Design a laboratory assay to distinguish whether an unknown antibiotic that inhibits bacterial growth acts like a beta-lactam or like vancomycin/bacitracin. Which of the following experimental approaches best differentiates these mechanisms?

Measure the accumulation of specific cell wall precursors: beta-lactam action leads to accumulation of uncross-linked muropeptides and activation of autolysins, whereas vancomycin-like agents cause accumulation of lipid II or UDP-MurNAc-peptide precursors or prevent incorporation of D-Ala-D-Ala termini. Assaying precursor pools and using radiolabeled precursors will distinguish the site of blockade.

Assess inhibition of bacterial protein synthesis using a 70S ribosome assay; both beta-lactams and vancomycin will show identical ribosomal inhibition profiles so matching patterns confirms a cell-wall mechanism.

Test whether the antibiotic induces the SOS DNA damage response; vancomycin triggers SOS whereas beta-lactams do not, and this single assay reliably identifies the drug class in all bacteria.

Sequence the host genome of the infected patient to find mutations associated with antibiotic response, because host genetics will directly reveal whether the bacterial target is transpeptidase or mycolic acid synthesizing enzymes.