In Greece, an increase in the rate of macrolide resistance among S. pneumoniae occurred after the introduction of newer macrolides in the 1990s and their extensive use thereafter. We have published the phenotypical and molecular analysis of the macrolide-resistant pneumococci recovered from young carriers in different geographic locations of Greece between 1995 and 1999 [32, 38, 39]. The overall rate of macrolide-resistant S. pneumoniae nasopharyngeal isolates was 18%, while these isolates belonged mainly to serotypes 23F, 6B, 19F, and 14 (in order of decreasing frequency). Subsequently, studies on clinical as well as colonizing isolates from Greece [R] have reported significantly higher rates of macrolide resistance (up to ~50%) than that found in our initial studies [28, 29, 40]. The highest rate of macrolide resistance has been reported in pneumococci recovered from children with non-invasive infections, particularly acute otitis media .
Across the 4 surveillance periods of the present study and in parallel to an increase in the number of children who were immunized with PCV7, the frequency of the macrolide-resistant isolates did not change significantly. Overall, 24% of the typeable S. pneumoniae isolates were macrolide-resistant. This result is in line with a study on carriage among day-care attendees in Lisbon . However, in 2009 a major shift in the serotype distribution of macrolide-resistant isolates occurred. Macrolide-resistant isolates of non-PCV7 serotypes replaced those belonging to PCV7 ones. This shift is in accordance with a recent French study . Vaccination against 7 serotypes of S. pneumoniae has led to the near extinction of vaccine serotypes in both asymptomatic carriage and disease [7, 11, 41]. In carriage, vaccine serotypes have been replaced by nonvaccine serotypes. Clonal expansion and/or serotype switching contribute to this replacement .
Overall, more than half (52.9%) of our macrolide-resistant isolates possessed erm(B) either alone or in combination with mef(E). A significant association was found between PCV7 serotypes and the presence of erm(B), either alone or in combination with mef(E) on the one hand and non-PCV7 serotypes and a mef gene as the sole resistance determinant on the other. Among pneumoccoci harboring a mef gene as the sole resistance determinant, the ratio of mef(E)- to mef(A)-positive isolates was 7.9:1. This ratio was significantly reversed from the one that we observed among carriers during 1995–1999, which was 1:2.3 , and the 1:5 ratio reported from Germany during 2005–2006 .
As an overall concept, drug efflux mediated by mef genes has been the most common mechanism in strains of S. pneumoniae in North America, whereas in most of the European countries and the Far East, the prevalent mechanism has been rRNA methylation encoded by erm(B) . Nevertheless, this pattern of macrolide resistance determinants is not static and may be changing due to clonal spread of S. pneumoniae of certain serotypes and horizontal transfer of mef elements among streptococci [23, 43]. Actually, in recent papers, mef(A) was the predominant macrolide resistance determinant in Norway (2001–2005)  and Germany (2005–2006) , whereas increased frequency of erm(B) as well as of the dual combination was found in the United States (2005–2008) [13, 45].
In the pre-PCV7 period, pneumococci of serotype 14 contributed significantly to macrolide resistance. Among children, serotype 14 had a higher frequency in invasive disease than observed in carriage and non-invasive disease . We have previously reported the circulation of mef(A)-positive macrolide-resistant, penicillin-susceptible serotype 14 isolates with a genotype identical to the international clone England-914 among young carriers in Greece . Similar isolates continued to circulate in our area during the first 2 years of the present study. In addition, in 2006 we found macrolide-resistant serotype 14 isolates possessing erm(B) and exhibiting penicillin-nonsusceptibility. Following the immunization with PCV7, a rapid decrease in the circulation of serotype 14 was noted and it is no longer a major macrolide-resistant serotype in Greece as well as in other countries [46, 47].
This study reports a high frequency of serotype 19F pneumococci with both erm(B) and mef(E) recovered from carriers in several day-care centers in Central Greece across all sampling periods. Since 2001, isolates with the dual resistance mechanism have been increasingly reported from many parts of the world [13, 23, 45, 48–50]. They have mainly been isolated from carriers or patients with non-invasive disease, particularly acute otitis media [45, 50]. Worldwide, most isolates with the dual resistance mechanism belong to serotypes 19F or 19A [45, 48]. Although serotype 19F is represented in the PCV7 vaccine, it affords low levels of protection against upper respiratory infections such as acute otitis media  and has been shown to be the least immunogenic of the vaccine serotypes . Moreover, little evidence shows that 19F provides cross-protection against serotype 19A. In Greece, antibiotic pressure may have also contributed to the persistence of these MDR isolates. In our country, isolates with the dual resistance mechanism in a low frequency were identified for the first time among carriers in Athens during 2003 . In the present study, which differentiated between mef(A) subclasses mef(A) and mef(E), only isolates carrying the mef(E) gene, but not mef(A), with erm(B) were observed, underscoring the different genetic background of mef(E) and mef(A). Our findings are in line with a recent study from the USA . S. pneumoniae isolates possessing the mef(A) subclass mef(A) gene, carried on transposon Tn1207.1, in combination with erm(B) have been described in a paper from Australia .