However, further mechanistic features of the combination that were shown (synergy, resistance suppression over longer periods of dosing, security level of sensitivity, etc.) will require substantially more screening to support the promising but initial activity observed in our highly aggressive neutropenic mouse model. Ccna2 We note that high resistance to meropenem or tazobactam slightly reduces the effectiveness of ME/PI/TZ, while maintaining its synergy, and our resistance evolution analysis cannot account for resistance genes acquired horizontally that could break the relationship between meropenem, piperacillin, and tazobactam. resistance to -lactam antibiotics 3. One of these genes, MRSA Polygalaxanthone III N315 31 from a group of fully genome-sequenced MDR strains of MRSA for this study. MRSA N315 contains the staphylococcal chromosome cassette (SCCmethicillin-resistance operon 32, as well as penicillinase plasmid pN315 comprising the -lactamase operon 33. From a focused combinatorial screen of these 23 antibiotic compounds, including representatives from every major drug class (Supplementary Table 1), we recognized the combination of ME/PI/TZ to display highly synergistic, bactericidal activity against MRSA N315 (4C8 g/ml), while tazobactam has no susceptibility breakpoint only, and is given clinically at a 1:8 percentage with piperacillin 36. The constituent double combinations ME/PI and PI/TZ were also synergistic against N315 with FICI = 0.44 and 0.22, Polygalaxanthone III respectively, while ME/TZ is less synergistic at 0.67. Based on the Loewe additivity model of synergy, medicines cannot be synergistic with themselves 30. Though the -lactams all target the cell-wall synthesis pathway, our use of the FICI method (Loewe additivity) confirms the non-additive nature of these interactions. In contrast to the high synergy of ME/PI/TZ seen in MRSA N315, the combination exhibits no additive activity (FICI = 1.12) in the methicillin-susceptible (MSSA) research strain ATCC 29213 36,37 (Supplementary Furniture 2b, c), and we hypothesize the necessity of PBP2a for synergy to occur. Open in a separate window Number 1 3D-Checkerboard synergy dedication showing isoboles of minimal inhibitory concentrations (MIC) and growth in solitary-, Polygalaxanthone III double-, or triple-drug conditions for ME/PI/TZColored lines/isoboles within each panel show MICs of two medicines in combination. Dashed lines show theoretical concentrations of additive relationships. Points indicate top sub-inhibitory concentrations of meropenem (ME), piperacillin (PI) and tazobactam (TZ) for each tested condition. The reddish triangle shows the MIC of all three medicines in combination (Each at 2 g/ml). We propose that the mechanism of synergy observed for ME/PI/TZ results from allosteric triggering of PBP2a by its constituents, akin to that reported for ceftaroline 8,9. Indeed, we identified that meropenem binds to the allosteric site of PBP2a having a dissociation constant (types displayed (Supplementary Furniture 3a, b). The MIC of the combination against the medical isolates ranged from 0.4C33.3 g/ml for each component, having a mean of 9.7 g/ml, and an MIC50 and MIC90 of 3.7 g/ml and 33.3 g/ml, respectively (Supplementary Table 4a). Class-specificity of -lactam synergy against MRSA We identified the observed synergy is not limited to the antibiotics assayed, but can be generalized to their respective -lactam classes, by screening MRSA N315 and representative medical MRSA isolates against additional carbapenem/penicillin/-lactamase inhibitor mixtures. We found that treatment of MRSA N315 with imipenem/piperacillin/clavulanate (IM/PI/CV) shows equal or higher synergism to ME/PI/TZ. Meropenem/amoxicillin/tazobactam (ME/AX/TZ) maintains high synergy in MRSA N315 only (FICI = 0.04), having a clinical MRSA isolate showing less synergy (FICI = 0.55) (Supplementary Table 2b). MICs for components of these substituted triples are all below the mean maximum human being plasma concentrations of these compounds gene product PBP2a with its attendant allosterism for synergy, due to lack of synergy of carbapenem/penicillin/-lactamase inhibitor mixtures in methicillin-susceptible and was sensitized to all tested -lactams (Supplementary Table 5). When meropenem, piperacillin, and tazobactam were tested against the antisense strain, only meropenem showed larger zones of inhibition under xylose induction, confirming PBP1 like a target of meropenem (Supplementary Table 5). For the antisense strain both meropenem and piperacillin showed improved performance under xylose induction, demonstrating that they each have some activity against PBP2 (Supplementary Table 5). We did not observe any.