| Abstract [eng] |
Investigation of Carbapenemase Genes by Real-Time Polymerase Chain Reaction Method Carbapenemases also known as β-lactamases are ferments that hydrolyze penicillins, cephalosporins, also, carbapenems and monobactams – nowadays the most important antibacterial pharmaceuticals against bacterial infections. The prevalence of Gram-negative bacteria, which is resistant to carbapenems, is growing around the world and causing more and more threat to the public health. This problem is related with the most common human pathogens which are responsible for majority of the public and nosocomial infections - Enterobacteriaceae, Pseudomonas spp. and Acinetobacter spp. The increasing use of carbapenems for the treatment of diseases, which were caused by these bacteria, resulted the selection and spreading of the pathogens that produce carbapenemases. In the past year, the carbapenemase-producing bacteria spread in almost all regions of the world. Bacteria that produce carbapenemases can hydrolyze almost all β-lactam antibiotics, and the genes encoding resistance, are transferred with plasmid between different species of bacteria. These genes are found in a lot of multiple resistance characterized isolates. Detection of such isolates from clinical material first of all starts from careful selection according to decreased sensitivity to carbapenems. After, there are made biochemical identification tests of the carbapenemases producers. In the end, the molecular methods are invoked for the carbapenemase gene identification, which remain the gold standards for the identification of carbapenemase genes. The aim of this work was to create the research protocol, based on real time PCR method of carbapenemase encoding genes (blaGIM, blaIMP, blaKPC, blaNDM, blaOXA-48, blaVIM) and evaluate these genes prevalence in VUH Santaros clinics from carbapenem-resistant Enterobacteriaceae family and Pseudomonas genus clinical isolates, who were grown in the Microbiology laboratory. BlaGIM gene encodes German imipenemase, blaIMP – imipenemase, blaKPC – Klebsiella pneumoniae carbapenemase, blaNDM – New Delhi metallo-β-lactamase, blaOXA-48 – carbapenem-hydrolyzing oxacillinase, and blaVIM – Verona integron-encoded metallo-β-lactamase. In the thesis were investigated 120 Enterobacteriaceae family and 143 Pseudomonas genus clinical isolates that correspondent the criteria of potential Carbapenemases producers according to the resistance to carbapenems. Typing was made by polymerase chain reaction method with TaqMan probes. The study shows that created TL-PGR protocol with TaqMan probes were suitable for detection of carbapenemase genes. According to the methods of statistical analysis it was assessed that the prevalence of blaNDM gene of VUH Santaros clinics reaches 1,52 %, and blaVIM – 23,78 %. The prevalence of blaGIM, blaIMP, blaKPC and blaOXA-48 genes reaches 0 %. Although, carbapenemases are the main resistance mechanism to antibiotics in the bacteria, however, this research results show that only the part of isolates are characterized by production of carbapenemases. The resistance to carbapenems of other investigated Enterobacteriaceae family and Pseudomonas genus clinical isolates should be related with other resistance mechanisms such as efflux pumps, porins or mutations which change the expression and functions of the penicillin binding proteins. |
