Manoharan Arthika, Das Theerthankar, Whiteley Gregory S, Glasbey Trevor, Kriel Frederik H, Manos Jim
Department of Infectious Diseases and Immunology, Central Clinical School, The University of Sydney, Sydney, Australia.
Whiteley Corporation, 19-23 Laverick Avenue, Tomago NSW 2319, Australia.
J Antimicrob Chemother. 2020 Jul 1;75(7):1787-1798. doi: 10.1093/jac/dkaa093.
The WHO declared Staphylococcus aureus as a 'pathogen of high importance' in 2017. One-fifth of all bloodstream-related infections in Australia and 12 000 cases of bacteraemia in the UK (2017-18) were caused by the MRSA variant. To address the need for novel therapies, we investigated several permutations of an innovative combination therapy containing N-acetylcysteine (NAC), an antibiotic and an enzyme of choice in eradicating MRSA and MSSA biofilms.
Biofilm viability (resazurin assay) and colony count methods were used to investigate the effect of NAC, antibiotics and enzymes on S. aureus biofilm disruption and killing. The effects of NAC and enzymes on the polysaccharide content of biofilm matrices were analysed using the phenol/sulphuric acid method and the effect of NAC on DNA cleavage was determined using the Qubit fluorometer technique. Changes in biofilm architecture when subjected to NAC and enzymes were visualized using confocal laser scanning microscopy (CLSM).
NAC alone displayed bacteriostatic effects when tested on planktonic bacterial growth. Combination treatments containing 30 mM NAC resulted in ≥90% disruption of biofilms across all MRSA and MSSA strains with a 2-3 log10 decrease in cfu/mL in treated biofilms. CLSM showed that NAC treatment drastically disrupted S. aureus biofilm architecture. There was also reduced polysaccharide production in MRSA biofilms in the presence of NAC.
Our results indicate that inclusion of NAC in a combination treatment is a promising strategy for S. aureus biofilm eradication. The intrinsic acidity of NAC was identified as key to maximum biofilm disruption and degradation of matrix components.
世界卫生组织在2017年宣布金黄色葡萄球菌为“高度重要的病原体”。在澳大利亚，五分之一的血流相关感染以及英国2017 - 2018年的12000例菌血症病例是由耐甲氧西林金黄色葡萄球菌（MRSA）变种引起的。为满足对新型疗法的需求，我们研究了一种创新联合疗法的几种排列组合，该联合疗法包含N - 乙酰半胱氨酸（NAC）、一种抗生素以及一种用于根除MRSA和甲氧西林敏感金黄色葡萄球菌（MSSA）生物膜的酶。
采用生物膜活力（刃天青测定法）和菌落计数法来研究NAC、抗生素和酶对金黄色葡萄球菌生物膜破坏和杀灭的影响。使用苯酚/硫酸法分析NAC和酶对生物膜基质多糖含量的影响，并使用Qubit荧光计技术测定NAC对DNA裂解的影响。使用共聚焦激光扫描显微镜（CLSM）观察NAC和酶作用下生物膜结构的变化。
单独测试时，NAC对浮游细菌生长显示出抑菌作用。含30 mM NAC的联合治疗导致所有MRSA和MSSA菌株的生物膜破坏率≥90%，处理后的生物膜中cfu/mL降低2 - 3个对数级。CLSM显示，NAC处理极大地破坏了金黄色葡萄球菌生物膜结构。在NAC存在的情况下，MRSA生物膜中的多糖产量也有所降低。
我们的结果表明，在联合治疗中加入NAC是根除金黄色葡萄球菌生物膜的一种有前景的策略。NAC的固有酸性被确定为最大程度破坏生物膜和降解基质成分的关键。
