Invasive Group A Streptococcal Penicillin Binding Protein 2× Variants Associated with Reduced Susceptibility to β-Lactam Antibiotics in the United States, 2015-2021

Abstract

All known group A streptococci [GAS] are susceptible to β-lactam antibiotics. We recently identified an invasive GAS (iGAS) variant (emm43.4/PBP2x-T553K) with unusually high minimum inhibitory concentrations (MICs) for ampicillin and amoxicillin, although clinically susceptible to β-lactams. We aimed to quantitate PBP2x variants, small changes in β-lactam MICs, and lineages within contemporary population-based iGAS. PBP2x substitutions were comprehensively identified among 13,727 iGAS recovered during 2015-2021, in the USA. Isolates were subjected to antimicrobial susceptibility testing employing low range agar diffusion and PBP2x variants were subjected to phylogenetic analyses. Fifty-five variants were defined based upon substitutions within an assigned PBP2x transpeptidase domain. Twenty-nine of these variants, representing 338/13,727 (2.5%) isolates and 16 emm types, exhibited slightly elevated β-lactam MICs, none of which were above clinical breakpoints. The emm43.4/PBP2x-T553K variant, comprised of two isolates, displayed the most significant phenotype (ampicillin MIC 0.25 μg/ml) and harbored missense mutations within 3 non-PBP genes with known involvement in antibiotic efflux, membrane insertion of PBP2x, and peptidoglycan remodeling. The proportion of all PBP2x variants with elevated MICs remained stable throughout 2015-2021 (<3.0%). The predominant lineage (emm4/PBP2x-M593T/ermT) was resistant to macrolides/lincosamides and comprised 129/340 (37.9%) of isolates with elevated β-lactam MICs. Continuing β-lactam selective pressure is likely to have selected PBP2x variants that had escaped scrutiny due to MICs that remain below clinical cutoffs. Higher MICs exhibited by emm43.4/PBP2x-T553K are probably rare due to the requirement of additional mutations. Although elevated β-lactam MICs remain uncommon, emm43.4/PBP2x-T553K and emm4/PBP2x-M593T/ermT lineages indicate that antibiotic stewardship and strain monitoring is necessary.

Keywords: PBP2x catalytic site; amino substituted β-lactams; clinical susceptibility; group A streptococcal lineages; penicillin binding protein 2×; transpeptidase domain.

FIG 1

Depiction of the Pbp2x full-length protein and the transpeptidase encompassing region indicating, 3 conserved catalytic site motifs, and substitutions within 19 residues (23 substitutions) associated with decreased susceptibility in this study to β-lactam antibiotics. The catalytic serine in motif I that covalently binds to β-lactam antibiotics is indicated in red. The substitution associated with the most significant MIC, T553K, is highlighted in yellow. Substitutions at 4 positions indicated in red font accounted for 322/340 (94.7%) of the isolates with reduced susceptibility to β-lactams and occurred at positions M593 (205 isolates), P601 (42 isolates), A397 (48 isolates), and K410 (23 isolates). Corresponding substitutions in the GBS PBP2x associated with reduced susceptibility or nonsusceptibility to β-lactams are indicated below. Substitutions reported in other studies (25–27, 29) to be associated with reduced β-lactam susceptibility in naturally occurring isolates or through allelic exchange experiments are indicated with an asterisk.

FIG 2

Core phylogeny of emm4 isolates (all 390 recovered in 2015 to 2019 are characterized). All isolates are within clonal complex CC39. Legends for this and all other phylogenies depicted employ same scheme as follows: Innermost circle is color coded for PBP2x substitution, the second circle from inside is color coded for resistance determinants for erythromycin and clindamycin (both constitutive and inducible), the third circle from inside is depicting state, and the fourth circle from inside is depicting year of isolation. Figure depicts well established emm4/PBP2x10 sublineage recovered in multiple states and years and is the principal component of a single major branch within the overall invasive emm4 phylogeny. The emm4/ermT-positive/PBP2x1 isolates are situated on the same main branch, potentially indicating that the pbp2x10 missense mutation originated within an emm4/ermT background.

FIG 3

Core phylogeny of emm1 sequence type ST28 lineage, average distance 45 SNPs (all 1619 characterized). PBP2x types of this lineage associated with reduced beta-lactam susceptibility and represented by 15 or more isolates were characterized. Figure depicts three distinct tight phylogenetic clusters of PBP2x10, PBP2x18, and PBP2x47 collected within multiple years and states.

FIG 4

Core phylogeny of emm75 sequence type ST49 lineage associated with four reduced-susceptibility PBP2x variant types (all 111 characterized). Figure depicts tight phylogenetic clusters of emm75/PBP2x10 collected during multiple years, with highest incidence in Minnesota and more recently emerged emm75/PBP2x20 within Colorado and New Mexico. Legends for this and all other phylogenies depicted employ same scheme as follows: innermost circle is color coded for PBP2x substitution, the second circle from inside is depicting state, and the third circle from inside is depicting year of isolation.