異硫氰酸酯和螯合劑合併使用於清除化學誘導細菌持久細胞的評估

Evaluation of the Combined Application of Isothiocyanates and Chelating Agents for Eliminating Chemically Induced Bacterial Persisters

細菌抗藥性在目前為嚴重的公共衛生問題。細菌可以透過發展持久性及抗藥性來抵禦抗生素以提高生存機會。細菌持久細胞(bacterial persisters cell)其特徵是少部分細菌細胞會處於休眠狀態，導致其代謝活動降低，並提高對抗生素的耐藥性，雖然與其他細菌種群在表型上不同，但在基因上相同的細菌型態，在細菌產生抗藥性及慢性感染復發中扮演著重要角色。因此我們計畫透過合併ethylenediaminetetraacetic acid (EDTA)來提高異硫氰酸酯(isothiocyanates,ITCs)類藥物的抗菌效果。其中EDTA是一種螯合劑，已證實可以增加細菌外膜的滲透性。而ITCs是天然抗菌分子，可有效抑制許多病原菌的生長。我們同時評估EDTA與ITCs聯合使用在對抗細菌持久性和細菌生長之抑制作用。實驗中細菌對抗菌劑的敏感性是由最低抑菌濃度(minimum inhibitory concentration, MIC)測定。化學誘導細菌持久細胞則是利用carbonyl cyanide m-chlorophenylhydrazone(CCCP)誘導使Pseudomonas aeruginosa進入持久性表型。並在單獨sub-MICITCs與合併EDTA的情況下，透過存活數來評估對細菌持久細胞的殺菌效果。最後以生長抑制作用，觀察前述抗菌藥物對細菌之抑制效果。結果顯示在CCCP誘導下P. aeruginosa所形成的細菌持久細胞數量比未經處理的細菌多約100倍，以MIC確認細菌持久細胞對ITCs類藥物並未產生抗藥性，存活數試驗中可見sub-MIC的Benzyl isothiocyanate (BITC)或indole-3-carbinol (I3C)合併EDTA使用後以劑量和時間依賴性方式測定，發現亦顯著降低細菌持久細胞的存活數。因此最後結果顯示，ITCs合併EDTA可有效地根除CCCP誘導的P. aeruginosa細菌持久細胞。這結果提供一種對抗細菌持久細胞的策略，亦可能有助於防止細菌之反覆感染。

Infections caused by multidrug-resistant (MDR) bacteria pose a significant threat to public health. Bacterial persisters, which exhibit reduced metabolic activity and heightened resistance to antibiotics while being genetically identical to the rest of the bacterial population, are a distinct phenotype. These persistent bacteria play a crucial role in the recurrence of chronic infections. The study aimed to evaluate the combined effectiveness of EDTA and ITCs in combating bacterial persisters and preventing bacterial biofilm formation. EDTA acts as a chelating agent that enhances the permeability of the bacterial outer membrane, while ITCs are natural antibacterial molecules known to inhibit the growth of various pathogens. Bacterial persisters were enriched using a chemical-induced method involving exposure to Carbonyl cyanide m-chlorophenylhydrazone (CCCP). The susceptibility of bacteria to antimicrobial agents was determined through minimal inhibitory concentration (MIC) testing. The survival of bacterial persisters was evaluated by exposing them to sub-MIC levels of ITCs with or without EDTA. The number of persistent cells of Pseudomonas aeruginosa strain induced by CCCP was 100 times higher than that of untreated bacteria. There was no obvious difference in MIC values of antimicrobial compounds between CCCP-treated and untreated bacteria. However, the combined use of sub-MIC levels of benzyl isothiocyanate (BITC) or indole-3-carbinol (I3C) with EDTA led to a substantial reduction in the survival of persistent cells in a doseand time-dependent manner. These findings suggest that the combined application of ITCs and EDTA effectively eliminated CCCP-induced bacterial persisters, offering a promising strategy to combat these persisters and potentially prevent the recurrence of bacterial infections.