Table_1_Regulation of anti-phage defense mechanisms by using cinnamaldehyde as a quorum sensing inhibitor.xlsx

BackgroundMultidrug-resistant bacteria and the shortage of new antibiotics constitute a serious health problem. This problem has led to increased interest in the use of bacteriophages, which have great potential as antimicrobial agents but also carry the risk of inducing resistance. The objective of the present study was to minimize the development of phage resistance in Klebsiella pneumoniae strains by inhibiting quorum sensing (QS) and thus demonstrate the role of QS in regulating defense mechanisms.ResultsCinnamaldehyde (CAD) was added to K. pneumoniae cultures to inhibit QS and thus demonstrate the role of the signaling system in regulating the anti-phage defense mechanism. The QS inhibitory activity of CAD in K. pneumoniae was confirmed by a reduction in the quantitative expression of the lsrB gene (AI-2 pathway) and by proteomic analysis. The infection assays showed that the phage was able to infect a previously resistant K. pneumoniae strain in the cultures to which CAD was added. The results were confirmed using proteomic analysis. Thus, anti-phage defense-related proteins from different systems, such as cyclic oligonucleotide-based bacterial anti-phage signaling systems (CBASS), restriction–modification (R–M) systems, clustered regularly interspaced short palindromic repeat-Cas (CRISPR-Cas) system, and bacteriophage control infection (BCI), were present in the cultures with phage but not in the cultures with phage and CAD. When the QS and anti-phage defense systems were inhibited by the combined treatment, proteins related to phage infection and proliferation, such as the tail fiber protein, the cell division protein DamX, and the outer membrane channel protein TolC, were detected.ConclusionInhibition of QS reduces phage resistance in K. pneumoniae, resulting in the infection of a previously resistant strain by phage, with a significant increase in phage proliferation and a significant reduction in bacterial growth. QS inhibitors could be considered for therapeutic application by including them in phage cocktails or in phage-antibiotic combinations to enhance synergistic effects and reduce the emergence of antimicrobial resistance.

背景：多重耐药细菌的出现以及新型抗生素的短缺构成了一项严重的公共卫生问题。这一问题促使人们加大对噬菌体应用的研究兴趣，噬菌体作为抗菌剂具有巨大的潜力，但也存在诱导耐药性的风险。本研究旨在通过抑制群感应（QS）来最小化肺炎克雷伯菌菌株对噬菌体的耐药性发展，从而验证QS在调节防御机制中的作用。结果：香草醛（CAD）被添加到肺炎克雷伯菌培养物中以抑制QS，并以此展示信号系统在调节抗噬菌体防御机制中的角色。通过lsrB基因（AI-2途径）的定量表达降低以及蛋白质组学分析证实了CAD在肺炎克雷伯菌中的QS抑制活性。感染实验表明，噬菌体能够感染先前对CAD敏感的肺炎克雷伯菌菌株。使用蛋白质组学分析对结果进行了确认。因此，不同系统中的抗噬菌体防御相关蛋白，如基于环状寡核苷酸的细菌抗噬菌体信号系统（CBASS）、限制-修饰（R-M）系统、成簇规律间隔的回文重复序列-Cas（CRISPR-Cas）系统和噬菌体控制感染（BCI），存在于噬菌体培养物中，而不存在于噬菌体与CAD共存的培养物中。当QS和抗噬菌体防御系统被联合治疗抑制时，检测到了与噬菌体感染和增殖相关的蛋白质，例如尾纤维蛋白、细胞分裂蛋白DamX和外膜通道蛋白TolC。结论：抑制QS可降低肺炎克雷伯菌的噬菌体耐药性，导致噬菌体感染先前耐药菌株，噬菌体增殖显著增加，细菌生长显著减少。QS抑制剂可以考虑作为治疗应用，通过将其纳入噬菌体混合物或噬菌体-抗生素组合中，以增强协同作用并减少抗菌耐药性的产生。