Clinical Isolates of the Spain14-5 clone of Streptococcus pneumoniae carry a recombinant rpoB gene.

A characterization of 61 rifampin (RIF)-resistant Streptococcus pneumoniae isolates revealed two isolates (Rif-15 and Rif16) of the Spain-5 clone with identical recombinant rpoB genes (5) that were collected at the Hospital Donostia (HD) from two human immunodeficiency virus-infected patients. Given that the Spain-5 clone has been extensively studied in HD (10, 11), we have analyzed in this work 10 RIF-susceptible isolates representative of each pbp2b restriction fragment length polymorphism allelic profile among 93 isolates of the Spain-5 clone from the HD (Table 1). The MICs, pulsedfield gel electrophoresis patterns, and rpoB sequences were determined as previously described (5). The two RIF-resistant strains carried, in addition to the H499N change responsible for RIF resistance (5), several residue changes common to five RIF-susceptible isolates (Fig. 1) and to the Streptococcus mitis NCTC 12261 type strain (5), changes that are therefore not involved in RIF resistance. Comparisons of the rpoB sequences of the 12 strains with that of S. pneumoniae R6 (Fig. 1) revealed three types of isolates: four isolates that were nonrecombinant (0.15% variation) whose sequences were identical to that of the type strain, seven recombinant isolates (R2 and R3; 4.1% variation) that had identical sequences (excluding the mutation responsible for the H499N change), and one recombinant isolate (R1; 1.2% variation) that featured two blocks of divergence, the first (L42 to T472) being identical to the sequence of S. pneumoniae R6 and the second (A473 to T700) being identical to that of strains R2 and R3. Recombination events between S. pneumoniae and the viridans streptococci of the mitis group (VSM), which have presumably occurred in the face of antibiotic selection pressure and have led to the acquisition of resistance, have been described for the genes encoding the targets of penicillin (7), fluoroquinolones (1, 4), and rifampin (5). Furthermore, recombination in genes not involved in antimicrobial resistance and therefore not subjected to selective pressure has also been detected (3, 8, 9). Given the great genetic diversity of neutral VSM genes (12), fluoroquinolone target genes (1, 6), and rpoB (M. J. Ferrándiz et al., unpublished), the most plausible explanation for the identity of the sequences of the rpoB recombinant isolates is that they derive from an ancestral isolate that underwent recombination with an RIF-susceptible VSM at, or before, 1987. Isolate Rif-16 would have acquired RIF resistance by point mutation after recombination in the presence of RIF selection (this patient had a Mycobacterium avium-disseminated infection and was treated with RIF). Transmission among patients infected with Rif-16 and Rif-15 could have occurred, since there is no evidence of RIF treatment in the patient infected with Rif-15 and both patients were admitted in the same unit of HD. However, in other cases, RIF resistance could be acquired directly by recombination (5), given that, under the ideal laboratory conditions, the frequency of transformation is several orders of magnitude greater than that of spontaneous mutation, being 5 10 9 (2; our unpublished results), and the frequencies of transformation are 1 10 2 (5) and 2 10 4 for chromosomal DNAs from S. pneumoniae and VSM, respectively (data not shown). The frequency of