Multiplex PCR to determine Streptococcus pneumoniae serotypes causing otitis media in the Republic of Ireland with further characterisation of antimicrobial susceptibilities and genotypes

  • I. VickersEmail author
  • D. O’Flanagan
  • M. Cafferkey
  • H. Humphreys


The purpose of this study was to determine the serotypes, genotypes and antimicrobial susceptibilities of Streptococcus pneumoniae causing otitis media (OM) in children in Dublin, Ireland. S. pneumoniae isolates (n = 28) from spontaneously discharging OM were studied. Serotyping was performed using a previously undescribed multiplex polymerase chain reaction (PCR) scheme in combination with serological methods. Multilocus sequence typing (MLST) was performed using standard procedures. Antimicrobial susceptibility testing was performed using the Etest method. Fourteen different S. pneumoniae serotypes were identified. The five most common serotypes were 3, 19F, 19A, 14 and 6A, which accounted for 68% of all infections. The 7-valent pneumococcal conjugate vaccine (PCV7), 10-valent pneumococcal conjugate vaccine (PHiD-CV) and 13-valent pneumococcal conjugate vaccine (PCV13) provided potential coverages of 43%, 46% and 86%, respectively. Reduced susceptibility to penicillin was evident for 25% of isolates and was associated with serotypes 14, 19A, 19F and 9V. A total of 21 different sequence types (STs) were identified. Pneumococcal Molecular Epidemiology Network (PMEN) clones or their variants represented 54% (15/28) of all isolates. Continued monitoring and characterisation of S. pneumoniae causing OM in Ireland is warranted in order to guide future vaccine and treatment policies.


Otitis Medium Multiplex Polymerase Chain Reaction Tigecycline Pneumococcal Conjugate Vaccine Penicillin Resistance 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



We are grateful to the staff of the Children’s University Hospital, Temple St., Dublin, for their assistance with the collection of the isolates used in this study. We also wish to thank the Health Protection Surveillance Centre, which financially supports the laboratory surveillance of pneumococcal disease in Ireland. We acknowledge the use of the pneumococcal MLST database, which is located at Imperial College London and funded by the Wellcome Trust.


