Transcriptional responses to tetracycline differ in highly syntenous strains of probiotic Bifidobacterium animalis subsp. lactis.

Commercial probiotic bacteria must be tested for acquired antibiotic resistance elements to avoid potential transfer to pathogens. The European Food Safety Authority (EFSA) recommends testing resistance using microdilution culture techniques, which have been used to establish inhibitory thresholds for the Bifidobacterium genus. Many Bifidobacterium animalis subsp. lactis strains exhibit increased resistance to tetracycline, historically attributed to the ribosomal protection gene tet(W). However, some strains that harbor genetically identical tet(W) genes exhibit various inhibition levels suggesting that other genetic elements contribute as well. In this report, we adapted several molecular assays to confirm the inhibition of B. lactis strains Bl-04 and HN019, and then employed RNA-seq to assess the transcriptional differences related to genomic polymorphisms. We detected specific stress responses to the antibiotic by correlating ATP concentration to viable genome copies from droplet digital PCR, and found that the bacteria were still metabolically active in high concentrations of the drug. Transcriptional analyses revealed that several polymorphic regions, particularly a novel multi-drug efflux transporter, were differentially expressed between the strains in each experimental condition, likely having phenotypic effects. We also found that the tet(W) gene was upregulated only during sub-inhibitory concentrations of tetracycline, while two novel tetracycline resistance genes were upregulated during high concentrations. Furthermore, many genes involved in amino acid metabolism and transporter function were upregulated while genes for complex carbohydrate utilization, protein metabolism, and CRISPR-Cas were down-regulated. These results provide high-throughput means for assessing antibiotic resistance and show the network of genetic elements that contribute to the global tetracycline response between two highly related probiotic strains.

商用益生菌需针对其获得性抗生素耐药元件开展检测，以避免耐药基因向病原菌发生潜在转移。欧洲食品安全局（European Food Safety Authority, EFSA）推荐采用微量稀释培养技术进行耐药性检测，该方法已被用于确立双歧杆菌属（Bifidobacterium genus）的抑菌阈值。诸多动物双歧杆菌乳亚种（Bifidobacterium animalis subsp. lactis）菌株对四环素表现出增强的耐药性，这一现象既往被认为由核糖体保护蛋白基因tet(W)介导。然而，部分携带遗传一致的tet(W)基因的菌株却呈现出不同的抑菌耐受水平，提示尚有其他遗传元件共同参与耐药过程。本研究对多种分子检测手段进行优化适配，以验证乳双歧杆菌菌株Bl-04与HN019的抑菌特性，随后借助RNA测序（RNA-seq）技术分析与基因组多态性相关的转录差异。我们通过将ATP浓度与液滴数字PCR（droplet digital PCR）检测的存活基因组拷贝数进行关联分析，检测到菌株针对该抗生素的特异性应激反应；同时发现，即便在高浓度四环素环境中，这些细菌仍保持代谢活性。转录组分析结果显示，在各实验条件下，两菌株间存在多个多态性区域的差异表达，其中尤以一种新型多药外排转运体（multi-drug efflux transporter）最为显著，此类差异或对菌株表型产生影响。此外我们还发现，tet(W)基因仅在亚抑菌浓度（sub-inhibitory concentrations）四环素处理下发生上调，而两种新型四环素耐药基因则在高浓度四环素环境中呈现表达上调。进一步分析显示，诸多参与氨基酸代谢与转运蛋白功能的基因呈现表达上调，而参与复杂碳水化合物利用、蛋白质代谢以及CRISPR-Cas系统的基因则出现表达下调。本研究结果为抗生素耐药性评估提供了高通量检测手段，同时揭示了两株亲缘关系密切的益生菌菌株中，参与四环素全局应答的遗传元件调控网络。