In a viable but nonculturable (VBNC) state, bacteria are no longer culturable on standard laboratory media, but still, remain a pathogenic potential and present possible health risks. In this study, we investigated ampicillin's ability, which is commonly used in dairy cattle disease treatment, to induce Cronobacter sakazakii into the VBNC state. After treatment with ampicillin, the counts of culturable cells decreased from 10⁸ CFU/mL to an undetected level 7–30 days post-treatment. Meanwhile, viable cells were still approximately 10⁴–10⁵ cells/mL, and could be resuscitated under appropriate conditions. Fluorescence microscopy showed that VBNC cell maintained apparent cellular integrity, but that the morphology of VBNC cells differed visibly from that of normal cells. Moreover, the respiratory chain activity of VBNC cells were confirmed by flow cytometry (FCM) analysis, suggesting that cells in a VBNC state were physiologically active. Finally, transcriptomics analysis and real-time PCR (qPCR) validation were used to explore the underlying mechanisms of VBNC cell formation. Over-expression of relA, lon, ppx, and ppk in the toxin-antitoxin (TA) trigger system contributed to VBNC cell formation. In the TA trigger system, RelA and exopolyphosphatases/guanosine pentaphosphate phosphohydrolases (PPX/GPPA) synthesize ppGpp, which activates polyphosphate kinase (PPK), the cellular enzyme that accumulates plyphosphate (PolyP). PolyP combines with and stimulates Lon to degrade the antitoxins, thereby activating the toxins that induce a VBNC state. The results of our research will facilitate a better understanding of the survival strategies that bacteria develop to deal with ampicillin pressure and the health risks associated with VBNC Cronobacter sakazakii induced by antibiotics.

在可行但不可培养（VBNC）的状态下，细菌不再可以在标准实验室培养基上进行培养，但仍然具有潜在的致病性，并存在健康隐患。在这项研究中，我们调查了通常在奶牛疾病治疗中使用的氨苄西林诱导阪崎肠杆菌进入VBNC状态的能力。用氨苄西林治疗后，可培养细胞的数量从10 ⁸  CFU / mL降至治疗后7-30天未检测到的水平。同时，存活细胞仍约为10 ⁴ –10 ⁵细胞/ mL，可在适当条件下复苏。荧光显微镜显示VBNC细胞保持明显的细胞完整性，但是VBNC细胞的形态与正常细胞明显不同。此外，通过流式细胞术（FCM）分析证实了VBNC细胞的呼吸链活性，这表明处于VBNC状态的细胞具有生理活性。最后，转录组学分析和实时PCR（qPCR）验证被用来探索VBNC细胞形成的潜在机制。relA，lon，ppx和ppk的过表达在毒素-抗毒素（TA）触发系统中的作用有助于VBNC细胞的形成。在TA触发系统中，RelA和胞外多磷酸酶/鸟苷五磷酸鸟苷磷酸水解酶（PPX / GPPA）合成ppGpp，从而激活聚磷酸激酶（PPK），聚磷酸激酶（PPK）是一种积累聚磷酸（PolyP）的细胞酶。PolyP与Lon结合并刺激Lon降解抗毒素，从而激活诱导VBNC状态的毒素。我们的研究结果将有助于人们更好地理解细菌为应对氨苄青霉素压力而开发的生存策略以及与抗生素诱发的阪崎VBNC克罗诺杆菌相关的健康风险。