Investigation of extended spectrum beta lactamase enzyme with PCR-RFLP in K. pneumoniae strains isolated from clinical samples

Extended-spectrum beta lactamases (ESBLs) are responsible for resistance to cephalosporins (ceftazidime, ceftriaxone, and cefotaxime) and aztreonam in gram-negative bacilli. Extended spectrum beta lactamase producing Klebsiella bacteria are a major problem for clinicians. These enzymes increase are cause of failure in treatment particularly paediatric patients and also in medical and surgical units. In the research ESBL was investigated by phenotypical (Double disc synergy test, Clavulonic acide combination) and genotypical methods (PCR-RFLP). Plasmid analysis of Klebsiella isolates were also studied in this research.In the research, one hundred twenty eight Klebsiella bacteria were collected from different microbiology laboratories in Ankara. These bacteria were identified with classical methods and API 20E. According to the results of identification, 103 K. pneumoniae (86 urine culture, 7 lesion culture, 5 blood culture, 3 sputum culture, 2 endotracheal aspirates culture, 2 aspirate fluid culture) and 25 K. oxytoca (21 urine culture, 2 lesion culture, 1 endotracheal aspirates culture, 1 aspirate fluid culture) were obtained. ESBL has been detected %66 in Klebsiella bacteria by DDST. It has been detected 59,37% in Klebsiella bacteria by disc method including Clavulonic acide combination. In the present study, SHV and TEM type ESBL was determined in Klebsiella isolates by PCR method using spesific primers. SHV type ESBL was found in 56,25% (72 of 128) of isolates and TEM type ESBL were only found in 39,84% (51 of 128) of isolates. Amplications of the expected size about 293bp and 966bp were observed, respectively. On the other hand; RFLP analysis of SHV and TEM specific PCR products were also studied. Result of the digestion showed that, any of the PCR products were digested by NheI restriction enzyme. Moreover; Plasmid DNA profiles of Klebsiella bacteria were determined and their molecular weights were found. The results indicated that fourty two isolates were carried plasmids and their molecular weights were ranged from 1,375 to >21,226 kb. It was also determined that other isolates were not carried any plasmid DNA. It was also determined that ESBL was detected in 32 of the 42 isolates that are carried plasmid DNA by PCR method or DDST.