In vivo development of carbapenem resistance in clinical isolates of Enterobacter aerogenes producing multiple β-lactamases

Abstract

Four clinical strains of extended-spectrum β-lactamase- and AmpC-producing Enterobacter aerogenes were isolated successively from a liver transplantation patient. Isolates C 1 and C 2 were isolated prior to carbapenem therapy, whilst isolates C 3 and C 4 were recovered after 40 days of carbapenem therapy. The homology of these strains was analysed by pulsed-field gel electrophoresis (PFGE). β-Lactamases were analysed by isoelectric focusing, polymerase chain reaction (PCR) and sequencing. Outer membrane proteins were analysed by PCR, sequencing, sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blot. Disruption of OmpE36 in C 1 in vitro was also performed by homologous gene recombination. The isolates demonstrated an indistinguishable PFGE pattern. Molecular characterisation revealed that, in addition to the pre-existing multiple β-lactamases (DHA-1, TEM-1, SHV-5, CTX-M-3 and CTX-M-14) found in C 1 and C 2, isolates C 3 and C 4 failed to express OmpE36 owing to insertional inactivation by an IS 903-like insertion sequence. Other resistance mechanisms, such as production of carbapenem-hydrolysing enzymes or expression of chromosomal efflux, were apparently not involved. Completely replacing OmpE36 by the kanamycin resistance gene ( kan) resulted in a significant increase in carbapenem minimum inhibitory concentrations of an ompE36 mutant. Thus, C 3 and C 4 were apparently derived from the previously imipenem-susceptible isolates C 1 and C 2. Following carbapenem exposure, depletion of OmpE36 expression resulted in the collateral effect of carbapenem resistance.