Diversity of penicillin-binding protein 2x in Streptococcus pyogenes from England and Wales, 2001 to 2015

Streptococcus pyogenes remains clinically susceptible to {beta}-lactam antibiotics, its first-line treatment. Yet recent reports from the US, Iceland, and Japan have identified isolates with PBP2x (a primary {beta}-lactam target) substitutions that reduced antibiotic susceptibility. To determine whether these PBP2x substitutions occurred in S. pyogenes from England and Wales, we re-analysed 2,970 invasive genomes from 2001 to 2015. We found 42 PBP2x sequence mutants in 34% (996/2,970) of the genomes, revealing an unexpected diversity in this antibiotic target. Nine emm12 isolates carried a novel double substitution, PBP2x G600A_P601H, that co-occurred with macrolide resistance genes. Experimentally, these isolates showed 2- to 4-fold elevated minimum inhibitory concentrations for four {beta}-lactam antibiotics, alongside erythromycin resistance. Other PBP2x substitutions, found across seven emm types without reduced antibiotic susceptibility, appeared driven by emm lineage expansion rather than antibiotic pressure. These findings establish a baseline for pbp2x variation, reinforcing the case for sustained genomic surveillance globally.