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 from the ing of cleared sonicates was performed with an LKB Multi- control strain of E. cloacae. They occurred at an apparent phor 2117 (LKB Instruments, Inc., Rockville, Md.) cooled frequency of between 10-6 and 10-7, and their resistance with a Lauda K-2/RD circulator as described previously (8). was stable over six subcultures in the absence of antibiotic. One hundred micrograms of protein from each sonicate were All mutants were rapidly positive by the nitrocefin screening focused in polyacrylamide gels containing ampholytes of pH test. Their antibiograms (Table 1) were identical, showing 3 to 10. Beta-lactamase bands were visualized by staining resistance to all the beta-lactam antibiotics tested. Stable with nitrocefin (8). The relative quantity of protein in each mutants were also selected from the cefamandole-intermedi- beta-lactamase band was also determined by staining with ate C. diversus 15 on plates containing 10 jig of cefotaxime Coomassie brilliant blue R-250. per ml. These showed cross-resistance to cefoperazone and

TABLE 1. Susceptibility patterns of study strains to beta-lactam antibiotics

StranNo. Wild type Diffusion test resultsb Rapid reaction Induced resistance tested mutanta MA CF FOX CFP CTX CB AM with nitrocefin cefoxitin E. cloacae 1 W S R R S S S R - + M R R R R R R R + NAC C. freundii 4 W S R R S S S R - + M R R R R R R R + NA C.freundii 26 1 W R R R R R I R + NA C. diversus 2 W S S S S S R R C. diversus 15 1 W I R S S S R R _ +d M R R S R R R R + NA a M, Mutant selected on brain heart infusion agar plates containing 64 jLg ofcefamandole per ml. C. diversus 15 M selected with 10 ,ug of ce- fotaxime per ml. W, Wild type. b Zone interpretations made according to standard references (14). Abbreviations: MA, cefamandole; CF, cephalothin; FOX, cefoxitin; CFP, cefoperazone; CTX, cefotaxime; CB, carbenicillin; AM, ampicillin; R, resistant; I, intermediate; S, susceptible. c NA, Not applicable. d Seen only when induced with a 1-,ug cefoxitin disk. VOL. 25, 1984 RESISTANCE TO CEPHALOSPORINS IN CITROBACTER SPP. 593

TABLE 2. Isoelectric points of beta-lactamases from wild-type, 2, C. freundii 3, and C. diversus 15, as well as the cefaman- induced wild-type, and mutant cultures dole-resistant C. freundii 26, were examined for beta-lacta- Isoelectric point mase activity in the spectrophotometric assay (15). The Strain Wild typea of induced wild Mutant results for the specific hydrolysis rates of five cephalospo- typeb rins are shown in Table 3. E. cloacae 2 8.0 8.0 8.0 The inducible enzymes of C. freundii 3 behaved in all C. freundii 3 9.0 9.0 9.0 regards like that of E. cloacae 2. Extracts from induced C. freundii 19 8.9 8.9 8.9 strains and mutants showed enhanced hydrolysis of cepha- C. freundii 20 9.1 9.1 9.1 lothin and activity against cefamandole, cefoxitin, and cefo- C. freundii 24 9.3 9.3 9.3 perazone. The beta-lactamase of the resistant C. freundii 26 C. freundii 26 9.7 9.7 NAc was not inducible but showed similar substrate specificity C. diversus 23 9.0 9.0 NA C. diversus 28 9.5 9.5 NA and activity to the enzymes from the resistant mutant of C. C. diversus 15 9.2 9.2 9.2 freundii 3 (Table 3). The beta-lactamases from the wild-type C. diversus 15 and a Mean of at least four determinations rounded to nearest 0.1 pH its induced derivative and mutant were comparable but unit. Values for 1 standard deviation range from 0.01 to 0.13. showed major differences in substrate specificity from those b Boldface numbers indicate greater density of beta-lactamase of the C. freundii species. They had very high activity bands by Coomassie brilliant blue stain. against cefamandole after induction, considerable activity c NA, Not applicable. against cefotaxime, but no activity against cefoxitin. C. diversus 15 thus appeared to possess a completely different inducible enzyme than that found in the E. cloacae and C. showed a higher level of resistance to cefamandole. They freundii strains studied. also gave a rapid nitrocefin test for beta-lactamase produc- The MICs of wild-type and mutant strains of E. cloacae 2, tion. In contrast, no resistant mutants were obtained from C. diversus 15, and C. freundii 3 were determined for inocula of 108 cells from the two cefamandole-susceptible cefoperazone, carbenicillin, cefamandole, and cefotaxime strains of C. diversus. with inocula of 105 and 107 CFU/ml (Table 4). Also shown are the MICs for the cefamandole-susceptible strain, C. Characterization of beta-lactamase in sonicates. The beta- diversus 28. The results show greatly increased MICs with lactamases in sonicates prepared from both induced and the higher inoculum for wild-type strains that yielded resist- uninduced wild-type strains and mutants were studied by ant mutants, reflecting the observed frequency of mutation isoelectric focusing (Table 2). The pl's of the major bands of to high beta-lactamase production. This phenomenon did not enzyme from induced wild-type and mutant cultures were occur with no mutants identical to those of the uninduced wild type but stained C. diversus 28, from which could be much more intensely with nitrocefin. The pl's of the Citro- obtained by direct selection. bacter beta-lactamases were considerably more basic than DISCUSSION those of the Enterobacter control, but fell within the range reported for other strains ofEnterobacter (7, 8, 22). Coomas- The results of this study show that stable mutants of sie brilliant blue-stained gels showed denser bands of beta- increased resistance to several second- and third-generation lactamase in sonicates prepared from mutant and cefoxitin- cephalosporin antibiotics occurred among wild-type cefa- induced wild types than from their uninduced wild-type mandole-susceptible C. freundii at an apparent frequency of strains. 10-6 to 10-7. These mutants produced greatly enhanced Cell-free sonicates of wild types and mutants ofE. cloacae amounts of beta-lactamase with the same pl's as those from

