Iron is essential for the formation, maturation and dispersal of bacterial biofilms, playing a crucial role in the physiological and metabolic functions of bacteria as well as in the regulation of virulence. Limited availability of iron can impair the formation of robust biofilms by altering cellular motility, hydrophobicity and protein composition of the bacterial surface. In this study, the antibiofilm activity of two natural iron chelating agents, kojic acid (5-hydroxy-2-hydroxymethyl-4H-pyran-4-one) and maltol (3-hydroxy-2-methyl-4-pyrone), were investigated against Staphylococcus aureus and Pseudomonas aeruginosa. In addition, the ability of these 2-hydroxy-4-pyrone derivatives in preventing and eradicating S. aureus and P. aeruginosa biofilms through the enhancement of the efficacy of two antibiotics (tobramycin and ciprofloxacin) was explored. The iron binding capacity of the kojic acid and maltol was confirmed by their affinity for iron (III) which was found to be about 90 %, comparable to the regular chelating agent ethylenediaminetetraacetic acid (EDTA, 89 %). The antibiofilm efficacy of 2-hydroxy-4-pyrone derivatives, alone and in combination with antibiotics, was evaluated by measuring the total biomass, metabolic activity, and culturability of biofilm cells. Furthermore, their impact on the membrane integrity of S. aureus biofilm cells was investigated using flow cytometry and epifluorescence microscopy with propidium iodide staining. It was also examined the ability of 2-hydroxy-4-pyrone derivatives and 2-hydroxy-4-pyrone derivate-antibiotic dual-combinations in inhibiting the production of virulence factors (total proteases, lipases, gelatinases and siderophores) by S. aureus. Regarding biofilm formation, the results showed that 2-hydroxy-4-pyrone derivatives alone reduced the metabolic activity of S. aureus biofilm cells by over 40 %. When combined with tobramycin, a 2-log (CFU cm⁻²) reduction in S. aureus biofilm cells was observed. Moreover, the combination of maltol and kojic acid with ciprofloxacin prevented P. aeruginosa biomass production by 60 %, compared to 36 % with ciprofloxacin alone. In pre-established S. aureus and P. aeruginosa biofilms, selected compounds reduced the metabolic activity by over 75 %, and a 3-log (CFU cm⁻²) reduction in the culturability of biofilm cells was noted when kojic acid and maltol were combined with antibiotics. Moreover, 2-hydroxy-4-pyrone derivatives alone and in combination with tobramycin, damaged the cell membranes of pre-established biofilms and completely inhibited total proteases production. Despite the increasing of reactive oxygen species production caused by the cellular treatment of maltol, both 2-hydroxy-4-pyrone derivatives showed good safe profile when tested in human hepatocarcinoma (HepG2) cells. The pre-treatment of HepG2 cells with both compounds was crucial to prevent the cellular damage caused by iron (III). This study demonstrates for the first time that the selected 2-hydroxy-4-pyrone derivatives significantly enhance the antibiofilm activity of tested antibiotics against S. aureus and P. aeruginosa, highlighting their potential as antibiotic adjuvants in preventing and eradicating biofilm-related infections.

中文翻译：

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揭示天然螯合剂在控制金黄色葡萄球菌和铜绿假单胞菌生物膜中的潜力


铁对于细菌生物膜的形成、成熟和扩散至关重要，在细菌的生理和代谢功能以及毒力的调节中起着至关重要的作用。铁的可用性有限会通过改变细菌表面的细胞运动、疏水性和蛋白质组成来损害稳健生物膜的形成。在这项研究中，研究了两种天然铁螯合剂曲酸 （5-hydroxy-2-hydroxymethyl-4H-pyran-4-one） 和麦芽酚 （3-hydroxy-2-methyl-4-pyrone） 对金黄色葡萄球菌和铜绿假单胞菌的抗生物膜活性。此外，还探讨了这些 2-羟基-4-吡喃酮衍生物通过增强两种抗生素（妥布霉素和环丙沙星）的疗效来预防和根除金黄色葡萄球菌和铜绿假单胞菌生物膜的能力。曲酸和麦芽酚的铁结合能力通过它们对铁的亲和力 （III） 得到证实，发现铁的亲和力约为 90%，与常规螯合剂乙二胺四乙酸（EDTA，89%）相当。通过测量生物膜细胞的总生物量、代谢活性和培养性来评估 2-羟基-4-吡喃酮衍生物单独和与抗生素联合使用的抗生物膜功效。此外，使用流式细胞术和落射荧光显微镜与碘化丙啶染色研究了它们对金黄色葡萄球菌生物膜细胞膜完整性的影响。还检查了 2-羟基-4-吡喃酮衍生物和 2-羟基-4-吡喃酮衍生物-抗生素双重组合抑制金黄色葡萄球菌产生毒力因子（总蛋白酶、脂肪酶、明胶酶和铁载体）的能力。 关于生物膜形成，结果表明，单独的 2-羟基-4-吡喃酮衍生物可将金黄色葡萄球菌生物膜细胞的代谢活性降低 40% 以上。当与妥布霉素联合使用时，观察到金黄色葡萄球菌生物膜细胞减少 2 对数 （CFU ^cm-2）。此外，麦芽酚和曲酸与环丙沙星的组合阻止了铜绿假单胞菌生物质的产生 60%，而单独使用环丙沙星则阻止了 36%。在预先建立的金黄色葡萄球菌和铜绿假单胞菌生物膜中，选定的化合物将代谢活性降低了 75% 以上，并且当曲酸和麦芽酚与抗生素联合使用时，观察到生物膜细胞的培养性降低了 3 对数 （CFU ^cm-2）。此外，2-羟基-4-吡喃酮衍生物单独使用和与妥布霉素联合使用，破坏了预先建立的生物膜的细胞膜，并完全抑制了总蛋白酶的产生。尽管麦芽酚细胞处理导致活性氧产生增加，但在人肝癌 （HepG2） 细胞中测试时，两种 2-羟基-4-吡喃酮衍生物均显示出良好的安全性。用这两种化合物对 HepG2 细胞进行预处理对于防止铁 （III） 引起的细胞损伤至关重要。本研究首次证明，选定的 2-羟基-4-吡喃酮衍生物显着增强了测试抗生素对金黄色葡萄球菌和铜绿假单胞菌的抗生物膜活性，突出了它们作为抗生素佐剂在预防和根除生物膜相关感染方面的潜力。