Restricted Sequence Variation in Streptococcus pyogenes Penicillin Binding Proteins

Abstract

A recent clinical report has linked Streptococcus pyogenes β-lactam antibiotic resistance to mutation in the penicillin binding protein (PBP) PBP2x. To determine whether this is an isolated case or reflects a broader prevalence of mutations that might confer reduced β-lactam susceptibility, we investigated the relative frequency of PBP sequence variation within a global database of 9,667 S. pyogenes isolates. We found that mutations in S. pyogenes PBPs (PBP2x, PBP1a, PBP1b, and PBP2a) occur infrequently across this global database, with fewer than 3 amino acid changes differing between >99% of the global population. Only 4 of the 9,667 strains contained mutations near transpeptidase active sites of PBP2x or PBP1a. The reported PBP2x T553K substitution was not identified. These findings are in contrast to those of 2,520 S. pneumococcus sequences where PBP mutations are relatively frequent and are often located in key β-lactam binding pockets. These data, combined with the general lack of penicillin resistance reported in S. pyogenes worldwide, suggests that extensive, unknown constraints restrict S. pyogenes PBP sequence plasticity. Our findings imply that while heavy antibiotic pressure may select for mutations in the PBPs, there is currently no evidence of such mutations becoming fixed in the S. pyogenes population or that mutations are being sequentially acquired in the PBPs. IMPORTANCE β-Lactam antibiotics are the first-line therapeutic option for Streptococcus pyogenes infections. Despite the global high prevalence of S. pyogenes infections and widespread use of β-lactams worldwide, reports of resistance to β-lactam antibiotics, such as penicillin, have been incredibly rare. Recently, β-lactam resistance, as defined by clinical breakpoints, was detected in two clinical S. pyogenes isolates with accompanying mutations in the active site of the penicillin binding protein PBP2x, raising concerns that β-lactam resistance will become more widespread. We screened a global database of S. pyogenes genome sequences to investigate the frequency of PBP mutations, identifying that PBP mutations are uncommon relative to those of Streptococcus pneumoniae. These findings support clinical observations that β-lactam resistance is rare in S. pyogenes and suggest that there are considerable constraints on S. pyogenes PBP sequence variation.

Genomic Surveillance of Streptococcus pyogenes Strains Causing Invasive Disease, United States, 2016–2017

“…We now plan to examine our PBP2x type variants carefully to determine exact phenotypic changes that have occurred and to continue to carefully monitor these variants. Although it does appear that pbp2x point mutants regularly appear in our surveillance as a consequence of beta lactam selective pressure, present data suggests that there are strict constraints upon sequence variation (Hayes et al, 2020).…”

Population Genomic Molecular Epidemiological Study of Macrolide-Resistant Streptococcus pyogenes in Iceland, 1995 to 2016: Identification of a Large Clonal Population with a pbp2x Mutation Conferring Reduced In Vitro β-Lactam Susceptibility

“…Given that most of the isolates in this cohort come from pharyngitis patients, this indicates that S. pyogenes strains with some beta-lactam susceptibility-altering mutations in pbp2x are sufficiently fit to be readily transmitted and cause abundant pharyngitis. This finding contradicts the recent analysis of Hayes et al (45) of PBPs among 9,667 GAS isolates, which found that "while heavy antibiotic selective pressure may select for mutations in the PBPs, there currently is no evidence of such mutations becoming fixed in the S. pyogenes population. "…”

“…That is, in terms of pbp2x allele/PBP2X variants, 712/713 emm4 isolates have the same pbp2x allele/PBP2X variant, 324/324 emm6 isolates are the same, and 327/332 emm12 isolates are the same. The emm4 isolates have the consensus PBP2X wild-type (WT) sequence that is most prevalent among S. pyogenes isolates of multiple emm types (12,44,45). The PBP2X variant of all 324 emm6 isolates have three substitutions (Ile 502 Val, Pro 676 Ser, and Lys 708 Glu), and 327/332 emm12 isolates have a single substitution (Met 593 Thr) relative to the PBP2X WT 751-amino acid sequence.…”

Molecular Characteristics of Streptococcus pyogenes Isolated From Chinese Children With Different Diseases

“…No penicillin-resistant strains or mutations in pbp2x were found. Nonetheless, a relationship exists between pbp2x gene variations and MIC values ( Hayes et al, 2020 ; Musser et al, 2020 ; Vannice et al, 2020 ). Our WGS and MIC results showed that the vanB + vanrG + vanuG– and vanB + vanrG + vanuG + strains were susceptible to vancomycin.…”