Antibiotics. Disks for agar diffusion tests were purchased from Diagnostics Pasteur. Antibiotic powders were provided

Transcription

1 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Nov. 1988, p /88/ $02.00/0 Copyright X 1988, American Society for Microbiology Vol. 32, No. 11 Comparative Study of a Novel Plasmid-Mediated 3-Lactamase, CAZ-2, and the CTX-1 and CAZ-1 Enzymes Conferring Resistance to Broad-Spectrum Cephalosporins CATHERINE M. CHANAL,1* DANIELLE L. SIROT,1 ROGER LABIA,2 AGNtS PETIT,' ANNICK MORAND,2 JACQUES L. SIROT,1 AND ROGER A. CLUZEL' Service de Bacteriologie, Facultes de Medecine et Pharmacie, Clermont-Ferrand,' and Museum National d'histoire Naturelle, Centre National de la Recherche Scientifique Unite Associee 401, Paris Cedex 05,2 France Received 5 April 1988/Accepted 25 August 1988 Infections caused by strains of KkebsieUa pneumoniae resistant to broad-spectrum cephalosporins have been observed recently in hospitals in Clermont-Ferrand, France. P-Lactam resistance resulted primarily from the plasmid-mediated, expanded-spectrum CTX-1,B-lactamase. Furthermore, since 1987 some K. pneumoniae isolates more resistant to ceftazidime than to other cephalosporins have been observed. This new resistance phenotype was the result of the production of ceftazidimase CAZ-1 and, more recently, CAZ-2. As in CTX-1-producing strains, resistance to l-lactams resulting from CAZ-2 was associated with resistance to aminoglycosides except gentamicin, sulfonamide, and tetracycline and was transferable to Escherichia coli by conjugation. Agarose gel electrophoresis of plasmid DNA from wild-type strains and transconjugants indicated that CAZ-2 production was mediated by a plasmnid of 85 kilobases highly related to plasmid pcff04 coding for CTX-1 I-lactamase. The isoelectric point, close to 6.0, of this novel enzyme differed from those of CTX-1 and CAZ-1. Like CAZ-1, the CAZ-2 enzyme efficiently hydrolyzed ceftazidime and aztreonam, but as with CTX-1, cefotaxime gave the maximal reaction rate. For each expanded-spectrum,b-lactamase, the activity of broad-spectrum cephalosporins was restored by clavulanic acid or sulbactam. Since 1984, we have observed a nosocomial outbreak of infections primarily caused by Klebsiella pneumoniae and then by other genera of the family Enterobacteriaceae resistant to all '-lactams except cephamycins (cefoxitin, moxalactam) and imipenem.,-lactam resistance resulted from CTX-1, a novel plasmid-mediated, expanded-spectrum,-lactamase with high hydrolytic activity against cefotaxime (13). Since January 1987, another plasmid-encoded,-lactamase has been characterized in K. pneumoniae strains isolated from patients hospitalized in the same intensive care unit. This enzyme hydrolyzed ceftazidime more than cefotaxime and was designated CAZ-1 (12, 15). Since July 1987, a third plasmid-mediated P-lactamase has been detected in K. pneumoniae isolates from patients hospitalized in another intensive care unit. These isolates were more resistant to ceftazidime than to cefotaxime, and the enzyme was designated CAZ-2. In this study, we compare this novel expanded-spectrum,3-lactamase with the two enzymes previously observed in the same hospital. MATERIALS AND METHODS Bacterial strains and plasmids. Three multiply resistant isolates of K. pneumoniae were examined: CF104 (CTX-1 producing), CF504 (CAZ-1 producing), and CF704 (CAZ-2 producing). K. pneumoniae CF004 (susceptible) and CF174 (TEM-2 producing) were included in this study for comparison. Rifampin- and nalidixic acid-resistant mutants of Escherichia coli K-12 C600 were used in transfer experiments (2). Plasmids of known molecular weights used as controls were pip55 (150 kilobases [kb]), pip186 (111 kb), and