Research On Mechanisms Of Acinetobacter Baumanmii Drug Tolerant Persister Formation

Acinetobacter baumanmii is an important nosocomial pathogens that can cause a serious respiratory infection, urinary tract infections and etc. The antimicrobial resistance to A.baumannii has been one of the biggest obstacles to eveloping of an effective therapy. The formation of drug tolerant persister of A.baumannii is an important factor leading to treatment failure and relapse. Recent investigation has revealed the importance of antimicrobial-resistance persister cells as a major treat of nosocomial infections. In-depth understanding of the regulatory mechanisms and characteristics of persister cells of A.baumannii is urgently required, as it would not only provide the possible management regime, and also serves as a study model for other persister in bacteria.In this study, the ATCC 19606 was chosen as a model strain to explore the essential features of A. baumannii persister and the underlying molecular mechanisms of persister formation. The growth curve and the minimum inhibitory concentration (MIC) were examined. The growth curve of A.baumannii was used to determine the time frame required in diferent stages (logarithmic, stationary phase), and also serves as the background knowledge in the subsequent experiments. The results showed that ATCC 19606 was susceptible to most antibiotics (9 of 13 antibiotics), resistance only to ampicillin (AMP) and cefepime (CPM).In order to reveal the characteristics and the mechanisms of persister formation, the methods involved in separation and quantification of A. baumannii persisters were optimized, and the persistence levels in different growth stages against different levels of antimicrobial pressures were compared. After being treated with different antibiotics, A. baumannii followed a distinct biphasic killing pattern similar to other bacteria, in which, an initial rapid killing of the bacterial population was observed, and the remaining viable drug tolerant persister cells were found after reached plateau phase. The persistence level of A. baumannii was shown to be closely related to different growth staged, as the level of persistence was found significantly higher in the stationary phase over samples collected during logarithmic phase. Moreover, the formation of persister cells appeared to be dose-depend to antibiotics, and the death curve can vary whilst different antibiotics were administered. In addition, polymyxin B (PB) and tobramycin (TOB) were shown to be able to kill persister cells. These results uncovered some fundamental characteristics of the formation A. baumannii persister cells.Bioinformatics tools have been used to search for type II Toxin-Antitoxin (TA) systems in 721 A. baumannii genomes which have been sequenced and open-accessed. The results showed that type II TA systems were highly diversed in A. baumannii, comprising at least 15 pairs of the TA systems. The distribution of five pairs of functional TA systems in 44 clinical MDR A. baumannii namely RelB/RelE, HicA/HicB, GP49/Cro (HigB/HigA), HTH/GNAT and DUF497/COG3514 were analyszed. The results showed that, HTH/GNAT, GP49/Cro (HigB/HigA) and DUF497/COG3514 were widely distributed, which accounted for 61%,56% and 40%, respectively. On the other hand, real-time PCR was used to confirm the relative expression of type Ⅱ TA system in persister and non-persister. The results revealed that three type Ⅱ TA systems GP49/Cro (HigB/HigA), HTH/GNAT and DUF497/COG3514 expression levels were statistic significantly higher than non-persister. The results suggested that these three type II TA systems might play an important role in the formation of A. baumannii persisters.In order to understand the main genes, pathways and regulatory mechanisms in the process of persister formation and transformation. The genes exhibited statistic significant expression differences, and their corresponding pathways at transcriptome level were analysed by Mi-Seq.The results showed that the expression levels of numerous genes changed at the first hour post-treatment and the fifith hour after sub-culture. The number of gene expressions with two-fold change were 898 and 704, respectivly. The genes involve in carbohydrate metabolism and energy metabolism pathways were shown to play an important role in the process of persister formation and transformation; whereas, xenobiotics biodegradation pathway expressed only in persister and it might be a way to survive in hostile environment. Cluster analysis found some genes up-regulated in persister and down-regulated in non-persister. In addition, the result of expression of four TA systems indicated that the expression level of GP49(HigB)/Cro(HigA) and DUF1044/RelB increased significantly in persister (E1-E3). In conclusion, the formation of A. baumannii persisters were shown to be controlled by a complex network of gene regulatory mechanisms, numerous genes were up-and down-regulated at different stages of normal growth or under the pressure of antimicrobials. Further investigations are required to narrow down the amount of information obtained in this study, which could potentially be used to develop an effective control method for A. baumannii infections.