Design a two-step laboratory assay that distinguishes whether tetracycline resistance in an isolate is due to an efflux pump versus target protection (ribosomal polypeptide preventing binding). Which approach best separates these mechanisms?

Treat bacteria with a combination of high salt and low pH and observe growth; if growth persists, call it enzymatic inactivation because efflux pumps and target protection are both inhibited by pH shifts equally and thus indistinguishable.

Only perform a disk diffusion test with a single tetracycline concentration; if a zone of inhibition is absent, conclude target protection because disk diffusion cannot identify efflux contributions reliably.

Measure intracellular accumulation of radiolabeled tetracycline with and without an efflux pump inhibitor (e.g., CCCP); if accumulation increases with inhibitor, efflux is involved. Then perform a ribosomal protection assay or PCR to detect known protection genes; if pump inhibition doesn't restore sensitivity and protection gene is present, target protection is the mechanism.

Sequence the whole genome and if any mutation is found anywhere, attribute resistance to efflux because genome sequencing is always sufficient to separate molecular mechanisms without functional assays.