Target Fidelity and Failure: Structure–Activity Relationship of High-Molecular-Mass Penicillin-Binding Proteins (HMM-PBPs) in Refractory Granulicatella adiacens Endocarditis

<b>Background/Objectives</b>: <i>Granulicatella adiacens</i> infective endocarditis is conventionally managed with penicillin, ampicillin, or ceftriaxone in combination with gentamicin, although double beta-lactam regiments have been proposed a safer alternative to reduce aminoglycoside-associated nephrotoxicity. To date, the High-Molecular-Mass Penicillin-Binding Proteins (HMM-PBPs) of <i>G. adiacens</i> and their affinities for beta-lactam antibiotics have not been previously characterized. This study investigated the HMM-PBP profile of <i>G. adiacens</i>, with particular interest on sequence alterations and beta-lactam binding properties, both as single agents and in combination. <b>Methods:</b> Beta-lactam activity, synergistic interactions and PBP binding affinities were evaluated in a clinical isolate (IS 48) and compared with those in the reference strain ATCC 49175. Binding of PBPs to ampicillin, ceftriaxone, and ceftobiprole, alone or in combination, was investigated by Bocillin-FL labeling. PBP homology and conserved active-sites motifs were assessed by sequence alignment, and <i>pbp</i> gene mutations were identified by whole-genome sequencing. <b>Results:</b> The clinical isolate was non-susceptible to ampicillin, resistant to ceftriaxone and exhibited higher minimum inhibitory concentrations (MICs) for ceftobiprole relative to the fully susceptible ATCC reference strain. Five HMM PBPs with high enterococcal homology, were identified. In the IS 48 isolate, the class A PBP showed distinct amino acid substitutions in proximity to the catalytic centers. Despite these alterations, PBP1A and PBP2A were strongly inhibited by the tested beta-lactams, whereas PBP2 and PBP2B demonstrated low acylation rates. Combination of ampicillin with either ceftobiprole or ceftriaxone resulted in enhanced acylation of the three bifunctional HMM PBPs compared with monotreatment. IC₅₀ values were consistently higher for the IS 48 clinical isolate, suggesting decreased target availability and/or reduced beta-lactam affinity under clinical conditions. <b>Conclusions:</b> The resistance phenotype of <i>G. adiacens</i> clinical isolate appears to be primarily associated with altered PBP beta-lactam interactions. Nonetheless, beta-lactam combination regimes remain effective by achieving substantial inhibition of key HMM-PBPs involved in peptidoglycan synthesis, thereby supporting the rationale for dual beta-lactam therapy in this setting.