Population structure and MURm gene analysis of early and contemporary high penicillin resistant Streptococcus pneumoniae isolates in Spain

Abstract number: 1134_02_229

Del Campo R., Morosini M.I., Fenoll A., Gómez G-de la Pedrosa E., Liñares J., Cafini F., Baquero F., Cantón R., Prieto J.

Background:

Sequential alterations in PBP sequences constitute the classical mechanism to acquire penicillin resistance in S. pneumoniae, that is reflected in changes in the cell wall peptidoglycan structure. murM gene controls the addition of the first amino acid of the dipeptide bridge of the pneumococcal muropeptide. Mutations in this gene are apparently required for high penicillin and cefotaxime resistance. The aim of this study was to compare the murM gene sequence within the earlier and the latest highly penicillin resistant pneumococcal populations recovered in Spain.

Material and methods:

A total of 56 S. pneumoniae isolates (10 of them from 1987–88 and 46 from 2002–04) with resistance to penicillin (MIC 2–8 mg/ml) were included. Susceptibility to different b-lactams, macrolides, tetracycline, levofloxacin, chloramphenicol, and trimethoprim-sulfamethoxazole (SxT) were determined by the agar dilution method. All isolates were grouped according to serotype and pulsotype (PFGE-SmaI pattern) and the murM gene was sequenced.

Results:

A high percentage of resistance (intermediate plus resistant) was observed for cefotaxime (100%) and SxT (96.6%). All isolates remained susceptible to telithromycin but not to levofloxacin (93% of susceptibility). All isolates recovered during 1987–88 were genetically unrelated and mainly belonged to serotypes 23F (40%) and 6B (40%). In contrast, the most frequent serotypes during 2002–04 were 14 (30%), 9V (26%), and 6B (17%). In this period, a predominant Spain9V-3clone was detected in 10 out of 14 isolates of serotype 14, and in 11 out of 12 isolates of serotype 9V (capsular switch). All 8 isolates of serotype 6B belonged to a single clone (Spain6B-2). The analysis of murM sequences of isolates from both periods revealed the existence of different alleles. Homologous sequences to the murMA (identical to that described for R6 penicillin susceptible strain), were replaced in part by murMB5, and murMB6 (different variants of these alleles were detected in our collection).

Conclusion:

Early high penicillin resistant S. pneumoniae isolates in Spain were genetically unrelated and corresponding to 23F and 6B serotypes. On the contrary, recent isolates showed dissemination of selected clones, particularly Spain9V-3. Many S. pneumoniae isolates with high-level resistance to penicillin retain (or reacquired) the murM gene of susceptible strains, but different allelic variants was also detected.