Comparative genomics analysis of aminoglycoside resistance in multi-drug resistant Klebsiella pneumoniae JM45 isolate.. Klebsiella pneumoniae JM45

Infections caused by K. pneumoniae are discovered throughout the world. This organism is a major cause of urinary tract infection and an important source of nosocomial infection. To date, the spread of multidrug-resistant, even pandrug-resistant, K. pneumoniae has developed into a serious clinical threat. Furthermore, carbapenemases production has recently emerged as an important mechanism for carbapenems resistance. Disturbingly, more and more novel carbapenemases and their variants were continuously discovered. In 2011, we also reported a carbapenem-resistant strain of K. pneumoniae which carried a KPC-2 variant4. In 2010, we isolated another carbapenem-resistant K. pneumoniae strain and then performed bacteriological experiments, PCR and whole-genome sequencing. The genomic sequence data provided us the cause of the carbapenem resistance of this strain. K. pneumoniae JM45 was an isolate from the blood of a 72-year-old male with cerebral infarction after colon carcinoma resection hospitalized in the intensive care unit, the Second Affiliated Hospital of ZheJiang University School of Medcine on April 7, 2010. Species identification was carried out by using Vitek Gram-negative identification cards (bioMérieux, Marcy l’Etoile, France) and by 16S rDNA and gyrA sequencing for BLAST searches on Genbank. The minimum inhibitory concentrations(MICs) were determined by Etest (AB bioMe´rieux, Solna, Sweden) with the results interpreted according to Clinical and Laboratory Standards Institute(CLSI) guidelines. For rapid detection of the presence of AmpC-derepressed, extended-spectrum β-lactamases(ESBL) and metallo-β-lactamases(MBL), a new system (Cica-Beta-Test, Kanto Chemical, Japan) was utilized. The Modified Hodge Test was carried out to detect carbapenemases. We isolated a pandrug-resistant K. pneumoniae strain from the blood of a patient with cerbral infarction after colon carcinoma resection in the intensive care unit. According to Cica-Beta-Test and Modified Hodge Test, it was considered to harbor various kinds of β-lactamases including carbapenemase. However, we only detected four class A β-lactamase genes with no carbapenemase genes by PCR unexpectedly. To explain the inconsistency of these results, we subsequently conducted whole-genome sequencing for K. pneumoniae JM45. As a result, we also detected four homologous class A β-lactamase genes, and in addition, discovered two novel genes encoding metallo-β-lactamase domain protein locating on the chromosome simultaneously. This is the first report of two non-cassette-mediated metallo-β-lactamase domain protein sequences for carbapenem resistance in the same strain. We will carry out further research on these two novel genes by cloning and expression analysis followed by enzyme kinetics analysis.