Imipenem Resistance in Klebsiella pneumoniae Is Associated to the Combination of Plasmid-Mediated CMY-4 AmpC β-Lactamase and Loss of an Outer Membrane Protein

This study was conducted to identify the molecular mechanisms of imipenem resistance in a Klebsiella pneumoniae (Kp16137) isolate recovered in August 2008 at the University Hospital Sahloul, Sousse, Tunisia. The strain was identified with the API 20E system; antibiotic-containing disks were used for detection of antibiotic susceptibility by a disk diffusion assay. We investigated the presence of β-lactamases by PCR, using specific primers for bla TEM , bla SHV , bla CTX-M , bla OXA , bla CMY , bla ACC , bla FOX , bla IMP , bla KPC , bla VIM , and by sequencing. Extraction of plasmid DNA from Kp16137 and the transconjugant was performed by the method of Kado. Southern transfer was performed on nylon. The membrane was hybridized with a specific probe for the bla CMY-2 gene. Outer membrane proteins were isolated and were examined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis on 12% polyacrylamide gel. K. pneumoniae Kp16137 was resistant to all available β-lactams, including third generation cephalosporins and carbapenems. The screening of β-lactamases showed the presence of three β-lactamases: TEM-1, SHV-61, and CMY-4. The CMY-4 β-lactamase was located on an 80-kb plasmid. An analysis of the outer membrane proteins of this isolate revealed that it lacked a porin of 42 kDa. The loss of this outer membrane protein band correlated with imipenem resistance in this strain. In K. pneumoniae 16137, synthesis of a plasmid-mediated β-lactamase: AmpC CMY-4, together with alteration in permeability led to resistance to all available β-lactams and carbapenems.