University Grenoble Alpes, CEA, LETI, Technologies for Healthcare and Biology division, Microfluidic Systems and Bioengineering Lab, Grenoble, France.
Departamento de Microbiología, Facultad de Medicina, Universidad de Zaragoza, Zaragoza, Spain.
PLoS One. 2019 Sep 3;14(9):e0220684. doi: 10.1371/journal.pone.0220684. eCollection 2019.
Bacterial resistance to antibiotics is widely regarded as a major public health concern with last resort MRSA treatments like vancomycin now encountering resistant strains. TFDs (Transcription Factor Decoys) are oligonucleotide copies of the DNA-binding sites for transcription factors. They bind to and sequester the targeted transcription factor, thus inhibiting transcription of many genes. By developing TFDs with sequences aimed at inhibiting transcription factors controlling the expression of highly conserved bacterial cell wall proteins, TFDs present as a potential method for inhibiting microbial growth without encountering typical resistance mechanisms. However, the efficient protection and delivery of the TFDs inside the bacterial cells is a critical step for the success of this technology. Therefore, in our study, specific TFDs against S. aureus were complexed with two different types of nanocarriers: cationic nanostructured lipid carriers (cNLCs) and chitosan-based nanoparticles (CS-NCs). These TFD-carrier nanocomplexes were characterized for size, zeta potential and TFD complexation or loading efficiency in a variety of buffers. In vitro activity of the nanocomplexes was examined alone and in combination with vancomycin, first in methicillin susceptible strains of S. aureus with the lead candidate advancing to tests against MRSA cultures. Results found that both cNLCs and chitosan-based carriers were adept at complexing and protecting TFDs in a range of physiological and microbiological buffers up to 72 hours. From initial testing, chitosan-TFD particles demonstrated no visible improvements in effect when co-administered with vancomycin. However, co-delivery of cNLC-TFD with vancomycin reduced the MIC of vancomycin by over 50% in MSSA and resulted in significant decreases in viability compared with vancomycin alone in MRSA cultures. Furthermore, these TFD-loaded particles demonstrated very low levels of cytotoxicity and haemolysis in vitro. To our knowledge, this is the first attempt at a combined antibiotic/oligonucleotide-TFD approach to combatting MRSA and, as such, highlights a new avenue of MRSA treatment combining traditional small molecules drugs and bacterial gene inhibition.
细菌对抗生素的耐药性被广泛认为是一个主要的公共卫生问题，而万古霉素等最后的 MRSA 治疗方法现在也遇到了耐药菌株。TFD（转录因子诱饵）是转录因子 DNA 结合位点的寡核苷酸拷贝。它们与靶转录因子结合并将其隔离，从而抑制许多基因的转录。通过开发针对控制高度保守细菌细胞壁蛋白表达的转录因子的序列的 TFD，TFD 呈现出一种抑制微生物生长而不遇到典型耐药机制的潜在方法。然而，TFD 在细菌细胞内的有效保护和递送是该技术成功的关键步骤。因此，在我们的研究中，针对金黄色葡萄球菌的特定 TFD 与两种不同类型的纳米载体复合：阳离子纳米结构化脂质载体（cNLCs）和壳聚糖基纳米粒子（CS-NCs）。这些 TFD-载体纳米复合物的大小、ζ电位以及 TFD 复合或负载效率在各种缓冲液中进行了表征。单独和与万古霉素联合使用时，体外活性首先在耐甲氧西林的金黄色葡萄球菌敏感株中进行了测试，先导候选物推进了对 MRSA 培养物的测试。结果发现，cNLCs 和壳聚糖载体都能够在一系列生理和微生物缓冲液中复合和保护 TFD 长达 72 小时。从初步测试来看，壳聚糖-TFD 颗粒与万古霉素联合使用时，在效果上没有明显改善。然而，cNLC-TFD 的共递送使 MSSA 中的万古霉素 MIC 降低了 50%以上，并且与单独使用万古霉素相比，MRSA 培养物的活力显著降低。此外，这些负载 TFD 的颗粒在体外表现出非常低的细胞毒性和溶血水平。据我们所知，这是首次尝试将抗生素/寡核苷酸-TFD 联合用于对抗 MRSA，因此强调了一种新的 MRSA 治疗途径，将传统小分子药物与细菌基因抑制相结合。
