Purification And Genetic Characterization Of A Novel Metallo-β-lactamase Gene, Bla_IMP-38,and Virulence Factors Of Klebsiella Pneumoniae Strains Producing Bla_IMP-38

Objective （1） To obtain and purify a novel metallo-β-lactamase gene, blaIMP-38-（2） To investigate genetic characterization of a novel metallo-β-lactamase gene, blaIMP-38.（3） To detect virulence factors and capsular types of blaIMP-38-producing Klebsiella pneumonia. Methods （1） The blaIMP-38gene was amplified by PCR and ligated into a vector, PUC19, in order to create recombinant plasmid PUC19/IMP-38, which could verify the sequence of blaIMP-38gene.（2） The blaIMP-38gene was expressed, purified and refolded by the construction of recombinant plasmid PET-28a（+）/IMP-38.（3） The genome DNA of blaIMP-38-producing K. pneumonia isolates was extracted, then PCR, long PCR and overlap extension PCR were used to investigate the relationship between blaIMP-38and integron, and the relationship between blaIMP-38and transposon.（4） blaIMP-38-producing K. pneumonia were collected, their genome DNA were extracted, then PCR was used to investigate capsular types and virulence factors. Results （1） The recombinant plasmid PUC19/IMP-38was constructed successfully, the sequence of blaIMP-38was totally correct, blaIMP-38was a novel metallo-β-lactamase gene.（2） The recombinant plasmid PET-28a（+）/IMP-38was constructed successfully, the Recombinant protein was overexpressed, the purified enzyme was obtained.（3） blaIMP-38was found on class I integron, which also located on a Tn402-like transposon. There were also other transoposons （mer1696, Tn5051） and other mobile genetic element （tnp513, IS26, tnpU and IS5075） found on blaIMP-38-producing K. pneumonia..（4） FimH1, mrkD, ycfm, kpn, entB, irp-2, uge, wabG and ureA were found in 4blaIMP₃8-producing K. pneumonia isolates. The other virulence genes and capsular types gene were not detected. Conclusions （1） blaIMP-38was a novel metallo-β-lactamase gene.（2） Recombinant plasmid PET-28a （+）/IMP-38was constructed successfully, the purified enzyme was obtained.（3） blaIMP-38was found on class Ⅰ integron, which is also located on a Tn402-like transposon. There were also other transoposons and insertion sequence genetic marks.（4） It is very necessary for clinical laboratory to detect virulence factors, because the same clone type does not mean they have same virulence genes.