pip135 (80 kb). * Corresponding author. Antibiotics. Disks for agar diffusion tests were purchased from Diagnostics Pasteur. Antibiotic powders were provided as follows: cephalothin and moxalactam, Eli Lilly & Co.; cefoxitin and imipenem, Merck Sharp & Dohme; ceftazidime, Glaxo Pharmaceuticals, Ltd.; cefotaxime, Hoechst- Roussel; aztreonam, E. R. Squibb & Sons; clavulanic acid, Beecham Laboratories; and sulbactam, Pfizer Inc. Determination of MICs. The MICs of the 1-lactams were determined in Mueller-Hinton broth by a microdilution technique (Autodiluter II; Dynatech Laboratories, Inc.). Inocula of 105 to 106 CFU/ml were distributed with a multipoint inoculator (MIC 2000; Dynatech). Agar disk diffusion susceptibility tests (6) were done on Mueller-Hinton agar. Transfer of resistance characters. Conjugation experiments were performed for 40 min at 37 C as previously described (10). Plasmids were transferred to mutants of E. coli K-12 C600 resistant to rifampin or nalidixic acid. Transconjugants were selected on Mueller-Hinton agar containing rifampin (300,ug/ml) or nalidixic acid (150,ug/ml) plus cefotaxime (4,g/ml), ceftazidime (8,ug/ml), or kanamycin (20,ug/ml). The frequency of transfer was expressed relative to the number of donor cells. Tests for incompatibility. Tests for incompatibility were performed as described previously (7). Plasmid DNA isolation. Plasmid DNA of clinical isolates and transconjugants was extracted as previously described (11) by a protocol which is a modification of the method initially described by Bimboim and Doly (5). DNA agarose gel electrophoresis. DNA electrophoresis was performed in 0.7% agarose gels for 2 h at 12 V/cm. Gels were stained with ethidium bromide and photographed with Polaroid film with a UV light source. Restriction endonuclease analysis. Plasmid DNA obtained from crude preparations was digested with restriction endonuclease EcoRI according to the recommendations of the 1660

2 VOL. 32, 1988 EXPANDED-SPECTRUM P-LACTAMASES FROM K. PNEUMONIAE 1661 TABLE 1. Antibiotic susceptibilities of K. pneumoniae isolates CF104 (CTX-1) CF504 (CAZ-1) CF704 (CAZ-2) Antibiotic (disk potency; ~g) Zone diam Interpretationa Zone diam Interpretation Zone diam Interpretation (mm) (mm) (mm) P-Lactams Amoxycillin (25) <7 R <7 R <7 R Amoxycillin-clavulanate (20/10) 20 I 14 I 18 I Ticarcillin (75) <7 R <7 R <7 R Ticarcillin-clavulanate (75/10) 23 S 17 I 17 I Mezlocillin (75) <7 R <7 R <7 R Piperacillin (100) <7 R <7 R 10 R Mecillinam (25) 13 R 8 R 18 I Cephalothin (30) <7 R <7 R 10 R Cefotiam (30) 15 I 17 I 23 S Cefuroxime (30) 10 R <7 R 12 R Cefamandole (30) 10 R <7 R 16 I Cefoxitin (30) 22 S 13 R 16 I Cefoperazone (30) 12 R 11 R 16 I Cefotaxime (30) 15 I 19 I 20 I Ceftriaxone (30) 15 I 20 I 20 I Ceftazidime (30) 10 R 9 R <7 R Moxalactam (30) 27 S 25 S 23 S Aztreonam (30) 19 I 15 R 15 R Imipenem (10) 30 S 31 S 28 S Aminoglycosides Kanamycin (30) <7 R <7 R <7 R Tobramycin (10) 8 R 12 R <7 R Dibekacin (10) <7 R 12 R <7 R Gentamicin (10) 20 S <7 R 20 S Sisomicin (10) 12 R <7 R 13 R Amikacin (30) 13 R 25 S 14 R Netilmicin (30) 10 R 19 S 11 R Others Chloramphenicol (30) 17 R <7 R <7 R Tetracycline (30) <7 R <7 R <7 R Nalidixic acid (30) <7 R <7 R <7 R Pefloxacin (5) 12 R 10 R <7 R Trimethoprim (5) 20 S <7 R 23 S Sulfonamide (200) <7 R <7 R <7 R a R, Resistant; I, intermediate; S, susceptible; according to the recommendations of the Comite de l'antibiogramme de la Societe Frangaise de Microbiologie (1). manufacturer (Bethesda Research Laboratories, Inc., Gaithersburg, Md.). After digestion, the samples were applied to 1% agarose gels, electrophoresed, stained, and photographed as described above. I8-Lactamase preparation. Sonic extracts were prepared from cultures