Abstract 941
Poster Session IV, Tuesday, 5/4 (poster 160)
Through active surveillance of all major tertiary care Canadian pediatric centers in 1991-7, the mean annual caseload of invasive penicillin-highly-resistant S. pneumoniae (PHRP) was 238 (range 197-277). PHRP isolates slowly increased in frequency, from 2.5% in 1991 to 9.4% in 1997. PHRP defined as penicillin MIC > 1 mg / ml, comprised 32% of the 1997 penicillin-resistant isolates (3% of all cases). These isolates were sporadically encountered at 10 centers. Thirteen of the PHRP isolates were obtained for genotyping. The goal of this study was to determine the molecular epidemiology of PHRP isolates from across Canada. A simple and robust random amplified polymorphic DNA (RAPD) method was developed for this purpose. A total of 100 ten-base oligonucleotide primers were screened using S. pneumoniae strains R6. Twelve primers produced a genetic fingerprint containing more then ten bands. These primers were screened further using ten unrelated S. pneumoniae strains, and two primers produced the most discriminatory patterns. The reproducibility of the method was confirmed and results analyzed by Molecular Analyst Fingerprinting Software (BIORAD). Each strain produced a different but reproducible RAPD pattern. In addition, four mutants of strain R6 were screened by these primers, and all had the same RAPD pattern. Fifty-two isolates which had been evaluated by pulsed field gel electrophoresis (PFGE), were tested in a blinded comparison, in order to validate the RAPD method. S. pneumoniae isolates were separated by serotyping to 5 groups, by PFGE into 22 groups, and by RAPD into 25 groups. The agreement between the latter two methods was 88.8% (46/52). RAPD analysis of the 13 PHRP isolates from blood or CSF was able to differentiate between the serotypes and identify two different patterns within serotype 23F. All seven serotype 9V isolates from Alberta and Quebec displayed identical RAPD profiles which were distinct from all other isolates. This study shows that RAPD analysis can reliably distinguish among different invasive serotypes and clones of S. pneumoniae and can be used as a robust method for epidemiological screening. These data show that a single clone of S. pneumoniae may be responsible for the type 9V PHRP found in different provinces of Canada. Further molecular typing of isolates collected from the Canadian pediatric invasive pneumococcal infections monitoring study is being undertaken on the basis of this preliminary study.
