 2002; 40:3671–3680 [View Article] [PubMed]
     [Google Scholar]
  5. Silva LMG, Baums CG, Rehm T, Wisselink HJ, Goethe R et al. Virulence-associated gene profiling of Streptococcus suis isolates by PCR. Vet Microbiol 2006; 115:117–127 [View Article] [PubMed]
     [Google Scholar]
  6. Chen C, Zhang W, Zheng H, Lan R, Wang H et al. Minimum core genome sequence typing of bacterial pathogens: a unified approach for clinical and public health microbiology. J Clin Microbiol 2013; 51:2582–2591 [View Article] [PubMed]
     [Google Scholar]
  7. Estrada AA, Gottschalk M, Rendahl A, Rossow S, Marshall-Lund L et al. Proposed virulence-associated genes of Streptococcus suis isolates from the United States serve as predictors of pathogenicity. Porcine Health Manag 2021; 7:22 [View Article] [PubMed]
     [Google Scholar]
  8. Estrada AA, Gottschalk M, Gebhart CJ, Marthaler DG. Comparative analysis of Streptococcus suis genomes identifies novel candidate virulence-associated genes in North American isolates. Vet Res 2022; 53:23 [View Article] [PubMed]
     [Google Scholar]
  9. Dong X, Chao Y, Zhou Y, Zhou R, Zhang W et al. The global emergence of a novel Streptococcus suis clade associated with human infections. EMBO Mol Med 2021; 13:e13810 [View Article] [PubMed]
     [Google Scholar]
  10. Wileman TM, Weinert LA, Howell KJ, Wang J, Peters SE et al. Pathotyping the zoonotic pathogen Streptococcus suis: novel genetic markers to differentiate invasive disease-associated isolates from non-disease-associated isolates from England and Wales. J Clin Microbiol 2019; 57:e01712-18 [View Article] [PubMed]
     [Google Scholar]
  11. Murray GGR, Hossain ASMM, Miller EL, Bruchmann S, Balmer AJ et al. The emergence and diversification of a zoonotic pathogen from within the microbiota of intensively farmed pigs. Proc Natl Acad Sci U S A 2023; 120:e2307773120 [View Article] [PubMed]
     [Google Scholar]
  12. Kerdsin A, Akeda Y, Hatrongjit R, Detchawna U, Sekizaki T et al. Streptococcus suis serotyping by a new multiplex PCR. J Med Microbiol 2014; 63:824–830 [View Article] [PubMed]
     [Google Scholar]
  13. Okura M, Osaki M, Nomoto R, Arai S, Osawa R et al. Current taxonomical situation of Streptococcus suis. Pathogens 2016; 5:45 [View Article] [PubMed]
     [Google Scholar]
  14. Tohya M, Arai S, Tomida J, Watanabe T, Kawamura Y et al. Defining the taxonomic status of Streptococcus suis serotype 33: the proposal for Streptococcus ruminantium sp. nov. Int J Syst Evol Microbiol 2017; 67:3660–3665 [View Article] [PubMed]
     [Google Scholar]
  15. Lacouture S, Okura M, Takamatsu D, Corsaut L, Gottschalk M. Development of a mismatch amplification mutation assay to correctly serotype isolates of Streptococcus suis serotypes 1, 2, 1/2, and 14. J Vet Diagn Invest 2020; 32:490–494 [View Article] [PubMed]
     [Google Scholar]
  16. Okuhama-Yoshida E, Nakayama M, Hattori M, Takamatsu D, Okura M. Improvement of the mismatch amplification mutation assay-PCR for discrimination between Streptococcus suis serotypes 2 and 1/2. J Microbiol Methods 2023; 214:106828 [View Article] [PubMed]
     [Google Scholar]
  17. Matiasovic J, Zouharova M, Nedbalcova K, Kralova N, Matiaskova K et al. Resolution of Streptococcus suis serotypes 1/2 versus 2 and 1 versus 14 by PCR-restriction fragment length polymorphism method. J Clin Microbiol 2020; 58:e00480-20 [View Article] [PubMed]
     [Google Scholar]
  18. Sint D, Raso L, Traugott M. Advances in multiplex PCR: balancing primer efficiencies and improving detection success. Methods Ecol Evol 2012; 3:898–905 [View Article] [PubMed]
     [Google Scholar]
  19. Goyette-Desjardins G, Auger JP, Xu J, Segura M, Gottschalk M. Streptococcus suis, an important pig pathogen and emerging zoonotic agent-an update on the worldwide distribution based on serotyping and sequence typing. Emerg Microbes Infect 2014; 3:e45 [View Article] [PubMed]
     [Google Scholar]
  20. Kerdsin A, Takeuchi D, Nuangmek A, Akeda Y, Gottschalk M et al. Genotypic comparison between Streptococcus suis isolated from pigs and humans in Thailand. Pathogens 2020; 9:50 [View Article] [PubMed]
     [Google Scholar]
  21. Gottschalk M. Streptococcus suis infection in pigs. MSD Veterinay Manua. n.d https://www.msdvetmanual.com/generalized-conditions/streptococcal-infections-in-pigs/streptococcus-suis-infection-in-pigs) accessed 7 November 2024
  22. Estrada AA, Gottschalk M, Rossow S, Rendahl A, Gebhart C et al. Serotype and genotype (multilocus sequence type) of Streptococcus suis isolates from the United States serve as predictors of pathotype. J Clin Microbiol 2019; 57:e00377-19 [View Article] [PubMed]
     [Google Scholar]
  23. Hatrongjit R, Fittipaldi N, Gottschalk M, Kerdsin A. Genomic epidemiology in Streptococcus suis: Moving beyond traditional typing techniques. Heliyon 2024; 10:e27818 [View Article] [PubMed]
     [Google Scholar]
  24. Liu Z, Zheng H, Gottschalk M, Bai X, Lan R et al. Development of multiplex PCR assays for the identification of the 33 serotypes of Streptococcus suis. PLoS One 2013; 8:e72070 [View Article] [PubMed]
     [Google Scholar]
  25. Pan Z, Ma J, Dong W, Song W, Wang K et al. Novel variant serotype of Streptococcus suis isolated from piglets with meningitis. Appl Environ Microbiol 2015; 81:976–985 [View Article] [PubMed]
     [Google Scholar]
  26. Huang J, Liu X, Chen H, Chen L, Gao X et al. Identification of six novel capsular polysaccharide loci (NCL) from Streptococcus suis multidrug resistant non-typeable strains and the pathogenic characteristic of strains carrying new NCLs. Transbound Emerg Dis 2019; 66:995–1003 [View Article] [PubMed]
     [Google Scholar]
/content/journal/jmm/10.1099/jmm.0.001950
Loading
Modified multiplex PCR for serotyping and pathotyping of Streptococcus suis
J. Med. Microbiol. 74, 001950 (2025); https://doi.org/10.1099/jmm.0.001950
/content/journal/jmm/10.1099/jmm.0.001950
/content/journal/jmm/10.1099/jmm.0.001950
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF

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