- Background: The major aim of this study was to establish the molecular epidemiology dynamics of Streptococcus pneumoniae serotype 6A in acute otitis media, before the introduction of the 7-valent pneumococcal conjugate vaccine. Methods: Acute otitis media in Jewish and Bedouin children <5 years old undergoing tympanocentesis during 1999 to 2006, were studied. Serotype 6A was identified by the Quellung reaction and by polymerase chain reaction (PCR) of the wciN gene, to differentiate between 6A and 6C. Antibiogram and molecular typing by pulsed field gel electrophoresis were performed on all 6A isolates. Multilocus sequence typing was performed on representative isolates of each clone. The 7-valent conjugate vaccine had not yet been licensed in Israel during the study period. Results: Serotype 6A constituted 5.8% (254/4408) of all pneumococcal acute otitis media episodes. The yearly proportion of serotype 6A among the Jewish children showed no distinct trend, whereas among the Bedouin children serotype 6A exhibited a significant increase, from 3.0% in 1999 to 7.6% in 2006. Among the Jewish children a single penicillin-nonsusceptible and erythromycin-resistant clone, ST-473, constituted 73.6% of the strains and dominated throughout the study period. Among the Bedouin children, the proportions of the most common, penicillin-nonsusceptible clone, ST-1988, gradually decreased, from 44.1 % in 1999 to 2000 to 21.4% in 2005 to 2006, concurrently with the expansion of a multidrug-resistant clone, ST-457, from 5.9% in 1999 to 2000 to 28.6% in 2005 to 2006. Conclusions: The expansion of multidrug-resistant serotype 6A clone occurred before the introduction of the vaccine. Continued surveillance following vaccine introduction is warranted to further investigate its efficacy on vaccine-related serotypes.