Kuyucuklu Gülcan, Kaynak Onurdağ Fatma, Eryıldız Canan
Trakya University Faculty of Medicine, Department of Medical Microbiology, Edirne, Turkey.
Trakya University Faculty of Pharmacy, Department of Pharmaceutical Microbiology, Edirne, Turkey.
Mikrobiyol Bul. 2021 Apr;55(2):125-145. doi: 10.5578/mb.20219902.
Biofilms are often responsible for the difficulties in the treatment of infectious diseases due to their properties that facilitate escape from antibiotic effect and their antiphagocytic effects. At least 65% of all infectious diseases are associated with biofilm-forming bacteria. As Staphylococcus aureus and Staphylococcus epidermidis are among the most common agents of hospital infections and the infections are mostly biofilm-related, they pose an important problem. In infectious isolates, the minimum biofilm eradication concentration (MBEK) values of biofilm forms are much higher than the minimum inhibition concentration (MIC) values of planktonic forms. This situation requires the use of much higher doses of antibiotics in the treatment of infections and causes an increase in antibiotic resistance. The N-acetylcysteine (NAC) molecule is known to be effective against biofilm by disrupting mature biofilms and reducing the adhesion of bacteria to surfaces. In this study, it was aimed to demonstrate i) the biofilm-forming abilities ii) the change in ampicillin and vancomycin MIC values in the presence of NAC molecules, iii) the change in the MBEK values of these antibiotics in the presence of NAC molecule and iv) the change in the expression levels of genes thought to be related to biofilm formation in the presence of the NAC molecule among S.aureus (n= 38) and S.epidermidis (n= 12) isolates isolated from various clinical specimens in Trakya University Health Research and Application Center. In this study, microplate crystal violet method was used to demonstrate the biofilm formation in staphylococci. Broth microdilution and checkerboard method were used to demonstrate the change in the presence of NAC molecule of the MIC and MBEC values of ampicillin and vancomycin. The effect of NAC on the expression of intercellular binding proteins A and D (icaA, icaD) and Staphylococcus regulatory protein A (sarA) genes, which are the genes involved in biofilm formation in staphylococci, was determined by quantitative real-time Polymerase Chain Reaction (qRt-PCR) method. The Student-t test was used to compare the control and experimental groups (concentrations detected with synergy and additive effect); p˂ 0.05 was accepted as the limit value of significance. In this study, when the NAC molecule was used together with ampicillin and vancomycin, it was determined that this combination lowers the MIC values of staphylococcus isolates and staphylococcal biofilm MBEK values; and also the expression levels of icaA, icaD and sarA which were effective in biofilm formation in staphylococci have not changed and decreased. As a result, in this study, it has been determined that the NAC molecule can be a new alternative for combined drug therapy and is promising in terms of bringing a new approach to treatment. In addition, it is thought that it is possible to use the NAC molecule together with different microorganisms and antimicrobial agents, and the results obtained in this study are considered to be guiding for further studies on this subject.
生物膜因其具有促进逃避抗生素作用的特性及其抗吞噬作用,常常导致传染病治疗困难。所有传染病中至少65%与形成生物膜的细菌有关。由于金黄色葡萄球菌和表皮葡萄球菌是医院感染最常见的病原体之一,且这些感染大多与生物膜相关,它们构成了一个重要问题。在感染分离株中,生物膜形式的最低生物膜根除浓度(MBEK)值远高于浮游形式的最低抑菌浓度(MIC)值。这种情况在感染治疗中需要使用高得多剂量的抗生素,并导致抗生素耐药性增加。已知N - 乙酰半胱氨酸(NAC)分子通过破坏成熟生物膜和减少细菌对表面的粘附而对生物膜有效。在本研究中,旨在证明:i)来自恰纳卡莱大学健康研究与应用中心各种临床标本分离的金黄色葡萄球菌(n = 38)和表皮葡萄球菌(n = 12)分离株的生物膜形成能力;ii)在NAC分子存在下氨苄西林和万古霉素MIC值的变化;iii)在NAC分子存在下这些抗生素的MBEK值的变化;iv)在NAC分子存在下被认为与生物膜形成相关的基因表达水平的变化。在本研究中,使用微孔板结晶紫法证明葡萄球菌中的生物膜形成。使用肉汤微量稀释法和棋盘法证明在NAC分子存在下氨苄西林和万古霉素的MIC和MBEC值的变化。通过定量实时聚合酶链反应(qRt - PCR)方法确定NAC对细胞间结合蛋白A和D(icaA、icaD)以及葡萄球菌调节蛋白A(sarA)基因表达的影响,这些基因是葡萄球菌中参与生物膜形成的基因。使用Student - t检验比较对照组和实验组(协同和相加效应检测的浓度);p˂0.05被接受为显著性的极限值。在本研究中,当NAC分子与氨苄西林和万古霉素一起使用时,确定这种组合降低了葡萄球菌分离株的MIC值和葡萄球菌生物膜的MBEK值;并且在葡萄球菌生物膜形成中起作用的icaA、icaD和sarA的表达水平没有变化且有所降低。结果,在本研究中,已确定NAC分子可以成为联合药物治疗的新选择,并且在带来新的治疗方法方面很有前景。此外,认为有可能将NAC分子与不同的微生物和抗菌剂一起使用,并且本研究中获得的结果被认为对该主题的进一步研究具有指导意义。