Kumar Deepak, Singhal Chaitali, Yadav Manisha, Joshi Pooja, Patra Priyanka, Tanwar Subhash, Das Amitava, Kumar Pramanik Sumit, Chaudhuri Susmita
Translational Health Science and Technology Institute (THSTI), Faridabad, India.
CSIR-Central Salt and Marine Chemicals Research Institute, Bhavnagar, India.
Front Microbiol. 2023 Jan 4;13:1006604. doi: 10.3389/fmicb.2022.1006604. eCollection 2022.
A novel nano-formulation (NF) that sensitizes (AB) to otherwise ineffective colistin is described in the present study. Infections due to multidrug resistant (MDR) AB represent a major therapeutic challenge, especially in situations of pre-existing colistin resistance (colR). Subsequently, boosting the effectiveness of colistin would be a better alternative tactic to treat AB infections rather than discovering a new class of antibiotics. We have previously demonstrated an NF comprising self-assembled guanidinium and ionic silver nanoparticles [AD-L@Ag(0)] to have anti-biofilm and bactericidal activity. We report NF AD-L@Ag(0) for the very first time for the potentiation of colistin in Gram-negative colistin-resistant bacteria. Our results implied that a combination of clinically relevant concentrations of colistin and AD-L@Ag(0) significantly decreased colistin-resistant AB bacterial growth and viability, which otherwise was elevated in the presence of only colistin. In this study, we have described various combinations of minimum inhibitory concentration (MIC) of colistin (MICcol, MICcol, and MICcol) and that of AD-L@Ag(0) [MICAD-L@Ag(0), MICAD-L@Ag(0), and MICAD-L@Ag(0)] and tested them against MDR AB culture. The results (in broth as well as in solid media) signified that AD-L@Ag(0) was able to potentiate the anti-microbial activity of colistin at sub-MIC concentrations. Furthermore, the viability and metabolic activity of bacterial cells were also measured by CTC fluorescence assay and ATP bioluminescence assay. The results of these assays were in perfect concordance with the scores of cultures (colony forming unit and culture turbidity). In addition, quantitative real-time PCR (qRT-PCR) was performed to unveil the expression of selected genes, DNAgyrA, DNAgyrB, and dac. These genes introduce negative supercoiling in the DNA, and hence are important for basic cellular processes. These genes, due to mutation, modified the Lipid A of bacteria, further resisting the uptake of colistin. Therefore, the expression of these genes was upregulated when AB was treated with only colistin, substantiating that AB is resistant to colistin, whereas the combinations of MICcol + MICAD-L@Ag(0) downregulated the expression of these genes, implying that the developed formulation can potentiate the efficiency of colistin. In conclusion, AD-L@Ag(0) can potentiate the proficiency of colistin, further enhancing colistin-mediated death of AB by putatively disrupting the outer membrane (OM) and facilitating bacterial death.
本研究描述了一种新型纳米制剂（NF），它能使耐多药鲍曼不动杆菌（AB）对原本无效的黏菌素敏感。耐多药AB引起的感染是一个重大的治疗挑战，尤其是在已存在黏菌素耐药（colR）的情况下。因此，提高黏菌素的有效性将是治疗AB感染的更好替代策略，而不是发现一类新的抗生素。我们之前已证明一种由自组装胍盐和离子银纳米颗粒[AD-L@Ag(0)]组成的NF具有抗生物膜和杀菌活性。我们首次报道了NF AD-L@Ag(0)可增强黏菌素对革兰氏阴性耐黏菌素细菌的作用。我们的结果表明，临床相关浓度的黏菌素与AD-L@Ag(0)联合使用可显著降低耐黏菌素AB细菌的生长和活力，而仅使用黏菌素时其生长和活力会升高。在本研究中，我们描述了黏菌素的最低抑菌浓度（MICcol、MICcol和MICcol）与AD-L@Ag(0)的最低抑菌浓度[MICAD-L@Ag(0)、MICAD-L@Ag(0)和MICAD-L@Ag(0)]的各种组合，并对它们进行了耐多药AB培养测试。结果（在肉汤和固体培养基中）表明，AD-L@Ag(0)能够在亚MIC浓度下增强黏菌素的抗菌活性。此外，还通过CTC荧光测定法和ATP生物发光测定法测量了细菌细胞的活力和代谢活性。这些测定结果与培养物的评分（菌落形成单位和培养物浊度）完全一致。另外，进行了定量实时PCR（qRT-PCR）以揭示选定基因DNAgyrA、DNAgyrB和dac的表达。这些基因在DNA中引入负超螺旋，因此对基本细胞过程很重要。由于突变，这些基因改变了细菌脂多糖A，进一步抵抗黏菌素的摄取。因此，仅用黏菌素处理AB时，这些基因的表达上调，证实AB对黏菌素耐药，而MICcol + MICAD-L@Ag(0)组合下调了这些基因的表达，这意味着所开发的制剂可以增强黏菌素的效率。总之，AD-L@Ag(0)可以增强黏菌素的效能，通过可能破坏外膜（OM）并促进细菌死亡，进一步增强黏菌素介导的AB死亡。
