Poster Presentation Australian Society for Microbiology Annual Scientific Meeting 2013

Detection of two common clonal complexes among S.pneumoniae serotype 19A and 19F isolates, using allele specific PCR (#242)

Shahin Oftadeh 1 , Vishal Ahuja 1 , Fanrong Kong 1 , Gwendolyn Gilbert 1
  1. Pneumococcal Reference Laboratory, Centre for Infectious Diseases and Microbiology (CIDM), Institute of Clinical Pathology and Medical Research (ICPMR), Westmead Hospital, Westmead, New South Wales, Australia

Background: Reliable methods are required for large scale monitoring of changes in pneumococcal serotype distribution, antibiotic susceptibility and molecular epidemiology. The most commonly used method to assess molecular epidemiology of S.pneumoniae is multi-locus sequence typing (MLST), based on alleles of seven house-keeping genes (aroE, gdh, gki, recP, ddl, xpt, spi).  After introduction of the pneumococcal 7-valent conjugate vaccine (PCV-7) in Australia in 2005, serotype 19A emerged as the most frequently identified antibiotic resistant serotype among invasive isolates, while serotype 19F rapidly decreased.

Aim: We developed a simple screening test to identify the most common clonal complexes (CC) among serotype 19A, in Australia, to determine whether its increased incidence was due to expansion of CC199 (previously most prevalent among 19A and 19F) or replacement with the resistant CC320, which is widely distributed in North America.

Methods: Based on published sequences and our previous analysis, CC199 and CC320 have different aroE and gki alleles;CC199 contains aroe8-gki14 and CC320 contains aroe4-gki19. We designed allele-specific primers for CC19919A and CC32019A targeting the corresponding alleles. All isolates of serotype 19A (n=244) and 19F (n=125) in our collection were screened, using four real-time PCRs. The proportions of CC199 and CC320 were compared among seotype19F and 19A isolates collected before (2002-4) and after (2005-11) introduction of PCV-7. 

Results: After 2004 there was a significant fall in the proportion of 19F (from 82% to 17%) and increase in that of 19A (18% to 83%) among invasive isolates. The proportion of CC320 among 19A and 19F isolates, combined, decreased from 19% of 95 isolates before to 11% of 274 isolates after 2004 whereas that of CC199 remained largely unchanged (38% vs 36%) but accounted for 41% of serotype 19A.

Conclusion: The increase in serotype 19A appears to have been largely due to expansion of CC199, which is currently its dominant CC.