숨겨진 위협: VBNC 상태 Streptococcus parauberis의 소생과 병원성 회복
Hidden but harmful: resuscitation and pathogenic rebound of VBNC Streptococcus parauberis
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The persistence of bacterial pathogens in aquaculture environments under stress conditions raises growing concern regarding disease recurrence and diagnostic limitations. This study focused on Streptococcus parauberis, a key etiological agent of streptococcosis in olive flounder, and its ability to transition into and recover from the viable but non-culturable (VBNC) state under environmental challenges. Filtered sterile seawater microcosms were employed to simulate low-nutrient conditions at three temperatures (4, 10, 25°C). The entry into the VBNC state was monitored via culturability loss, PMA-qPCR, and fluorescence microscopy. Notably, the timing and stability of the VBNC state varied with temperature, with more rapid induction at 25°C and prolonged viability at 10°C. Resuscitation trials revealed that recovery was strictly dependent on both nutrient supplementation and temperature elevation to 28°C, with defined temporal limits depending on the prior induction condition. In parallel, a PMA-qPCR assay targeting the cpn60 gene was optimized to selectively detect viable S. parauberis cells. The assay proved sensitive across a wide range of live-to-dead ratios and effectively suppressed signals from non-viable cells. While VBNC cells alone exhibited no detectable pathogenicity in fish infection assays, their resuscitated counterparts induced significant mortality, underscoring the risk of undetected VBNC populations in aquaculture systems. This study highlights the adaptive plasticity of S. parauberis and presents a refined molecular approach for mon- itoring its hidden presence during low-activity phases, with practical implications for fish health surveil- lance and risk management strategies.
Abstract
The persistence of bacterial pathogens in aquaculture environments under stress conditions raises growing concern regarding disease recurrence and diagnostic limitations. This study focused on Streptococcus parauberis, a key etiological agent of streptococcosis in olive flounder, and its ability to transition into and recover from the viable but non-culturable (VBNC) state under environmental challenges. Filtered sterile seawater microcosms were employed to simulate low-nutrient conditions at three temperatures (4, 10, 25°C). The entry into the VBNC state was monitored via culturability loss, PMA-qPCR, and fluorescence microscopy. Notably, the timing and stability of the VBNC state varied with temperature, with more rapid induction at 25°C and prolonged viability at 10°C. Resuscitation trials revealed that recovery was strictly dependent on both nutrient supplementation and temperature elevation to 28°C, with defined temporal limits depending on the prior induction condition. In parallel, a PMA-qPCR assay targeting the cpn60 gene was optimized to selectively detect viable S. parauberis cells. The assay proved sensitive across a wide range of live-to-dead ratios and effectively suppressed signals from non-viable cells. While VBNC cells alone exhibited no detectable pathogenicity in fish infection assays, their resuscitated counterparts induced significant mortality, underscoring the risk of undetected VBNC populations in aquaculture systems. This study highlights the adaptive plasticity of S. parauberis and presents a refined molecular approach for mon- itoring its hidden presence during low-activity phases, with practical implications for fish health surveil- lance and risk management strategies.