(M1230-04-26) Flow Cytometry and Confocal Microscopy to Quantify β-lactam Penetration in a Double β-lactamase and Double Efflux Pump Knockout Strain of Acinetobacter baumannii (AB)

Purpose: AB is one of the most clinically challenging Gram-negative pathogens. However, there are significant gaps in our understanding of the periplasmic target site penetration of β-lactams in AB. The β-lactams inactivate penicillin binding proteins (PBPs), which yields rapid and extensive morphological changes. This study aimed to characterize the extent of periplasmic target site penetration and PBP binding based on distinct morphological changes using latest flow cytometry and confocal microscopy.

Methods: Static in vitro time-kill experiments (inoculum: ~5.3 log₁₀ CFU/mL) over 5 h were performed in a quadruple knockout AB strain AL3681 (lacking two efflux pumps and both β-lactamases, Δbla_ADC Δbla_OXA-51, ΔadeABC, ΔadeIJK). Based on our PBP IC₅₀ and MIC data on 50 drugs in lysed AL3681, we identified the lowest concentration that showed morphological changes via flow cytometry and confocal microscopy for 34 β-lactams in monotherapy. Live/dead staining was performed using Syto9 and propidium iodide. We calculated the penetration factor (PF) for each drug by dividing the lowest concentration yielding a noticeable morphological change in flow cytometry by the highest affinity PBP IC₅₀.

Results: Inactivation of PBP3 (or of PBP1a) yielded filaments, whereas PBP2 inactivation led to spheres. Imipenem and meropenem required 8x higher concentrations to yield spheres compared to their PBP2 IC₅₀ (yielding a PF of 8). In contrast, mecillinam yielded spheres at 2x the PBP2 IC₅₀. The PF was lowest (0.25) for sulbactam and tazobactam which both targeted PBP3, 1a and 1b with IC₅₀s of 2 to 4 mg/L, whereas 4-methoxy β-lactams had the highest PF of 64 and 256. There was a trend (r²=0.7) of lower PF for drugs with higher PBP IC₅₀ for their primary target(s).

Conclusion: This novel flow cytometry and confocal microscopy approach could efficiently quantify the periplasmic penetration and PBP binding of β-lactams and β-lactamase inhibitors based on morphological changes. Our data suggested that mass balance in periplasm was important, as drugs with higher primary target IC₅₀s tended to require lower multiples of this IC₅₀ to yield distinct morphology changes.