in Trypticase soy broth (BBL Microbiology Systems, Cockeysville, Md.) as previously described (8). Induction of P-lactamases was attempted by growing the cultures in broth containing subinhibitory concentrations of ceftazidime or cefotaxime. f-lactamase activity was revealed by adding 10,ul of crude bacterial extract to 10,ul of a 500-,g/ml solution of chromogenic cephalosporin CM32150 (Sanofi Clin-Midy). Analytical isoelectric focusing. Extracts prepared by ultrasonic disintegration were applied on filter paper strips to commercially obtained polyacrylamide gels containing ampholines with ph ranges of 3.5 to 10 and 5 to 6.5 (LKB Products). Electrofocusing was done by the procedure recommended by the manufacturer, using the LKB 2117 Multiphor apparatus. P-Lactamases of known pi (TEM-1, pi 5.4; TEM-2, 5.6; TEM-4, 5.9) were focused in parallel with the extracts. The enzyme activities were located in the gels with chromogenic cephalosporin CM Determination of j8-lactamase kinetic constants. The Km and Vmax constants of the P-lactamases were obtained by the computerized microacidometric method described by Labia et al. (9). The Km and Vmax values of plasmid-mediated,b-lactamases CTX-1, CAZ-1, and CAZ-2 were determined by using partially (close to 50-fold) purified extracts (3) obtained by using cultures of the E. coli transconjugants and were compared with those of P-lactamase TEM-2. The relative Vmax rates of hydrolysis for amoxycillin, carbenicillin, piperacillin, cephalothin, cefotaxime, ceftazidime, imipenem, and aztreonam were compared with that for benzylpenicillin, which was taken as 100%. RESULTS Resistance phenotypes of K. pneumoniae isolates. The resistance phenotypes of the three strains of K. pneumoniae were determined by the disk diffusion assay (Table 1). The isolates were resistant to all penicillins, first- and second-generation cephalosporins, and, most importantly, broad-spectrum cephalosporins and aztreonam. Strains CF504 (CAZ-1) and CF704 (CAZ-2) were more resistant to ceftazidime (zone diameters, 9 and <7 mm) than to cefotaxime (zone diame-

3 1662 CHANAL ET AL. ANTIMICROB. AGENTS CHEMOTHER. TABLE 2. Susceptibilities of K. pneumoniae isolates and their transconjugants to P-lactams MIC (pg/ml) for: Antibiotic(s) CF104 CF504 CF704 CF174 CF004 E. coli (CTX-1) (CAZ-1) (CAZ-2) (TEM-2) (susceptible) K-12 C600a Cephalothin 256 (128)b 1,024 (256) 32 (32) 64 (64) CAc 16 (8) 64 (8) 8 (8) 4 (16) SUld 16 (8) 512 (32) 4 (8) 64 (64) 4 16 Ceftazidime 64 (16) 128 (64) 64 (64) 0.5 (0.25) CA 2 (0.5) 2 (1) 0.25 (0.25) 0.25 (0.25) Sul 1 (0.5) 64 (4) 0.5 (0.25) 0.5 (0.25) Cefotaxime 32 (8) 16 (16) 4 (4) 0.06 (0.06) CA 0.5 (0.12) 1 (0.25) 0.06 (0.06) 0.06 (0.06) Sul 0.5 (0.12) 8 (1) 0.06 (0.06) 0.06 (0.06) Aztreonam 16 (8) 32 (8) 8 (4) 0.12 (0.12) CA 0.25 (0.25) 0.5 (0.12) 0.12 (0.06) 0.06 (0.06) Sul 0.5 (0.25) 16 (0.5) 0.12 (0.06) 0.25 (0.12) Cefoxitin 8 (4) 32 (4) 8 (4) 4 (2) CA 8 (4) 32 (4) 4 (2) 2 (2) Sul 8 (4) 32 (4) 4(2) 4 (2) 4 4 Moxalactam 0.5 (0.5) 4 (1) 0.5 (0.25) 0.12 (0.25) CA 0.5 (0.25) 2 (0.5) 0.12 (0.12) 0.12 (0.12) Sul 0.25 (0.25) 2 (0.5) 0.12 (0.12) 0.12 (0.25) Imipenem 0.5 (0.5) 1 (0.5) 0.12 (0.5) 0.25 (0.25) CA 0.5 (0.5) 0.25 (0.5) 0.25 (0.25) 0.12 (0.25) Sul 0.25 (0.5) 0.25 (0.5) 0.12 (0.12) 0.5 (0.25) a Plasmid free. b MICs for transconjugants are shown in parentheses. c CA, Clavulanic acid (2,ug/mi). d Sul, Sulbactam (2,ug/ml). ters, 19 and 20 mm). The three strains were always susceptible to moxalactam and imipenem. Synergism was observed when clavulanic acid (10,ug) was combined with amoxycillin or ticarcillin. Resistance to aminoglycosides, chloramphenicol, tetracycline, and sulfonamide was also noted in all the isolates. Table 2 shows the MICs of