Molecular Characterization of SHV-2a ESBL from Clinical Isolate Ofklebsiellapneumoniae SLA55 in Baghdad, Iraq.

Abstract

SHV-producing K. pneumoniae is mainly related to the development of plasmid-mediated extended-spectrum β-lactamases and more often involved with different infections, particularly among hospitalized patients. Fifty-nine isolates of K. pneumoniae collected from various hospitals from Baghdad city, Iraq. The MICs towards 17 antimicrobial agents were measured using the Vitek-2 system. A combination disc method was used for phenotypic detection of ESBL-producing isolates. All ESBLs producers were subjected to PCR-based technique to detect the existence bla_SHV gene using specific primers, followed by sequencing of entire genes in selected bla_SHV-containing isolates. The bla_SHV-2a gene was cloned to the pTriEx vector using the In-Fusion process, expressed in E. coli BL21-pLysS cells and confirmed with Coomassie Blue stain. 54 (91.53%) isolates were ESBLs producers and 48 (81.36 %) of isolates possessed the bla_SHV gene using the PCR-based method; the sequencing of the entire bla_SHV gene of selected isolates, confirmed the existence of this gene in the amplified-PCR products. The success of cloning was determined by double digestion and sequencing of the cloned bla_SHV-2a. The expressed SHV-2 protein was detected after 1-hour induction by IPTGand was stable after four hours induction. To the best of our knowledge, this is the first report of the molecular characterization of plasmid-borne-bla_SHV-2a-containing K. pneumoniae clinical isolate from Baghdad, Iraq, that provides evidence of the rapid spread of these enzymes among pathogens in health settings.