Cloning of the extended spectrum beta-lactamases of klebsiella pneumoniae and escherichia coli strains

Objective: To perform cloning of blaCTX-M, blaSHV and blaTEM genes which are found in extended spectrum beta-lactamases Klebsiella pneumoniae and Escherichia coli strains isolated from blood cultures. Methods: Twenty one K. pneumoniae and E. coli strains isolated from blood cultures at Dokuz Eylul University Hospital in 2004 were taken in to the study. blaCTX-M, blaSHV, blaTEM presence was sought by PCR. Six isolates producing different ESBL types were selected among these isolates and these strains were taken in to the cloning studies. InsTAclone? PCR Cloning Kit was used for T/A cloning. Colony PCR was performed to ascertain that the correct genes was cloned. Plasmid extraction was performed to reveal the presence of cloned genes before sequencing. Sequencing was performed on both strands to determine nucleotide sequences of blaCTX-M, blaSHV, blaTEM genes from selected strains. Results and Conclusion: ESBL pattern was detected in 21 strains in our study. According to the result of specific PCR for bla genes, eighteen isolates had blaCTX-M, nine isolates had blaSHV and eleven of them had blaTEM.. As a result of cloning, vectors with PCR product which had inactive lacZ gene produced lactose negative, lucid colonies on LB agar. On the other hand vectors without PCR product produced lactose positive, blue colonies on LB agar. Clones were found positive for the transfered genes. The sequence analysis revealed that; CTX-M-3 and CTX-M-15 were found in the CTX-M clones, SHV-5 and SHV-12 were found in the SHV clones and TEM-1 were found in the TEM clones. According to the results, there was a failure in selecting of TEM clones producing GSBL.