Functional analysis of Colistin resistance associated genes in Acinetobacter sp. ADP1

• Main Authors: Chun-hui Chung, 鍾俊輝
• Other Authors: Kai-Chih Chang
• Format: Others
• Language: zh-TW
• Online Access: http://ndltd.ncl.edu.tw/handle/46374426323412826599

碩士 === 慈濟大學 === 醫學生物技術研究所 === 96 === Multi-drug resistant Acinetobacter baumannii had been verified in making severe nosocomial infection. In the past, there was no any appropriate antibiotic to cure it, but recently, presented by some review papers, indicated that colistin is capable of cure from the infections of multi-drug resistant gram-negative bacteria. Unfortunately, the multi-drug resistant Acinetobacter baumannii was easy to convert into colistin- resistant lineage when colistins existed surrounding its environment. There was no any related study about the mechanism of colistin resistance by Acinetobacter spp. presented before. Therefore, we were intended to study and focus on the analysis of colistin-resistant genes in Acinetobacter spp. First, we produced the colistin-resistant (CR) bacterial lineages, 33305CR and 17978CR, from induction of ATCC33305 (Acinetobacter sp. ADP1) and ATCC17978 (Acinetobacter baumannii) respectively by the method that increasing the colistin concentration gradually. Then, we used RT-PCR to analyze the gene ACIAD0727 and found that the RNA amount of ACIAD0727 gene expressed was higher in resistant bacterial lineage 33305CR than the sensitive lineage. Further, we tried to destroy the ACIAD0727 genes in both ATCC33305 and 33305CR lineages for additional analyses. The results showed that the standard bacterial lineage ATCC33305 with ACIAD0207 gene destroyed was more difficult to be induced to become the colistin-resistant one and the minimal inhibitory concentration (MIC) against colistin was obviously decreased in 33305CR lineage with ACIAD0207 gene destroyed. Whereas, with performing the complementary experience of ACIAD0727 gene, we found that the MIC against colistin was compensated for only a little degree by the complementary lineage. This maybe result from the amount of RNA expressed by the complementary lineage was less than original 33305CR lineage. By contrasting with the database, we could find the gene corresponding to the ACIAD0727 gene of Acinetobacter sp. ADP1 in ATCC17978 lineage, the A1S_0749 gene, with up to 94 % similarity. We also found the MIC against colistin was decreased in 17978CR lineage when destroying the A1S_0749 gene. Based on the results above, we could deduce that both the ACIAD0727 and A1S_0749 genes were related to the colistin resistance. Finally, we performed the functional analyses for both genes and obtained the result that they were not regulated by the concentration of magnesium (Mg2+) ion. Furthermore, destruction of both genes would make the bacterial lineage become mucoid form from non-mucoid one. Thus, we deduced the ACIAD0727 and A1S_0749 genes may be concerned with the exopolysaccharide regulation in Acinetobacter spp.