Güldaş Nevcivan, Bayrakal Vahide, Bahar I Hakkı
Dokuz Eylul University Faculty of Medicine, Research Laboratory, Izmir, Turkey.
Mikrobiyol Bul. 2013 Jan;47(1):19-26. doi: 10.5578/mb.4011.
The frequency of infections caused by Staphylococcus aureus strains and the rate of antibiotic resistance among these strains are gradually increasing. Accordingly, serious problems emerge in the treatment of community or hospital acquired S.aureus infections. This study was aimed to determine the role of MIC and sub-MIC concentrations of gentamicin on biofilm and coagulase forming effects of S.aureus in in vitro test systems and cell cultures. A standard S.aureus ATCC 25923 strain and two clinical S.aureus strains isolated from blood cultures (C1 and C2) were included in the study. Gentamicin MIC values of the strains were determined with microdilution method at the cation-adjusted Mueller Hinton broth according to CLSI standards. For each strain, MIC, 50% MIC and 25% MIC values of gentamicin were determined separately. At the determined MIC values, biofilm formations of strains were determined with crystal violet method spectrophotometrically. Also, coagulase activities of the strains were evaluated in glass tubes. Human origin epithelial cell cultures namely HEp-2 cell lines, were infected with the standard and clinical S.aureus strains (Multiplicity of infection: 50/1) and left for incubation for two hours. After all, MIC, 50% MIC and 25% MIC values of gentamicin, were added to infected cell lines and incubated for 18 hours. Cells were blown up with distilled water and then bacteria were collected. Biofilm formation and coagulase production of these bacteria were evaluated. When S.aureus ATCC 25923 strain and C1 strains' biofilm formation was evaluated before (in vitro) and after incubation in cell culture, no difference was observed. However in C2 strain, under the effect of MIC level gentamicin, biofilm formation was occurred after interaction with the cell. In the same way, when coagulase responses were evaluated, after interaction with the cell, coagulase production of C2 strain was inhibited. These results indicated that, phenotypic characteristics such as biofilm formation and coagulase production might change during the process of bacterial adaptation to microenvironment. Further advanced experimental modelling designed with different combinations of antibiotics and different cell lines may provide data about the causes and timing of these phenotypic changes and shed light on the development of new treatment policies.
金黄色葡萄球菌菌株引起感染的频率以及这些菌株中的抗生素耐药率正在逐渐上升。因此，社区或医院获得性金黄色葡萄球菌感染的治疗中出现了严重问题。本研究旨在确定庆大霉素的最低抑菌浓度（MIC）和亚MIC浓度在体外测试系统和细胞培养中对金黄色葡萄球菌生物膜形成和凝固酶形成的作用。研究纳入了标准金黄色葡萄球菌ATCC 25923菌株以及从血培养物中分离出的两株临床金黄色葡萄球菌菌株（C1和C2）。根据CLSI标准，采用微量稀释法在阳离子调节的 Mueller Hinton肉汤中测定菌株的庆大霉素MIC值。对于每株菌株，分别测定庆大霉素的MIC、50% MIC和25% MIC值。在测定的MIC值下，采用结晶紫法通过分光光度法测定菌株的生物膜形成情况。此外，在玻璃管中评估菌株的凝固酶活性。用人源上皮细胞培养物即HEp-2细胞系，用标准和临床金黄色葡萄球菌菌株进行感染（感染复数：50/1），并孵育两小时。之后，将庆大霉素的MIC、50% MIC和25% MIC值添加到感染的细胞系中，并孵育18小时。用蒸馏水裂解细胞，然后收集细菌。评估这些细菌的生物膜形成和凝固酶产生情况。当评估金黄色葡萄球菌ATCC 25923菌株和C1菌株在细胞培养孵育前后（体外）的生物膜形成情况时，未观察到差异。然而，在C2菌株中，在MIC水平庆大霉素的作用下，与细胞相互作用后发生了生物膜形成。同样，当评估凝固酶反应时，与细胞相互作用后，C2菌株的凝固酶产生受到抑制。这些结果表明，生物膜形成和凝固酶产生等表型特征在细菌适应微环境的过程中可能会发生变化。用不同抗生素组合和不同细胞系设计的进一步先进实验模型可能会提供有关这些表型变化的原因和时间的数据，并为新治疗策略的开发提供线索。
