Low-affinity PBP4, historically linked to penicillin resistance in Enterococcus faecalis, may still have affinity for novel cephalosporins. Ceftobiprole (BPR) is a common therapeutic choice, even with PBP4-related overexpression and amino acid substitution due to mutations. Our study aims to explore the interaction between BPR and High-Molecular-Mass (HMM) low-reactive PBPs in Penicillin-Resistant-Ampicillin-Susceptible/Ceftobiprole Non-Susceptible (PRAS/BPR-NS) E. faecalis clinical isolates. We conducted competition assays examining class A and B HMM PBPs from four PRAS/BPR-NS E. faecalis strains using purified membrane proteins and fluorescent penicillin (Bocillin FL), in treated and untreated conditions. Interaction strength was assessed calculating the 50% inhibitory concentration (IC₅₀) values for ceftobiprole, by analyzing fluorescence intensity trends. Due to its low affinity, PBP4 did not display significant acylation among all strains. Moreover, both PBP1a and PBP1b showed a similar insensitivity trend. Conversely, other PBPs showed IC₅₀ values ranging from 1/2-fold to 4-fold MICs. Upon higher BPR concentrations, increased percentages of PBP4 inhibition were observed in all strains. Our results support the hypothesis that PBP4 is necessary but not sufficient for BPR resistance, changing the paradigm for enterococcal cephalosporin resistance. We hypothesize that cooperation between class B PBP4 and at least one bifunctional class A PBP could be required to synthesize peptidoglycan and promote growth.

(1) Background: Granulicatella adiacens is a former nutritionally variant streptococci (NVS). NVS infective endocarditis (IE) is generally characterized by a higher rate of morbidity and mortality, partially due to difficulties in choosing the most adequate microbiological culture method and the most effective treatment strategy, and partially due to higher rates of complications, such as heart failure, peripheral septic embolism, and peri-valvular abscess, as well as a higher rate of valve replacement. Depending on the affected valve (native valve endocarditisNVE, or prosthetic valve endocarditisPVE), the American Heart Association (AHA) 2015 treatment guidelines (GLs) suggest penicillin G, ampicillin, or ceftriaxone plus gentamicin (2 weeks for NVE and up to 6 weeks for PVE), while vancomycin alone may be a reasonable alternative in patients who are intolerant of β-lactam therapy. The European Society of Cardiology (ESC) 2023 GLs recommend treating NVE with penicillin G, ceftriaxone, or vancomycin for 6 weeks, suggesting combined with an aminoglycoside (AG) for at least the first 2 weeks only for PVE; likewise, the same recommendations for IE due to Enterococcus faecalis. (2) Methods: Starting from the case of a 51-year-old man with G. adiacens aortic bio-prosthesis IE who was successfully treated with aortic valve replacement combined with double beta-lactams, an AG-sparing regimen, we performed microbiology tests in order to validate this potential treatment change. (3) Results: As for E. faecalis IE, we found that the combination of ampicillin plus cephalosporines (like ceftriaxone or ceftobiprole) showed a synergistic effect in vitro, probably due to wider binding to penicillin-binding proteins (PBPs), thus contributing to enhanced bacterial killing and good clinical outcome, as well as avoiding the risk of nephrotoxicity due to AG association therapy. (4) Conclusions: Further studies are required to confirm this hypothesis, but double beta-lactams and an adequate sourcecontrol could be a choice in treating G. adiacens IE.

Background/Objectives: Granulicatella adiacens 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 G. adiacens and their affinities for beta-lactam antibiotics have not been previously characterized. This study investigated the HMM-PBP profile of G. adiacens, with particular interest on sequence alterations and beta-lactam binding properties, both as single agents and in combination. Methods: 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 pbp gene mutations were identified by whole-genome sequencing. Results: 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. Conclusions: The resistance phenotype of G. adiacens 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.