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.
Infections with Multiresistant Bacteria to Antibiotics (UR/29/04), Department of Microbiology, University Hospital of Sahloul, Sousse, Tunisia. safia_dah@yahoo.fr
Safia
S
Dahmen
Wejdène
W
Mansour
Karama
K
Charfi
Noureddine
N
Boujaafar
Guillaume
G
Arlet
Olfa
O
Bouallègue
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics*; D005260:Female; D006801:Humans; D015378:Imipenem / Q000494:pharmacology*; D007711:Klebsiella pneumoniae / Q000187:drug effects* / Q000235:genetics / Q000302:isolation & purification; D008826:Microbial Sensitivity Tests; D008875:Middle Aged; D010957:Plasmids / Q000235:genetics*; D016133:Polymerase Chain Reaction; D014416:Tunisia / Q000453:epidemiology; D018440:beta-Lactam Resistance / Q000235:genetics*; D001618:beta-Lactamases / Q000235:genetics*
D000900:Anti-Bacterial Agents; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D015378:Imipenem; C048795:AmpC beta-lactamases; D001618:beta-Lactamases