ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, May 1984, p. 591-595 Vol. 25, No. 5 0066-4804/84/050591-05$02.00/0 Copyright C 1984, American Society for Microbiology Development of Resistance to Cephalosporins in Clinical Strains of Citrobacter spp. THOMAS D. GOOTZ,t* DAVID B. JACKSON, AND JOHN C. SHERRIS Department of Microbiology and Immunology, University of Washington, Seattle, Washington 98195 Received 28 September 1983/Accepted 21 February 1984 The predominant beta-lactam antibiogram of Citrobacterfreundii resembles that of Enterobacter cloacae in demonstrating resistance to cephalothin and cefoxitin with susceptibility to the newer cephalosporins. Four representative strains of C. freundii were reversibly induced to high-level beta-lactamase production by cefoxitin, and mutants with stable, high-level production were selected with cefamandole. The mutants were resistant to several second- and third-generation cephalosporins. Comparisons of isoelectric points and substrate profiles of beta-lactamases from wild-type, induced wild-type, and mutant organisms suggested a close relationship to those from E. cloacae and indicated that C. freundii mutants, like those of E. cloacae, were derepressed for production of beta-lactamase. One primary isolate of C. freundii resembled the mutants in all characteristics. In contrast, most strains of Citrobacter diversus were susceptible to all cephalosporins, and two representative strains showed neither inducible nor mutational resistance. Cefoxitin induction to enhanced beta-lactamase production was demonstrated in a cephalothin- resistant isolate, and a derepressed mutant was selected with cefotaxime. The beta-lactamase from this C. diversus strain differed substantially in substrate profile from that of E. cloacae and C. freundii. In recent studies, a mechanism responsible for resistance diversus strains isolated during 1982 in the Clinical Microbi- of Enterobacter species to many beta-lactam antibiotics has ology Laboratories of the University of Washington Medical been characterized (4, 7, 8, 21). These organisms have been Center, Seattle, were reviewed. Four strains of C. freundii shown to possess a chromosomally mediated group I beta- and two of C. diversus which represented the commonest lactamase (22), which appears to be normally under repres- antibiograms were studied in detail, as were one strain each sor control (7, 8). The small amounts of beta-lactamase of species resistant to cefamandole, cefotaxime, and cefo- produced in wild-type strains are sufficient to confer resist- perazone. A cefamandole-susceptible strain of Enterobacter ance to first-generation cephalosporins and to ampicillin but cloacae was studied in parallel. not to cefamandole and third-generation cephalosporins. Susceptibility test and disk induction procedures. The MICs Some cephalosporins to which Enterobacter species are of a number of beta-lactams against Citrobacter isolates resistant, notably cefoxitin, are able to reversibly induce were determined by a broth macrodilution procedure with high levels of enzyme by phenotypic derepression (6, 12, 18, Mueller-Hinton broth and inocula of 105 and 107 CFU/ml. 25). More importantly, from a clinical point of view, stably Diffusion susceptibility tests were performed by the method derepressed hyperproducers of beta-lactamase arise sponta- of Bauer et al. (2). The presence of inducible beta-lactamase neously from wild-type strains at a frequency of between was determined by a disk induction test (6, 18, 25), with a 10-6 and 10-7 (4, 7, 8). Sufficient beta-lactamase is produced cefoxitin disk as the inducer and cefamandole as the indica- by these mutants to confer resistance to cefamandole, cefo- tor. taxime, cefoperazone, and carbenicillin, with decreased Antibiotics and reagents. The following standard antibiotic susceptibility to moxalactam often observed (6). Evidence powders were obtained: cephaloridine and cefamandole (Eli suggests that such mutants can be selected in vivo as well as Lilly & Co., Indianapolis, Ind.), cefoxitin (Merck Sharp & in vitro (3, 5, 9, 11, 16, 17). Since emergence of resistance to Dohme, West Point, Pa.), cefoperazone (Pfizer Inc., New cefamandole has also involved organisms other than Entero- York, N.Y.), cefotaxime (Hoechst-Roussel Pharmaceuti- bacter species (9, 16), we examined two strains each of cals, Somerville, N.J.), and nitrocefin (Glaxo Ltd., Green- Serratia, Providencia, Morganella, and Citrobacter for gen- ford, Middlesex, England). Polyacrylamide and other iso- eration of mutants that hyperproduce beta-lactamase. Only electric focusing reagents were obtained from Bio-Rad the Citrobacter isolates demonstrated both an inducible laboratories (Richmond, Calif.). Ampholytes (pH 3 to 10) beta-lactamase and