Cao Chenxia, Wang Jicheng, Liu Yangshuo, Kwok Lai-Yu, Zhang Heping, Zhang Wenyi
Key Laboratory of Dairy Biotechnology and Engineering, Ministry of Education, Inner Mongolia Agricultural University, Huhhot, China.
Key Laboratory of Dairy Products Processing, Ministry of Agriculture, Inner Mongolia Agricultural University, Huhhot, China.
mSystems. 2020 Jan 28;5(1):e00853-19. doi: 10.1128/mSystems.00853-19.
The widespread use of antibiotics has caused great concern in the biosafety of probiotics. In this study, we conducted a 12-month adaptive laboratory evolution (ALE) experiment to select for antibiotics-adapted P-8, a dairy-originated probiotic bacterium. During the ALE process, the ampicillin MIC for the parental P-8 strain increased gradually and reached the maximum level of bacterial fitness. To elucidate the molecular mechanisms underlying the ampicillin-resistant phenotype, we comparatively analyzed the genomes and proteomes of the parental strain ( P-8) and two adapted lines ( 400g and 1600g). The adapted lines showed alterations in their carbon, amino acid, and cell surface-associated metabolic pathways. Then, gene disruption mutants were created to determine the role of six highly expressed genes in contributing to the enhanced ampicillin resistance. Inactivation of an ATP-dependent Clp protease/the ATP-binding subunit ClpL, a small heat shock protein, or a hypothetical protein resulted in partial but significant phenotypic reversion, confirming their necessary roles in the bacterial adaptation to ampicillin. Genomic analysis confirmed that none of the ampicillin-specific differential expressed genes were flanked by any mobile genetic elements; thus, even though long-term exposure to ampicillin upregulated their expression, there is low risk of spread of these genes and adapted drug resistance to other bacteria via horizontal gene transfer. Our study has provided evidence of the biosafety of probiotics even when used in the presence of antibiotics. Antibiotic resistance acquired by adaptation to certain antibiotics has led to growing public concerns. Here, a long-term evolution experiment was used together with proteomic analysis to identify genes/proteins responsible for the adaptive phenotype. This work has provided novel insights into the biosafety of new probiotics with high tolerance to antibiotics.
抗生素的广泛使用引发了对益生菌生物安全性的高度关注。在本研究中，我们进行了一项为期12个月的适应性实验室进化（ALE）实验，以筛选出适应抗生素的P-8，一种源自乳制品的益生菌。在ALE过程中，亲本P-8菌株对氨苄青霉素的最低抑菌浓度（MIC）逐渐增加，并达到了细菌适应性的最高水平。为了阐明耐氨苄青霉素表型背后的分子机制，我们对亲本菌株（P-8）和两个适应株系（400g和1600g）的基因组和蛋白质组进行了比较分析。适应株系在其碳代谢、氨基酸代谢和细胞表面相关代谢途径中表现出改变。然后，构建了基因敲除突变体，以确定六个高表达基因在增强氨苄青霉素抗性中的作用。ATP依赖性Clp蛋白酶/ATP结合亚基ClpL、一种小分子热休克蛋白或一种假定蛋白的失活导致了部分但显著的表型逆转，证实了它们在细菌适应氨苄青霉素过程中的必要作用。基因组分析证实，没有任何氨苄青霉素特异性差异表达基因侧翼存在任何可移动遗传元件；因此，即使长期暴露于氨苄青霉素会上调它们的表达，这些基因以及适应性耐药性通过水平基因转移传播到其他细菌的风险也很低。我们的研究提供了即使在抗生素存在的情况下使用益生菌时其生物安全性的证据。通过适应某些抗生素获得的抗生素抗性引发了公众越来越多的关注。在这里，一项长期进化实验与蛋白质组分析一起用于鉴定负责适应性表型的基因/蛋白质。这项工作为具有高抗生素耐受性的新型益生菌的生物安全性提供了新的见解。
