Molecular Characterization of CTX-M-15 β-Lactamase from Clinical Isolate of Klebsiella pneumoniae SJ16

Abstract

Background: “Klebsiella pneumoniae strains have become increasingly resistant to antibiotics, making it very difficult to
treat infection with these strains. Production of β-lactamase enzymes is the most generic and essential mechanisms of
bacterial resistance especially in Bacteria that are gram negative. The resistance of CTX-M beta-lactamase is primarily
associated with the manufacturing of β-lactamases plasmid-mediated extended spectrum. One of the most common
resistant markers found in enterobacteriaceae members is a bla CTX-M-15 gene.
Methods: We studied the presence of extended spectrum beta - lactamases in 55 clinical isolates of K. pneumonia collected
from various hospitals in the city of Baghdad, Iraq. Using the agar diffusion technique, β-lactam MICs were determined.
Combination disc diffusion test confirmed phenotypic confirmation of ESβLs; all ESβL-producing K.pneumoniae isolates
were tested for the entire bla CTX-M-15 gene by particular PCR primers followed by sequencing. The gene bla CTX-M-15
has been cloned using the In-Fusion method into the pTriEx vector in E. coli stellar cells and expression was conducted as a
receiving strain in E.coli BL21-pLysS cells. The entire bla CTX-M-15 gene linked to plasmid pTriEx NcoI and XhoI sites
was verified by the sequencing of colony PCR. Coomassie stain and Western blot have detected confirmation of
expression.
Results: 50 [90.90 percent] isolates were recognized as being ESβL positive by phenotypic characterization. 44 [80
percent] ESβL manufacturers were positive in bla CTX-M-15 gene PCR amplification, sequencing was used to detect
strains containing blaCTX-M-15 dna. A single 5.3 kb band corresponding to the pTriEx vector and a 876 bp band
corresponding to the blaCTX-M-15 gene was shown in double digestion. blaCTX-M-15 protein expression began after
two-hour induction and the protein remained stable after 4 hours of IPTG induction.
Conclusion: The successful cloning in the pTriEx1.1 vector has been verified by double digestion and sequencing, the
plasmid pTriEx 1.1 is specifically designed to allow rapid characterization of target genes in different expression systems.”