Transcriptional analysis of D, D-carboxypeptidase genes as secondary cephalosporin resistance reporters in Escherichia coli harbouring bla CTX-M

The emergence of novel plasmid-encoded resistance variants complicates antimicrobial resistance detection by conventional PCR-based methods. Therefore, endogenous secondary resistance genes expressed under antibiotic stresses to produce adaptive resistance mechanisms in bacteria may serve as reporters for AMR detection. This study investigated the transcriptional expression of D,D-carboxypeptidase genes and the transcriptional regulator bolA under oxyimino cephalosporin stress. Thirty clinical Escherichia coli isolates were tested for antibiotic susceptibility and extended-spectrum β-lactamase genes. Network analysis was performed using Cytoscape. D,D-carboxypeptidase genes were transformed into E. coli HST08 and B. subtilis RIK 1285 using pBE-S DNA; while blaCTX-M-harbouring plasmid was transformed into E. coli DH5α and subsequently eliminated from the parent isolate. MIC determination, growth assessment, and qRT-PCR were performed under ampicillin and oxyimino cephalosporins stress. Network analysis linked peptidoglycan synthesis, β-lactam resistance, and metabolic pathways with penicillin binding protein (PBP)-forming gene sets. Among 27 resistant isolates, 20 harboured blaCTX-M and 15 blaTEM. B. subtilis dacD and E. coli dacA clones showed elevated MICs. The study revealed consistent upregulation of dacB and dacD with host-specific effects on MICs, highlighting their potential as reporters for cephalosporin resistance in future diagnostics. Differential transcriptional responses observed among recombinant strains further suggested host-specific adaptive mechanisms during antibiotic exposure.