Abstract：
The World Health Organization has included the problem of antibiotic resistance among the top 10 important health problems in the world. Treatment of infectious diseases has become more difficult due to the spread of antibiotic resistance between bacteria via transposable elements. Vancomycin-resistant enterococci (VRE) are of critical medical and public health importance due to their association with serious nosocomial infections and high risk of death. One of the most important features of VREs is that they have multiple antibiotic resistance and treatment options are reduced. Therefore, new treatment methods are needed. The vanA gene constitutes the building block of the vancomycin resistance mechanism and causes high resistance to vancomycin. In this study, it was aimed to investigate the neutralization of the vancomycin resistance mechanism by creating vanA antisense RNA (asRNA). The vanA positive VRE50 strain in our culture collection which was isolated from the clinical sample, was used to amplify the vanA gene by polymerase chain reaction (PCR). The amplified vanA amplicon was inserted inversely into the pUC19 plasmid by means of the enzyme cutting sites in the primers used. The resulting plasmid was combined with the pAT392 plasmid which can replicate in gram-positive bacteria and a fusion plasmid was created. The fusion plasmid whose orientation was confirmed, was transferred to the wild strain VRE50 by electroporation method. Minimum inhibitory concentration (MIC) values of transformed VRE (tVRE50) and wild type VRE50 strains used as control were determined by the E-Test method. The vancomycin MIC value of the wild type VRE50 strain was determined as 1024 µg/mL and that of the tVRE50 strain was 32 µg/mL and it was determined that the vancomycin resistance of the tVRE50 strain decreased with asRNA (antisense RNA). Antisense RNA technology is an important method for neutralizing the expression of genes. This study showed that neutralization of the vancomycin resistance gene may provide a lower MIC value in a vancomycin-resistant enterococcus strain and lead to increased susceptibility. This new approach provides a new method for VRE treatment by neutralizing the vancomycin resistance mechanism. The result obtained in this study needs to be supported by in vivo tests.
摘要：
世界卫生组织已将抗生素耐药性问题列为世界十大重要健康问题之一。由于通过转座因子在细菌之间传播抗生素抗性,感染性疾病的治疗变得更加困难。耐万古霉素肠球菌（VRE）由于与严重的医院感染和高死亡风险有关，因此具有至关重要的医学和公共卫生重要性。VRE的最重要特征之一是它们具有多种抗生素抗性并且治疗选择减少。因此,需要新的治疗方法。vanA基因构成万古霉素抗性机制的组成部分，并导致对万古霉素的高抗性。在这项研究中,目的通过创建vanA反义RNA（asRNA）来研究万古霉素耐药机制的中和作用。我们的培养物中的vanA阳性VRE50菌株是从临床样本中分离的，通过聚合酶链反应（PCR）扩增vanA基因。通过所用引物中的酶切割位点将扩增的vanA扩增子反向插入pUC19质粒中。将所得质粒与可在革兰氏阳性细菌中复制的pAT392质粒组合,并产生融合质粒。方向被证实的融合质粒，通过电穿孔方法转移至野生菌株VRE50。通过E-Test方法测定转化的VRE(tVRE50)和用作对照的野生型VRE50菌株的最小抑制浓度(MIC)值。野生型VRE50菌株的万古霉素MIC值被确定为1024μg/mL，tVRE50菌株的MIC值为32μg/mL，并确定tVRE50菌株的万古霉素抗性用asRNA（反义RNA）降低。反义RNA技术是中和基因表达的重要方法。这项研究表明，万古霉素抗性基因的中和可能在万古霉素抗性肠球菌菌株中提供较低的MIC值，并导致敏感性增加。这种新方法为通过中和万古霉素耐药机制治疗VRE提供了一种新方法。在这项研究中获得的结果需要得到体内测试的支持。
