A new genetic element carrying the resistance determinant erm(TR) in Streptococcus pneumoniae

The rapid development of macrolide resistance in Streptococcus pneumoniae, particularly over the last decade, is of major clinical concern. The two main resistance mechanisms of macrolide resistance in S. pneumoniae are target site modification mediated by erm(B) and efflux mediated by mef genes. Recently the presence of another methylase gene, erm(A) subclass erm(TR), has been reported in few S. pneumoniae clinical cases from different part of the world. The erm(TR) gene is common in Streptococcus pyogenes but rare in S. pneumoniae. Although erm(TR) has been demonstrated to be transferable from S. pyogenes to other Gram-positive species, the genetic element carrying this resistance determinant has not been identified. Recently, a clinical S. pneumoniae isolate (AP200) carrying erm(TR) has been obtained from a patient with meningitis in Italy. In this work we have analysed the erm(TR)-carrying genetic element in AP200. This strain has been phenotypically and genotypically characterized. A DNA region of approximately 10kb, including erm(TR), was analysed by sequencing fragments obtained by inverse PCR and revealed 12 ORFs. Upstream erm(TR), an ORF coding for a regulatory protein of the TetR family, and two ORFs whose products are homologous to components of an efflux pump of the ABC type, were identified. Downstream erm(TR), 6 ORFs were found coding for products homolog respectively to a spectynomycin phosphotransferase, a cytidine deminase, a recombinase, two transposases and a relaxase. As we were sequencing the region carrying erm(TR) in AP200, the genomic sequence of a S. pyogenes strain (MGAS10750) carrying erm(TR) was deposited in GenBank. The sequence of MGAS10750 showed that erm(TR) was included in a genetic element of approximately 48 kb. PCR mapping of AP200 using primers based on MGAS10750 sequence, showed that the overall structure of the erm(TR)-element in AP200 was similar to that of MGAS10750. Analysis of the junction sequences suggested that the insertion of the element into the chromosome caused the same 3-nucleotides duplication both in AP200 and in MGAS10750. Examination of an erm(TR) deletion mutant indicated that the erythromycin resistance was mediated only by the presence of erm(TR) gene. The erm(TR)-element could be transferred from AP200 to an erythromycin susceptible pneumococcal strain by transformation but not by conjugation. In conclusion erm(TR) in S. pneumoniae AP200 appears to be contained in a genetic element similar to that of S. pyogenes MGAS10750. Probably this same genetic element may be responsible for dissemination of this macrolide resistance gene in the Streptococcus genus.