  1. 1.
    Abdelnour A, Soley C, Guevara S, Porat N, Dagan R, Arguedas A (2009) Streptococcus pneumoniae serotype 3 among Costa Rican children with otitis media: clinical, epidemiological characteristics and antimicrobial resistance patterns. BMC Pediatr 9:52CrossRefPubMedGoogle Scholar
  2. 2.
    Zhou F, Shefer A, Kong Y, Nuorti JP (2008) Trends in acute otitis media-related health care utilization by privately insured young children in the United States, 1997–2004. Pediatrics 121:253–260CrossRefPubMedGoogle Scholar
  3. 3.
    Fletcher MA, Fritzell B (2007) Brief review of the clinical effectiveness of PREVENAR against otitis media. Vaccine 25:2507–2512CrossRefPubMedGoogle Scholar
  4. 4.
    O’Halloran DM, Cafferkey MT (2005) Multiplex PCR for identification of seven Streptococcus pneumoniae serotypes targeted by a 7-valent conjugate vaccine. J Clin Microbiol 43:3487–3490CrossRefPubMedGoogle Scholar
  5. 5.
    Bennett D, Lennon B, Humphreys H, Cafferkey M (2003) Penicillin susceptibility and epidemiological typing of invasive pneumococcal isolates in the Republic of Ireland. J Clin Microbiol 41:3641–3648CrossRefPubMedGoogle Scholar
  6. 6.
    Llull D, López R, García E (2006) Characteristic signatures of the lytA gene provide a basis for rapid and reliable diagnosis of Streptococcus pneumoniae infections. J Clin Microbiol 44:1250–1256CrossRefPubMedGoogle Scholar
  7. 7.
    Obregón V, García P, García E, Fenoll A, López R, García JL (2002) Molecular peculiarities of the lytA gene isolated from clinical pneumococcal strains that are bile insoluble. J Clin Microbiol 40:2545–2554CrossRefPubMedGoogle Scholar
  8. 8.
    Whatmore AM, Efstratiou A, Pickerill AP, Broughton K, Woodard G, Sturgeon D et al (2000) Genetic relationships between clinical isolates of Streptococcus pneumoniae, Streptococcus oralis, and Streptococcus mitis: characterization of “Atypical” pneumococci and organisms allied to S. mitis harboring S. pneumoniae virulence factor-encoding genes. Infect Immun 68:1374–1382CrossRefPubMedGoogle Scholar
  9. 9.
    Brito DA, Ramirez M, de Lencastre H (2003) Serotyping Streptococcus pneumoniae by multiplex PCR. J Clin Microbiol 41:2378–2384CrossRefPubMedGoogle Scholar
  10. 10.
    Lawrence ER, Griffiths DB, Martin SA, George RC, Hall LM (2003) Evaluation of semiautomated multiplex PCR assay for determination of Streptococcus pneumoniae serotypes and serogroups. J Clin Microbiol 41:601–607CrossRefPubMedGoogle Scholar
  11. 11.
    Pai R, Gertz RE, Beall B (2006) Sequential multiplex PCR approach for determining capsular serotypes of Streptococcus pneumoniae isolates. J Clin Microbiol 44:124–131CrossRefPubMedGoogle Scholar
  12. 12.
    Smart LE, Dougall AJ, Girdwood RW (1989) Pneumococcal serotyping. J Infect 18:296–298CrossRefPubMedGoogle Scholar
  13. 13.
    Enright MC, Spratt BG (1998) A multilocus sequence typing scheme for Streptococcus pneumoniae: identification of clones associated with serious invasive disease. Microbiology 144:3049–3060CrossRefPubMedGoogle Scholar
  14. 14.
    Clinical and Laboratory Standards Institute (CLSI) (2008) Performance standards for antimicrobial susceptibility testing; eighteenth informational supplement. M100-S18. CLSI, Wayne, PAGoogle Scholar
  15. 15.
    Yu J, Carvalho Mda G, Beall B, Nahm MH (2008) A rapid pneumococcal serotyping system based on monoclonal antibodies and PCR. J Med Microbiol 57:171–178CrossRefPubMedGoogle Scholar
  16. 16.
    Park IH, Pritchard DG, Cartee R, Brandao A, Brandileone MC, Nahm MH (2007) Discovery of a new capsular serotype (6C) within serogroup 6 of Streptococcus pneumoniae. J Clin Microbiol 45:1225–1233CrossRefPubMedGoogle Scholar
  17. 17.
    Dagan R, Sikuler-Cohen M, Zamir O, Janco J, Givon-Lavi N, Fraser D (2001) Effect of a conjugate pneumococcal vaccine on the occurrence of respiratory infections and antibiotic use in day-care center attendees. Pediatr Infect Dis J 20:951–958CrossRefPubMedGoogle Scholar
  18. 18.
    del Castillo F, Baquero-Artigao F, García-Perea A (1998) Influence of recent antibiotic therapy on antimicrobial resistance of Streptococcus pneumoniae in children with acute otitis media in Spain. Pediatr Infect Dis J 17:94–97CrossRefPubMedGoogle Scholar
  19. 19.
