Molecular Identification Of Acinetobacter Baumannii And Its Quinolone Resistance Gene

Acinetobacter baumannii is the negative bacilli in the classification of Gram's bacteria,and it is a kind of conditional pathogenic bacteria.It exists not only in the hospital environment,but also in the natural ecological environment,which mainly caused infectious diseases,such as bacteremia,secondary meningitis and the infection of respiratory tract,urinary system,and the operative site.Since modern times,due to the incorrect use of antibiotics,multidrug-resistant and pan-resistant strains of Acinetobacter baumannii were found around the world,which caused the treatment of Acinetobacter baumannii faced to the impasse of infection drug selection or even no cured drugs.In recent years,quinolone-resistant strains were universal in Acinetobacter baumannii resistant strains.Therefore,a rapid detection method for Acinetobacter baumannii and its quinolone resistance gene was established,which provides a reliable method for rapid diagnosis and treatment in the clinic,and reached the purpose of accurate medication and timely treatment.At present,the main methods for the detection of Acinetobacter baumannii include traditional culture or automatic culture combined with drug sensitivity test,and mass spectrometry analysis.Culture methods with the disadvantages of time-consuming,low sensitivity,etc.Although mass spectrometry is fast and sensitive,the instrument used in this method is expensive and requires the pre-culture of the bacterium.Therefore,a method based on PCR and LAMP for the detection of Acinetobacter baumannii was established in this research,and PCR and multiplex PCR were used to detect the quinolone antibiotic resistance genes.Firstly,according to the principle of‘intraspecific common,interspecies specificity',screening the whole genome of Acinetobacter baumannii by local BLAST and online BLAST.And obtained a distinctive gene CP018677.1(2912288.2912548).PCR and LAMP identification systems were based on this gene.162 strains of Acinetobacter baumannii and 4 non-Acinetobacter baumannii including Pseudomonas aeruginosa,Escherichia coli,Klebsiella pneumoniae and Shigella were applied for specificity evaluation.The results showed that Acinetobacter baumannii could be identified by both PCR and LAMP and both with good specificity.Secondly,taking the gradient diluted positive cloning of specific genes and recultured Acinetobacter baumannii broth as template respectively for PCR reaction to the sensitivity evaluation of the system.The results showed that the sensitivity of positive plasmids could reach to 10~0 copy/reaction,and broth could reach to 10~1copies/reaction,which indicts the systems were with high sensitivity.Thirdly,a total of 3 quinolone resistance genes(qnrA,qnrB,qnr C)were found in Antibiotic Resistance Genes Database(ARDB).Download all kinds of resistant genes and their subtypes,and multiple sequence alignment of the downloaded drug resistance gene sequence by Align X multiple sequence alignment software.Design the primers of drug resistance genes based on the results of the comparison.According to the results of PCR,the detectable rate of qnr A,qnr B,qnr S in 162 Acinetobacter baumannii were41.36%,23.46%,and 89.51%respectively.Which is in accordance with the strains phenotype given by the hospital,and the results are accurate.Three positive plasmids with drug resistance genes were diluted by 10 times as a template for PCR reaction to test the sensitivity of the system.The results showed that sensitivity of positive plasmids with drug resistance genes qnrA,qnrS could be both reach to 1copy/reaction,and qnrB could be reach to 10 copy/reaction,respectively.The results showed that the system has high sensitivity.Lastly,multiplex PCR was used for the detection of specific gene CP018677.1(2912288.2912548)and three quinolone resistance genes qnr A,qnr B,qnrS.The results showed that the specific genes and three quinolones resistance genes can be detected in the multiplex PCR system at the same time.The detection results were consistent with PCR.In a word,a method based on PCR and LAMP in the detection of Acinetobacter baumannii was build in this research,and the primers used for the identified of specific genes and quinolone resistance genes were both the first time.Also,multiplex PCR system was built,which can quickly and efficiently detect Acinetobacter baumannii and its quinolone resistant genes.