Detection of Very High–Level Penicillin-Resistant Variants of the Tennessee 23F-4 Clone Via Single and Serial Transformations with Four Serotype 19A International Pneumococcal Clones

In the United States, penicillin-resistant variants of the Tennessee (Tenn) 23F-4 clone account for a substantial proportion of the very-high-level penicillin-resistant (MIC 8 µg/ml) infections in the 7-valent pneumococcal protein conjugate vaccine (PCV7) era. Serotype 19A strains account for an increasing proportion of penicillin-nonsusceptible Streptococcus pneumoniae infections. Sequential transformations of the Tenn 23F-4 clone (penicillin MIC 0.1 µg/ml) were performed with four penicillin-nonsusceptible serotype 19A international clones (penicillin MIC): S. Africa 19A-7 (0.5 µg/ml), Hungary 19A-6 (2 µg/ml), Slovakia 19A-11 (8 µg/ml), and South Africa 19A-13 (8 µg/ml). Fifty-two transformants were characterized by MICs, serogroup-specific PCR, pbp PCR restriction profile and sequence, pspA PCR restriction profile, and erm/mef PCR. A subset was analyzed with multilocus sequence typing (MLST) and pulsed-field gel electrophoresis. Serotype 23F transformants with penicillin MIC ≥ 8 µg/ml were detected through a single transformation with the Hungary 19A-6 clone or serial transformations using two to three different clones. Forty-four percent (14/32) of the transformants incorporated ≥1 new MLST allele. Using encapsulated donors, very-high-level penicillinresistant variants of the Tenn 23F-4 clone were detected. In addition to detecting stepwise increases in penicillin MIC, a 12-fold increase in penicillin MIC was achieved through a single transformation. This large increase in MIC may explain why this clone is commonly associated with very-high-level resistance in natural populations. Recombination within the MLST housekeeping genes was commonly detected in the transformants that had acquired penicillin resistance.