TABLE 3. Hydrolysis rates of cephalosporins by sonicates of Enterobacter spp. and Citrobacter spp. Specific beta-lactamase activity (nm/min per mg of protein) for the following antibiotics:a Strain Cephalo- Cefaman- Cefoxi- Cefoper- Cefotax- ridine dole tin azone ime E. cloacae 2 Wb 7 (0.2) 0 0 0 0 E. cloacae 2 W inducedc 3,850 (160) 24 (1) 3 (1) 29 (0.5) 0 E. cloacae 2 Md 10,200 (870) 56 (0.4) 4 (0.01) 76 (2) 1 (0.03) C. freundii 3 W 29 (1) 0 0 0 0 C. freundii 3 W induced 870 (11) 4 (0.1) 2 (0.4) 5 (1) 0 C. freundii 3 M 3,410 (180) 19 (2) 5 (0.3) 22 (3) 0 C. freundii 26 W 680 (5) 13 (0.5) 3 (0.2) 22 (1) 0 C. freundii 26 W induced 818 (3) 14 (1) 1 (0.6) 21 (0.3) 0 C. diversus 15 W 72 (1) 78 (3) 0 0 9 (1) C. diversus 15 W induced 387 (7) 463 (8) 0 20 (2) 29 (0.4) C. diversus 15 M 1,967 (45) 2,223 (16) 0 78 (2) 100 (5) a Mean of triplicate assays; values in parentheses equals 1 standard deviation. b W, Wild-type organism. c Cultures induced for 2 h with cefoxitin (25 ,ug/ml); C. diversus 15 induced with 5 jig/ml. d M, Mutant selected from wild-type population with cefamandole. C. diversus 15 M was selected with cefotaxime. 594 GOOTZ, JACKSON, AND SHERRIS ANTIMICROB. AGENTS CHEMOTHER.

