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

Four clinical strains of extended-spectrum β-lactamase- and AmpC-producing Enterobacter aerogenes were isolated successively from a liver transplantation patient. Isolates C1 and C2 were isolated prior to carbapenem therapy, whilst isolates C3 and C4 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 C1 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 C1 and C2, isolates C3 and C4 failed to express OmpE36 owing to insertional inactivation by an IS903-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, C3 and C4 were apparently derived from the previously imipenem-susceptible isolates C1 and C2. Following carbapenem exposure, depletion of OmpE36 expression resulted in the collateral effect of carbapenem resistance.