A 33-year-old man with acute myeloid leukemia was admitted to a hospital in Seattle, Washington, with neutropenic fever; he was initially treated with ceftazidime. His therapy was changed to acyclovir and imipenem because of unremitting fevers and oral lesions positive for herpes simplex virus type 1, whereupon his fever defervesced immediately. The fever recurred 2 weeks after treatment was begun, and imipenem-resistant E. cloacae was isolated from the blood. After the results of susceptibility testing were obtained, his therapy was changed to levofloxacin, and he exhibited a good clinical response.
The E. cloacae isolate was susceptible to piperacillin, piperacillin-tazobactam, ceftazidime, ceftriaxone, cefepime, ciprofloxacin, gentamicin, aztreonam, and trimethoprim-sulfamethoxazole and was resistant to ampicillin, amoxicillin-clavulanic acid, cefazolin, and cefoxitin. By both disk diffusion and E-test methods (AB Biodisk, Solna, Sweden), the zones of inhibition around the imipenem, meropenem, and ertapenem disks were indistinct, with inner colonies extending up to the disk or the highest concentration on the E-test strip (MIC >32 mug/mL) and were interpreted as resistant (Figure 1). Addition of 10 muL of 1,000 mug/mL clavulanic acid to the imipenem, meropenem, and ertapenem disks resulted in clearly defined and enlarged zones of inhibition and loss of the inner colonies (Figure 2). Using either cefoxitin or imipenem as inducers by the disk induction method resulted in the formation of blunted or D-shaped zones of inhibition to meropenem and ertapenem. We performed a carbapenem bioassay by incubating 100 muL of a 50 mug/mL solution of each carbapenem (imipenem, meropenem, or ertapenem) with 100 muL of either crude beta-lactamase extract or phosphate-buffered saline for 90 min at room temperature. The inactivating capacity of the enzyme was then tested by a disk diffusion assay that demonstrated hydrolysis of imipenem, meropenem, and ertapenem.
Isoelectric focusing (IEF) to determine isoelectric points (pIs) and general inhibitor characteristics with an ampholine polyacrylamide gel (pH range 3.5-9.5) on a flatbed apparatus (Multiphor LKB, Bromma, Stockholm, Sweden) indicated that this organism produced a beta-lactamase enzyme with a pI of 6.9 that was inhibited by clavulanic acid and not by cloxacillin. This enzyme hydrolyzed 1 mug/mL imipenem on the IEF gel overlay. Inducibility of the enzyme by imipenem (4 mg/L) and cefoxitin (8 mg/L) was confirmed by the broth induction method and IEF. After the broth induction procedure, the uninduced and induced preparations were serially diluted in 0.1 M potassium phosphate buffer, pH 7.0, permitting both qualitative and semiquantitative assessment of enzyme activity among the various beta-lactamases in the isolate, as determined by IEF. The broth induction enzyme preparation from a 100-mL, 3.5-h Mueller-Hinton broth produced a visible but light AmpC band, pI >9.0. The induction with cefoxitin and imipenem increased the levels of both the AmpC and the pI 6.9 carbapenem-hydrolyzing enzyme, as visualized in Figure 3. A cefoxitin-induced preparation of E. cloacae NOR-1 (that produces NmcA) was included as a control strain for IEF.
Chromosomal DNA was extracted from overnight cultures with the Qiagen DNeasy Tissue kit (QIAGEN Inc., Valencia, CA). Polymerase chain reaction (PCR)-amplification with a thermal cycler (MJ Research PTC - 200-DNA Engine, San Francisco, CA) was used to obtain a 2,122 base pair gene fragment consisting of the carbapenemase structural gene nmcA and its regulatory gene nmcR. The nucleotide sequences of the primers were 5'- TGC CAG CTT AAT TAT TTT CAG ATT AG -3' (nmcR positions 32-56) and 5'- ATT TTT TTC ATG ATG AAG TTA AGC C -3' (nmcA positions 2,129-2,154). PCR amplification of the nmcR/nmcA genes showed positive results with both NOR-1 and the strain under study, suggesting that the latter strain harbored a carbapenem-hydrolyzing enzyme similar to that of NOR-1. Subsequent sequence analysis of the amplified DNA-region showed a similarity of 100% (2,122 base pairs) between the sequence of our strain and the carbapenemase genes, regulatory (nmcR) and structural (nmcA) genes of the E. cloacae strain NOR-1.