    de Neeling AJ, Overbeek BP, Horrevorts AM, Ligtvoet EE, Goettsch WG (2001) Antibiotic use and resistance of Streptococcus pneumoniae in The Netherlands during the period 1994–1999. J Antimicrob Chemother 48:441–444CrossRefPubMedGoogle Scholar
  20. 20.
    Goossens H, Ferech M, Vander Stichele R, Elseviers M; ESAC Project Group (2005) Outpatient antibiotic use in Europe and association with resistance: a cross-national database study. Lancet 365:579–587PubMedGoogle Scholar
  21. 21.
    Rodgers GL, Arguedas A, Cohen R, Dagan R (2009) Global serotype distribution among Streptococcus pneumoniae isolates causing otitis media in children: potential implications for pneumococcal conjugate vaccines. Vaccine 27:3802–3810CrossRefPubMedGoogle Scholar
  22. 22.
    Porat N, Soley C, Marengolciene MM, Greenberg D, Givon-Lavi N, Trefler R et al (2008) An international serotype 3 clone causing pediatric noninvasive infections in Israel, Costa Rica, and Lithuania. Pediatr Infect Dis J 27:709–712CrossRefPubMedGoogle Scholar
  23. 23.
    Poolman J, Kriz P, Feron C, Di-Paolo E, Henckaerts I, Miseur A et al (2009) Pneumococcal serotype 3 otitis media, limited effect of polysaccharide conjugate immunisation and strain characteristics. Vaccine 27:3213–3222CrossRefPubMedGoogle Scholar
  24. 24.
    Pichichero ME, Casey JR (2007) Emergence of a multiresistant serotype 19A pneumococcal strain not included in the 7-valent conjugate vaccine as an otopathogen in children. JAMA 298:1772–1778CrossRefPubMedGoogle Scholar
  25. 25.
    Cohen R, Levy C, Bonnet E, Grondin S, Desvignes V, Lecuyer A et al (2010) Dynamic of pneumococcal nasopharyngeal carriage in children with acute otitis media following PCV7 introduction in France. Vaccine 28:6114–6121. doi: 10.1016/j.vaccine.2009.05.037 CrossRefPubMedGoogle Scholar
  26. 26.
    Fenoll A, Martín Bourgon C, Muñóz R, Vicioso D, Casal J (1991) Serotype distribution and antimicrobial resistance of Streptococcus pneumoniae isolates causing systemic infections in Spain, 1979–1989. Rev Infect Dis 13:56–60PubMedGoogle Scholar
  27. 27.
    Lefèvre JC, Bertrand MA, Faucon G (1995) Molecular analysis by pulsed-field gel electrophoresis of penicillin-resistant Streptococcus pneumoniae from Toulouse, France. Eur J Clin Microbiol Infect Dis 14:491–497CrossRefPubMedGoogle Scholar
  28. 28.
    Reinert RR, van der Linden M, Seegmüller I, Al-Lahham A, Siedler A, Weissmann B et al (2007) Molecular epidemiology of penicillin-non-susceptible Streptococcus pneumoniae isolates from children with invasive pneumococcal disease in Germany. Clin Microbiol Infect 13:363–368CrossRefPubMedGoogle Scholar
  29. 29.
    Lambertsen L, Brendstrup M, Friis H, Christensen JJ (2010) Molecular characterization of invasive penicillin non-susceptible Streptococcus pneumoniae from Denmark, 2001 to 2005. Scand J Infect Dis 42:333–340CrossRefPubMedGoogle Scholar
  30. 30.
    Hermans PW, Sluijter M, Elzenaar K, van Veen A, Schonkeren JJ, Nooren FM et al (1997) Penicillin-resistant Streptococcus pneumoniae in the Netherlands: results of a 1-year molecular epidemiologic survey. J Infect Dis 175:1413–1422CrossRefPubMedGoogle Scholar
  31. 31.
    Porat N, Amit U, Givon-Lavi N, Leibovitz E, Dagan R (2010) Increasing importance of multidrug-resistant serotype 6A Streptococcus pneumoniae clones in acute otitis media in southern Israel. Pediatr Infect Dis J 29:126–130CrossRefPubMedGoogle Scholar
  32. 32.
    Carvalho Mda G, Pimenta FC, Gertz RE Jr, Joshi HH, Trujillo AA, Keys LE et al (2009) PCR-based quantitation and clonal diversity of the current prevalent invasive serogroup 6 pneumococcal serotype, 6C, in the United States in 1999 and 2006 to 2007. J Clin Microbiol 47:554–559CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • I. Vickers
    • 1
    • 2
    Email author
  • D. O’Flanagan
    • 3
  • M. Cafferkey
    • 1
    • 2
  • H. Humphreys
    • 2
    • 4
  1. 1.Epidemiology and Molecular Biology Unit and Irish Meningococcal and Meningitis Reference LaboratoryChildren’s University HospitalDublinIreland
  2. 2.Department of Clinical Microbiology, RCSI Education and Research CentreBeaumont HospitalDublinIreland
  3. 3.Health Protection Surveillance CentreDublinIreland
  4. 4.Department of MicrobiologyBeaumont HospitalDublinIreland

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