New composite genetic element of the Tn916 family with dual macrolide resistance genes in a Streptococcus pneumoniae isolate belonging to clonal complex 271.

Clonal complex 271 (CC271) is one of the emerging multidrug-resistant clones of Streptococcus pneumoniae, spreading in many areas of the globe (3) and including serotype 19F and the nonvaccine serotype 19A (4). CC271 pneumococci often carry dual macrolide resistance genes erm(B) and mef(E), determining a macrolide resistance phenotype (MLSB) identical to that conferred by erm(B) alone (5). We examined a serotype 19F S. pneumoniae isolate (S43) belonging to CC271 (sequence type 1428), obtained in Hungary in 2003 from an infant with meningitis. S43 was multidrug resistant, showing resistance to penicillin (MIC 4 g/ml), erythromycin (MIC 256 g/ml), clindamycin (MIC 256 g/ml), and tetracycline (MIC 16 g/ml) and carrying erm(B), mef(E), and tet(M). We have previously demonstrated that CC271 dual-gene isolates carried the composite transposon Tn2010, a Tn916 family transposon including erm(B), mef(E), and tet(M) (1). To verify the presence of Tn2010 in S. pneumoniae S43, PCR mapping was performed (1). The presence of a Tn916like transposon including tet(M) and harboring mef(E) in a mega element integrated in orf6 (1) was confirmed. However, the erm(B) genetic element described in Tn2010 was not found. Since erm(B) can be carried by Tn917 (8), we investigated the presence of Tn917 and its physical linkage with the Tn916-like transposon in S43. Three sets of primer pairs were designed on the Tn917 sequence (GenBank accession no. M11180), as follows: TN7 (5 -GCAGGTGTAT TTCTTATCTATGG-3 ) and EB2 (1); O29 (5 -TCATCAA GCAATGAAACACG-3 ) and TN9 (5 -AGCTTCTTCTCC ATCTTGTAC-3 ); TN8 (5 -ACACGTGATTCACAAGAA CAG-3 ) and TN11 (5 -CACTAAACCAATGTTTCAAGG3 ). Amplicons of the expected size were obtained with primer pairs O29/TN9 and TN8/TN11, but not with TN7/ EB2 due to a 182-bp deletion from positions 240 to 421 of Tn917 sequence including primer TN7. This deletion in Tn917 had been already described in isolates from Japan (7). By using the primer pair EB2 and SG5 (1), an amplicon was obtained showing a physical linkage between Tn917 and mega. Sequence analysis of a 1,053-bp fragment (GenBank accession no. FJ208941) showed that the left end of Tn917 was inserted in orf9 of Tn916 in proximity to the insertion of mega in orf6 of Tn916 (Fig. 1). Tn917 was inserted in Tn916 at the identical site in which it is inserted to form Tn3872 (6). These findings confirmed the identification in S. pneumoniae S43 of a composite transposon of ca. 28.5 kb that was designated Tn2017, based on a new modular combination of Tn916, Tn917, and mega. The left and right junctions of Tn2017 in S43 were identical to those described for Tn2010 in CC271 isolates (2). In a previous study, we characterized three dual-gene isolates from Italy belonging to CC15 (1). Although these isolates contained Tn916, Tn917, and mega, they did not carry the newly described Tn2017 but, rather, carried the composite transposon Tn3872, with Tn917 inserted in Tn916, while mega was independently inserted in spr0166, a common mega insertion site (1). These findings indicate that in order to correctly trace the spreading of the antibiotic resistance determinants, it is