Plasmid-Mediated Resistance to Expanded-Spectrum Cephalosporins among Enterobacter aerogenesStrains

Bacterial strains.Among all E. aerogenes strains (n = 184) recovered from clinical specimens during an 18-month period (September 1993 to March 1995), 31 (16.8%) strains showing a resistance phenotype different from that observed with the derepressed mutants normally encountered at the Hunter Holmes McGuire Medical Center were selected for this study. The strains selected were shown to be intermediate to ceftriaxone but resistant to ceftazidime by the Vitek automated susceptibility system (bioMérieux Vitek, St. Louis, Mo.). Derepressed mutants previously isolated from this center were usually resistant to both ceftriaxone and ceftazidime.

Susceptibility testing.Antibiotic susceptibilities were determined by standard disk diffusion (21) and agar dilution (22) procedures. Disks were obtained from Becton Dickinson Microbiology Systems (Cockeysville, Md.). Disk diffusion susceptibilities to the following antibiotics were determined: ampicillin, amoxicillin clavulanic acid, aztreonam, cefazolin, cefoxitin, cefuroxime, cefotaxime, ceftriaxone, ceftazidime, cefepime, imipenem, gentamicin, trimethoprim-sulfamethoxazole, and ciprofloxacin. Standard powders of antimicrobial agents for MIC determinations were kindly provided by the following companies: Merck (Rahway, N.J.) (cefoxitin and imipenem), Hoechst-Roussel Pharmaceuticals Inc. (Somerville, N.J.) (cefotaxime), Glaxo Group Research Ltd. (Greenford, England) (ceftazidime), Bristol-Myers Squibb (Princeton, N.J.) (aztreonam and cefepime), and Schering-Plough (Liberty Corner, N.J.) (gentamicin). The following quality control strains were run simultaneously with the test organisms: E. coli ATCC 25922,Pseudomonas aeruginosa ATCC 27853, and E. coliATCC 35218. Throughout this study, results were interpreted with National Committee for Clinical Laboratory Standards criteria for disk diffusion (21) and broth dilution (22).

Double-disk potentiation test.This test, described by Jarlier et al. (15) with ceftazidime, ceftriaxone, cefotaxime, and aztreonam disks, was performed on the strains to screen for possible ESBL production. This test is a modification of the disk diffusion susceptibility test in that cefotaxime, ceftriaxone, ceftazidime, and aztreonam disks are placed 30 mm from disks containing amoxicillin-clavulanic acid. A potentiation of the zones of cefotaxime, ceftriaxone, ceftazidime, or aztreonam by clavulanic acid represented a positive test and was indicative of possible ESBL production.

β-Lactamase preparation, IEF, and assays.Overnight cultures in 5 ml of Mueller-Hinton broth were diluted with 95 ml of fresh broth and incubated with shaking for 90 min at 37°C. One-fourth of the cefoxitin MIC was added for induction, while sterile medium was used in the noninduced cultures and incubated for an additional 2 h. The induction process was stopped by the addition of 1 mM 8-hydroxyquinoline solution to each culture. Cells were harvested by centrifugation at 4°C, washed with 1 M potassium-phosphate buffer (pH 7.0), suspended, and sonicated. After sonication, crude extracts were obtained by centrifugation at 6,000 rpm for 1 h. The β-lactamases in the sonic extracts were assessed for isoelectric points (pIs) and substrate and inhibitor profiles in polyacrylamide gels with overlays of 0.75 μg of cefotaxime per ml, 1,000 μM clavulanic acid, and 1,000 μM cloxacillin prior to overlay with nitrocefin agar (1, 19, 28). Cephalothin hydrolysis rates were determined by UV spectrophotometric assay (23). Inducibility of the Bush group 1 β-lactamase was inferred from the intensity of isoelectric focusing (IEF) patterns for uninduced and induced β-lactamase extracts. As controls, crude β-lactamase preparations from the following organisms possessing different SHV enzymes were evaluated simultaneously with those obtained from theEnterobacter strains: SHV-1 [from E. coliJ53(R1010)], SHV-2 (from Klebsiella ozaenae 2180), SHV-3 [from E. coli J53-2(pUD18)], SHV-4 [from E. coli J53-2(pUD21)], and SHV-5 [from E. coli Cla Nal(pAFF2)].

Isolation of plasmids.The organisms were inoculated into 5 ml of Luria Bertani (LB) broth (Difco, Detroit, Mich.) and incubated for 20 h at 37°C with shaking. Cells from 1.5 ml of overnight culture were harvested by centrifugation in an Eppendorf centrifuge for 5 min. After the supernatant was decanted, the pellet was resuspended in 1% Triton X-100 in Tris-EDTA buffer for 10 min. Plasmid DNA was then isolated by the alkaline extraction method of Birnboim and Doly (3) and separated by electrophoresis in 0.8% agarose gel (Sigma) in TAE buffer (0.04 M Tris-acetate, 0.002 M EDTA [pH 8.5]). The gel was stained with ethidium bromide, and plasmid bands were visualized with UV light.

Conjugation experiments.To determine if the resistance was transferable, transconjugation experiments were performed with E. coli C600N (Nal^r) as the recipient (18). The filter paper mating technique with overnight incubation at 37°C was performed as described previously (25). Transconjugants were selected on LB agar (Difco) plates containing 12 μg of nalidixic acid per ml and 20 μg of ampicillin per ml.

DNA amplification by PCR.Organisms were inoculated into 5 ml of LB broth (Difco) and incubated for 20 h at 37°C with shaking. Cells from 1.5 ml of overnight culture were harvested by centrifugation at 13,000 rpm in an Eppendorf centrifuge for 5 min. After the supernatant was decanted, the pellet was resuspended in 500 μl of sterile deionized water. The cells were lysed by heating to 95°C for 10 min, and cellular debris was removed by centrifugation for 5 min at 13,000 rpm. The supernatant was used as the source of template for amplification. Oligonucleotide primers specific for SHV genes were selected from a consensus alignment sequence generated by the MacVector 4.5 (Kodak/IBI) software package from the published nucleotide sequences of SHV-1 (20), SHV-2 (13), SHV-5 (2), and SHV-7 (4). The sequences of the PCR primers used were A [5′-(CACTCAAGGATGTATTGTG)-3′] and B [5′-(TTAGCGTTGCCAGTGCTCG)-3′], which amplified a 781-bp fragment. Primer specificity controls included the TEM-1, MIR-1, and SHV-7 β-lactamase genes. PCR amplifications were carried out on a DNA Thermal Cycler 480 instrument (Perkin-Elmer Cetus, Norwalk, Conn.) with the GeneAmp DNA amplification kit containing AmpliTaqpolymerase (Perkin-Elmer, Roche Molecular Systems, Inc., Branchburg, N.J.). The composition of the reaction mixture was as follows: 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 0.1% Triton X-100, 1.5 mM MgCl₂, deoxynucleoside triphosphates (0.2 mM each), and 1.2 U of AmpliTaq in a total volume of 49 μl. A total of 1 μl of sample lysate was added to the reaction mixture, which was centrifuged briefly before 50 μl of mineral oil was layered onto the surface. The PCR program consisted of an initial denaturation step at 96°C for 30 s followed by 24 cycles of DNA denaturation at 96°C for 30 s, primer annealing at 50°C for 15 s, and primer extension at 72°C for 2 min. After the last cycle, the products were stored at 4°C. The PCR products were analyzed by electrophoresis with 1.4% agarose gels in TAE buffer. The gels were stained with ethidium bromide, and the PCR products were visualized with UV light.