Molecular Detection of acrAB and oqxAB Genes in Klebsiella pneumoniae and Evaluation the Effect of Berberine on their Gene Expression
One of the factors contributing to increase bacterial resistance to antimicrobial substances is the presence of multidrug efflux pumps. AcrAB and OqxAB are the most extensively researched efflux pumps genes in K. pneumoniae in relation to antibiotic resistance. The objectives of this study are Isolation and identification of K. pneumoniae from patient with urinary tract infections; detection the efflux pump genes (acrAB, oqxAB) in K. pneumoniae by Polymerase Chain Reaction using specific primers and then study the gene expression of one of the two pumps in the presence of the subinhibitory concentration of natural efflux pump inhibitor, Berberine. A total of 260 clinical specimens were collected from five of Baghdad hospitals. The identification of the isolates of K. pneumoniae depends on the biochemical tests. The Minimum Inhibitory Concentrations (MICs) of Berberine (plant – derived efflux pump inhibitors) against isolated bacteria was estimated with the microdilution broth method. Molecular Detection of acrA, acrB, oqxA and oqxB genes were carried out by polymerase chain reaction while the gene expression was done by real-time PCR. The results demonstrated that Klebsiella pneumoniae bacteria were found in 76 (29.2%) of all urine samples. The Minimum Inhibitory Concentrations (MICs) of Berberine HCL at the range 3.9-500 μg/ml. The detection of the efflux pump genes (acrAB, oqxAB) in K. pneumoniae revealed that all isolates have the 2 systems (100%). Relative quantification (RQ) was used to calculate the fold change in gene expression using the delta delta Ct value and the gene expression of the isolates was calculated before and after treatment with the subinhibitory concentration of each isolate. In oqxA gene, by using the sub-MIC values of Berberine in Klebsiella pneumoniae resistant isolates before treatment with Berberine, the fold of gene expression was (1), and the fold of the resistant isolates after Berberine treatment was low (0.1-0.04). For oqxB gene Before Berberine treatment, the fold of gene expression was slightly higher (1). After Berberine treatment, the fold of gene expression was low (0.2-0.0001). In conclusion, the obvious activity of Berberine as efflux pump inhibitor in K. pneumoniae may be contributed in the management and control the multidrug resistance especially in life threatening infections.