the ability to generate such stable mu- were obtained from Pharmacia Fine Chemicals (Uppsala, tants. We therefore focused our attention on strains of Sweden). Citrobacter species. Selection of resistant mutants. Cefamandole-susceptible strains were screened for the presence of resistant mutants MATERIALS AND METHODS by plating 108 wild-type cells from an overnight broth culture Bacterial strains. Laboratory records of routine suscepti- onto the surface of brain heart infusion agar plates contain- bility results for 46 Citrobacter freundii and 17 Citrobacter ing 64 p.g of cefamandole per ml (7). Any cefamandole- resistant mutants were counted after 18 h of incubation of the selection plates at 37°C, and three colonies of typical * Corresponding author. morphology were purified by passage on agar plates contain- t Present address: Pfizer Central Research, Eastern Point Road, ing antibiotic. The beta-lactamase reactivity of all mutants Groton, CT 06340. was compared with that of the original wild type by streaking 591 592 GOOTZ, JACKSON, AND SHERRIS ANTIMICROB. AGENTS CHEMOTHER. a single colony of each on a Whatman filter paper strip RESULTS impregnated with a solution of nitrocefin (500 ,ug/ml). Wild types produced a faint red color after several minutes. Susceptibility of wild-type Citrobacter strains. The predomi- Mutants that were hyperproductive of beta-lactamase gave nant antibiogram of the 46 C. freundii strains reviewed was an immediate color change. The stability of resistant mutants of resistance to ampicillin (61%), cephalothin (85%), and was determined after serial passages on drug-free medium. cefoxitin (85%) but susceptibility to cefamandole (74%), Preparation of cell-free sonicates. Samples for beta-lacta- carbenicillin (74%), cefotaxime (91%), cefoperazone (95%), mase assays and isoelectric focusing were prepared from and moxalactam (95%). Seventeen C. diversus strains dif- logarithmic-phase cells grown in 100 ml of brain heart fered in that the majority were susceptible to cephalothin infusion broth as previously described (6). Two culture (53%) and cefoxitin (95%) but resistant to carbenicillin flasks were inoculated from an overnight culture with each (88%). Eight representative strains were subsequently exam- of the wild-type strains, and each mutant was inoculated into ined for the production of inducible beta-lactamase and a flask. They were incubated with shaking (150 rpm) at 37°C generation of resistant mutants. Their antibiograms are until the cell density was approximately 108 CFU/ml. Cefoxi- shown in Table 1, together with results ofthe rapid nitrocefin tin was then added to one flask of the wild type to induce reaction and tests for induction of beta-lactamnase by cefoxi- beta-lactamase production (6). After an additional 2 h of tin. The four wild-type strains of C. freundii that represented incubation, cultures were centrifuged in a Sorvall GSA rotor the commonest antibiogram of the species contained an at 7,000 rpm for 15 min. The cell pellet was washed in 0.1 M inducible beta-lactamase but did not give a rapid nitrocefin phosphate buffer (pH 7.1) and centrifuged again. The pellet reaction in the absence of cefoxitin. The fifth strain (C. was resuspended in 7 ml of phosphate buffer and sonicated freundii 26) was resistant to all the beta-lactam antibiotics for 7.5 min (Branson Sonifier, Branson Sonic Power Co., tested and gave an immediate positive nitrocefin test. The Long Island, N.Y.). Cellular debris was removed by centrif- two wild-type strains of C. diversus that were susceptible to ugation at 14,000 rpm for 60 min in an SS-34 rotor at 4°C. The all the cephalosporin antibiotics did not demonstrate beta- protein content of the supernatants was determined by the lactamase induction with disks containing either 1 or 30 jig of method of Lowry et al. (10). cefoxitin and did not give a rapid nitrocefin test. C. diversus Measurement of beta-lactam hydrolysis. The specific beta- 15, which was resistant to cephalothin but sensitive to lactamase activity of cleared sonicates was measured against cefoxitin, showed a small inhibition zone with cefamandole cephalosporin substrates by the spectrophotometric assay of and was induced to complete cefamandole resistance only by O'Callaghan et al. (15). Hydrolysis of substrates at a final the 1-jig cefoxitin disk. concentration of 100 jiM was assayed at 37°C in phosphate Selection and characterization of resistant mutants. Resist- buffer (pH 7.2) by measuring the decrease in absorbance of ant mutants were obtained by the agar plate selection the beta-lactam ring of each compound (7, 8, 15). method with 64 jig of cefamandole per ml from the four Isoelectric focusing of cleared sonicates. Isoelectric focus- cefamandole-susceptible strains of C. freundii and