Background
The identification of polymorphisms causing decreased antibiotic susceptibility in bacterial pathogens has traditionally flowed from observed phenotype to defined mutant genotype. The availability of large collections of microbial genome sequences that lack antibiotic susceptibility metadata provides an important resource and opportunity to obtain new information about increased antimicrobial resistance by a reverse genotype-to-phenotype research approach. β-lactam antibiotics are the primary therapy used for S. pyogenes infection, but because β-lactam susceptibility testing is not routinely performed, the nature and extent of polymorphisms contributing to decreased β-lactam susceptibility are poorly characterized.
Methods
Using a cohort of 26,465 S. pyogenes whole genome sequences bioinformatic analyses were conducted to identify polymorphisms, infer phylogenetic relationships, and assess evolution due to mutation and recombination species wide. Working under the hypothesis that β-lactam therapy imparts strong evolutionary selection, amino acid substitutions in penicillin-binding proteins 1A, 1B, 2A, and 2X were evaluated for signatures of evolution under positive selection, as potential candidates for causing decreased β-lactam susceptibility.
Results
Hundreds of PBP variants (n = 830) were identified, including the first examples of horizontally acquired sequences with identity to S. equisimilis. Each PBP had hundreds of substituted sites, a minority with signatures of positive selection. Importantly 9 of 11 PBP2X substitutions associated with or proven with isogenic mutants to cause increased β-lactam MICs in-vitro had signatures of positive selection. Moreover, chimeric PBP2X strains exhibited 2-to-4 fold increased MICs for multiple β-lactams in-vitro.
Conclusions
The integrative reverse genotype-to-phenotype bioinformatic and experimental workflow taken successfully identified substitutions contributing to decreased S. pyogenes β-lactam susceptibility.