Propidium monoazide-quantitative PCR for antibiotic sensitivity testing and minimum inhibitory concentration testing of antibiotic-resistant bacteria

Abstract

Antibiotics are widely used in humans, livestock, and aquaculture, but they are not removed fully during wastewater treatment, leading to their accumulation in aquatic environments. This contributes to the spread of antibiotic-resistant bacteria (ARB), posing public health and ecosystem risks. Although antibiotic use has decreased, the transfer of antibiotic resistance genes via integrons among ARB remains a concern. Traditionally, methods such as antibiotic susceptibility testing (AST) and minimum inhibitory concentration (MIC) tests have been used to identify ARBs. However, these methods are limited by their variability, the necessity for time-consuming reculture, and the inability to detect viable but non-culturable (VBNC) bacteria. To overcome such limitations, the ability of propidium monoazide quantitative PCR (PMA-qPCR) to selectively detect only viable bacteria, excluding dead cells, was evaluated. This study assessed ARBs in oyster, sea pineapple, and fish farms along Korea's coast using both conventional AST and MIC tests alongside PMA-qPCR. Vibrio spp., frequently detected at all sites, were used as the target species. PMA-qPCR successfully detected VBNC bacteria that were undetectable in MIC tests, highlighting the potential for VBNC bacteria to be regenerated under favorable conditions and contribute to the spread of resistance genes. The discrepancies between MIC and PMA-qPCR results underscore the need for further development of PMA-qPCR methods to better assess antibiotic resistance and to determine optimal antibiotic dosages. © 2025 The Authors