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  • Title: In vivo selection of Enterobacter aerogenes with reduced susceptibility to cefepime and carbapenems associated with decreased expression of a 40 kDa outer membrane protein and hyperproduction of AmpC beta-lactamase.
    Author: Fernández-Cuenca F, Rodríguez-Martínez JM, Martínez-Martínez L, Pascual A.
    Journal: Int J Antimicrob Agents; 2006 Jun; 27(6):549-52. PubMed ID: 16697150.
    Abstract:
    The mechanism(s) of resistance or decreased susceptibility to cefepime (FEP) and/or imipenem (IMP) in three consecutive isolates of Enterobacter aerogenes (Ea1, Ea2 and Ea3) cultured from bronchial aspirates of the same patient after treatment with ceftriaxone and FEP were studied. Identification was performed with the VITEK 2 system. All three isolates showed identical pulsed-field gel electrophoresis patterns and were resistant (minimum inhibitory concentrations (MICs)) to cefoxitin (MIC, >1024 mg/L), cefotaxime (CTX; MIC, 32-128 mg/L) and ceftazidime (CAZ; MIC, 32-128 mg/L) but susceptible to meropenem (MIC, <or=0.5 mg/L) according to the National Committee for Clinical Laboratory Standards (NCCLS). MICs of FEP were 0.5 mg/L (Ea1), 2 mg/L (Ea2) and 16 mg/L (Ea3), whereas MICs of IMP were <or=0.5 mg/L (Ea1 and Ea3) and 8 mg/L (Ea2). Clavulanic acid (CLV) did not affect the MICs of CTX and FEP. In contrast, the MICs of CTX were reduced 32-128 times by BRL 42715 (BRL) or cloxacillin (CLX), whereas the MICs of FEP were reduced 2-128 times by BRL and 16-64 times by CLX. Production of extended-spectrum beta-lactamases (ESBLs) was not detected using the disk diffusion method (NCCLS) or Etest (CTX/CTX-CLV and CAZ/CAZ-CLV). TEM- or SHV-type ESBL genes were not detected by polymerase chain reaction amplification. The three isolates showed the same pattern of five beta-lactamases (isoelectric points 7.9-8.3, inhibited by CLX but not by CLV) by isoelectric focusing of crude extracts. Hydrolysis (nmol/mg) of cefaloridine (CF) was 3741.0 (Ea1), 4000.6 (Ea2) and 3797.4 (Ea3), suggesting that AmpC is hyperproduced. Hydrolysis of FEP was much lower than that of CF: 1.3 (Ea1), 2.1 (Ea2) and 17.3 (Ea3). The nucleotide sequences of the ampR-ampC genes of Ea1 and Ea2 were identical to that of E. aerogenes strain deposited in GenBank (accession no.). For Ea3, however, a point mutation in position 311 of ampC caused a change of Val-->Glu. Three outer membrane proteins (OMPs) of 51 kDa, 40 kDa and 38 kDa were observed in the three isolates by sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) (10% polyacrylamide gels with 4 M urea), although expression of the 40 kDa OMP was reduced in Ea2. In conclusion, decreased susceptibility to FEP and IMP in Ea2 is related to reduced expression of a 40 kDa OMP and hyperproduction of AmpC, whereas resistance to FEP in Ea3 is associated with hyperproduction of an altered AmpC.
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