Viability-based assessment of antibiotic resistance in Vibrio scophthalmi using propidium monoazide-qPCR

Vibrio scophthalmi is a marine fish pathogen commonly isolated from Paralichthys olivaceus and Scophthalmus maximus, two flatfish species primarily farmed in East Asia. Conventional antibiotic susceptibility testing (AST) and minimum inhibitory concentration (MIC) assays detect only culturable cells and may therefore underestimate bacterial survival under antibiotic stress, whereas quantitative PCR (qPCR) can overestimate bacterial abundance by amplifying DNA from nonviable cells. To address these methodological limitations, propidium monoazide (PMA)-qPCR was applied as a viability-based molecular approach to quantify membrane-intact V. scophthalmi cells in isolates obtained from wild marine fish. Six antibiotics were evaluated, and the results of AST, MIC, and PMA-qPCR were comparatively analyzed using paired t-tests and Bland-Altman analyses. PMA-qPCR consistently reduced amplification from membrane-compromised cells and yielded more conservative estimates of bacterial abundance than conventional qPCR. Among the tested antibiotics, tetracycline treatments showed limited reductions in PMA-qPCR signals despite growth suppression in MIC assays, indicating the persistence of membrane-intact cells under bacteriostatic conditions. These findings are consistent with previous reports describing tetracycline tolerance in V. scophthalmi. This study represents the first application of PMA-qPCR for antibiotic susceptibility assessment in V. scophthalmi within a viability-based framework. The results highlight the methodological potential of this approach to complement culture-based assays and support future studies of antibiotic-resistant bacteria in marine and aquaculture contexts within a One Health-oriented perspective.