A new beta-lactam antibiotic increases the intracellular concentration of Drug Z (an antibiotic that targets an intracellular enzyme). Which experimental observation would best support the hypothesis that the beta-lactam enhances uptake to produce synergy?

• A. Beta-lactam induces expression of a host immune modulator that enhances phagocytosis, and Drug Z remains at identical intracellular concentrations; enhanced clearance by immune cells rather than increased uptake accounts for improved efficacy.
• B. Serum protein binding of Drug Z decreases in the presence of beta-lactam, and time-kill curves show no change in intracellular concentrations but increased renal clearance, indicating a pharmacokinetic interaction driving synergy.
• C. The bacteria upregulate efflux pumps in response to the beta-lactam causing decreased intracellular Drug Z concentration, yet overall killing increases which suggests that the two drugs operate on completely unrelated pathways and that this is classical antagonism rather than synergy.
• D. Measured intracellular levels of Drug Z rise significantly when bacteria are pre-treated with the beta-lactam, and checkerboard testing shows FIC index ≤ 0.5 for the combination, indicating synergy.

Answer: (D)