TABLE 4. MICs for study strains The heterogeneity of Citrobacter beta-lactamase at the Wild type MICs (,ug/ml) for the following species level is apparent. Tajima et al. (23) have demonstrat- Strain or Inoculum antibiotics': ed that the chromosomal beta-lactamase from C. freundii mutanta (CFU/mI) MA CFP CTX CB GN7391 was antigenically related to that found in E. cloacae yet was unrelated to beta-lactamases obtained from Pseudo- E. cloacae 2 W 105 8 <1 <1 4 monas spp., Proteus spp., Serratia spp., and Bacteroides 107 >256 64 128 64 spp. More recent studies have shown significant heterogene- M 105 >256 128 128 256 ity to exist in the binding sites for cephalosporins in C. C. freundii 3 W 105 32 <1 2 4 freundii beta-lactamases (1). The accumulated evidence sug- 107 128 128 32 128 gests that these enzymes are continuing a divergent pattern M 105 64 32 64 128 of evolution, making classification based on physical-chemi- cal criteria difficult. In this sense, the maintenance of C. diversus 15 W 105 >256 8 <1 >256 regulatory genes for beta-lactamase would be expected to 107 >256 >256 >256 >256 influence the clinical efficacy of cephalosporin antibiotics M 105 >256 256 64 >256 against these strains. The strain of C. freundii 26 which was C. diversus 28 W 105 <1 <1 <1 256 resistant to several cephalosporins on primary isolation 107 8 8 4 256 behaved like resistant mutants selected in vitro. The beta- lactamase in this strain was not inducible, and this was likely a W, Wild type; M, mutant. a constitutively derepressed mutant. It was isolated as 450 b Abbreviations: MA, cefamandole; CFP, cefoperazone; CTX, CFU/ml in urine from a patient who had been treated with cefotaxime; CB, carbenicillin. cefazolin and ticarcillin during the 3 days before culture. It appeared not to have been clinically significant in that patient, but it is important to determine whether resistant their cefoxitin-induced and -uninduced wild types. The sub- Citrobacter mutants which are derepressed for either of the strate specificities of these beta-lactamases are indistinguish- two enzymes described here can cause acute infection and able from those previously described for Enterobacter sp., be spread among patients. and the pI's fall within the same range (7, 8, 22). Thus, many The striking inoculum effect in broth dilution tests de- C. freundii strains possess a beta-lactamase that can be scribed previously for Enterobacter spp. and cefamandole induced or stably derepressed mutationally in the same (4, 7, 8) applied to Citrobacter strains that produced resistant manner as occurs with Enterobacter species (4, 6-8, 25). mutants. It appeared to be largely, if not entirely, due to the Resistant strains of C. freundii were encountered in patients, mutation frequencies involved. Inocula of 105 CFU/ml gen- and the strain that we studied (C. freundii 26) behaved erally contained no mutants, whereas they were consistently identically to the resistant mutants selected in vitro. These present at an inoculum of 107 CFU/ml. Although susceptibil- results are consistent with those of previous studies in which ity testing under these conditions most efficiently detects the the existence of a repressor protein in the regulation of beta- presence of derepressed mutants in populations of wild-type lactamase production in C. freundii has been suggested (20, organisms, it fails to take into consideration the multitude of 26), although other mechanisms such as mutations affecting host factors that influence the emergence of resistance translation cannot be ruled out. during antibiotic therapy. In this regard, the judicious use of Comparison of the beta-lactamases found in Citrobacter the newer cephalosporins remains the most important factor strains indicate that there was a direct correlation between in determining the success of these compounds. the presence of an inducible enzyme and the development of beta-lactam resistance. Two C. diversus strains studied were ACKNOWLEDGMENTS not induced to cefamandole resistance by cefoxitin, and no cefamandole-resistant mutants were encountered in inocula This study was supported in part by Public Health Service grant of 108 bacteria. These strains represented the number 5 T32 A107149-05 from the National Institutes of Health. predominant David Jackson was supported by a stipend from the Medical Student susceptibility pattern found with this species, as they were Research Training Program at the University of Washington, Seat- susceptible to all cephalosporins tested. The third strain, C. tle. diversus 15, was of special interest in that increased beta- lactamase production was induced by low concentrations of LITERATURE CITED cefoxitin, despite susceptibility to this antibiotic. The pres- ence of this inducible enzyme was not detected with the 1. Arisawa, M., J. Ohshima, and H. B. Maruyama. 1983. Unusual mode of inhibition of Citrobacter freundii P-lactamases by standard disk induction assay with a 30-,ug cefoxitin disk ceftriaxone. Antimicrob. Agents Chemother. 23:317-319. (18). High-level beta-lactamase-producing mutants with an 2. Bauer, A. W., W. M. M. Kirby, J. C. Sherris, and M. Turck. increased resistance to cefamandole, cefoperazone, and 1966. Antibiotic susceptibility testing by a standardized single cefotaxime were selected from this strain with cefotaxime at disk method. Am. J. Clin. Pathol. 45:493-496. a frequency of 10-6 to 10-7. The beta-lactamase derived 3. Beckwith, D. G., and J. A. Jahre. 1980. Role of cefoxitin- from this mutant, its wild type, and its induced wild type inducible beta-lactamase in a case of breakthrough bacteremia. clearly differed from those of C. freundii by showing greater J. Clin. Microbiol. 12:517-520. activity against cefamandole, high activity against cefotax- 4. Findell, C. M., and J. C. Sherris. 1976. Susceptibility of ime and cefoperazone, but no activity against cefoxitin. The Enterobacter to cefamandole: evidence for a high mutation rate proposed barrier function of beta-lactamase (13, 19, 24) to resistance. Antimicrob. Agents Chemother. 9:970-974. 5. Goering, R. V., C. C. Sanders, and W. E. Sanders, Jr. 1978. apparently had no function against cefoxitin in this strain, Comparison of BL-S786 with cephalothin, cefamandole, and since no difference was observed in the zone size between cefoxitin in vitro and in treatment of experimental infections in the wild type and derepressed mutant for this drug. This mice. J. Antibiot. 31:363-372. suggests that the beta-lactamase in C. diversus 15 does not 6. Gootz, T. D., and C. C. Sanders. 1983. Characterization of P- bind cefoxitin and therefore cannot utilize it as a substrate. lactamase induction in Enterobacter cloacae. Antimicrob. VOL. 25, 1984 RESISTANCE TO CEPHALOSPORINS IN CITROBACTER SPP. 595

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