cephalothin, ceftazidime, cefotaxime, aztreonam, cefoxitin, moxalactam, and imipenem alone and combined with clavulanic acid (2,ug/ml) or sulbactam (2 jxg/ml). The MIC of cephalothin against CAZ- 2-producing K. pneumoniae was much lower (32,ug/ml) than it was against CTX-1- or CAZ-1-producing strains (256 and 1,024 plg/ml, respectively). The MICs of ceftazidime and cefotaxime were nearly the samne against CTX-1-producing K. pneumoniae (64 and 32,ug/ml, respectively), while the MICs of ceftazidime (128 and 64,g/ml) were much higher than the MICs of cefotaxime (16 and 4,ug/ml) against CAZ-1- and CAZ-2-producing strains. For the three strains, the MICs of aztreonam ranged from 8 to 32 jig/ml and the lowest MICs were those of moxalactam and imipenem. The impaired activity of all P-lactams except cephamycins was highly restored when the drugs were combined with clavulanic acid (2,ug/ml) against the three strains. However, the MIC of cephalothin against the CAZ-1 producer was still high (64,ug/ml). The efficacies of the combination with sulbactam (2,ug/ml) were equivalent in CTX-1- and CAZ-2- producing strains, whereas the efficacy was much weaker in the CAZ-1-producing strain. Plasmid-mediated resistance. The genes conferring resistance to P-lactams were transferable to E. coli with a frequency ranging from 1o-4 to Similar MICs were observed for K. pneumoniae isolates and their E. coli transconjugants with all,b-lactams except cefoxitin and moxalactam against the CAZ-1-producing strain; in this case, the wild type was more resistant (32 and 4,ug/ml) than its transconjugant (4 and 1,ug/ml) (Table 2). The effects of combinations with clavulanic acid or sulbactam against the transconjugants were similar to those observed with the clinical isolates: the efficacy of sulbactam was lower against the CAZ-1-producing strain. Resistance to aminoglycosides except gentamicin, streptomycin, and neomycin was cotransferred with the CTX-1 and CAZ-2,-lactamases; whereas only resistance to kanamycin, neomycin, and streptomycin was associated with the CAZ-1 P-lactamase. In the three strains, resistance to tetracycline and sulfonamide was also cotransferred with resistance to P-lactam antibiotics (Table 3). Plasmid DNA of the three wild-type strains and of the corresponding transconjugants was analyzed by agarose gel electrophoresis (Fig. 1). In K. pneumoniae CF104 and its transconjugant, CF204, P-lactamase CTX-1 was mediated by a 85-kb plasmid, pcff04. In K. pneumoniae CF504 and its transconjugant, CF604, P-lactamase CAZ-1 was mediated by a large plasmid (pcff14) with a molecular size of approximately 150 kb. In K. pneumoniae CF704 and its transconjugant, CF804, P-lactamase CAZ-2 was mediated, as in the CTX-1-producing strain, by a 85-kb plasmid, which was designated pcff34. Incompatibility testing of E. coli transconjugants CF204 and CF804 revealed that plasmids pcff04 and pcff34 both belonged to incompatibility group M or 7. Plasmid pcff14 did not belong to this group.

4 VOL. 32, 1988 EXPANDED-SPECTRUM P-LACTAMASES FROM K. PNEUMONIAE 1663 TABLE 3. Characteristics of three novel P-lactamase-specifying plasmids Enzyme pi Plasmid Incompatibility group Plasmid size (kb) Resistance markers' cotransferred Frequency of transfer CTX pcff04 M or 7 85 Ak Km Nt Su Tc Tm 10-4 CAZ pcff14? 150 Km Nm Sm Su Tc 10-6 CAZ pcff34 M or 7 85 Ak Km Nt Su Tc Tm 10-4 a Ak, Amikacin; Km, kanamycin; Nm, neomycin; Nt, netilmicin; Sm, streptomycin; Su, sulfonamide; Tc, tetracycline; Tm, tobramycin. Plasmids pcff04, pcff14, and pcff34 were digested with EcoRI restriction endonuclease and analyzed by agarose gel electrophoresis (Fig. 2). Very similar restriction patterns were observed for plasmids pcff04 (CTX-1) and pcff34 (CAZ-2). The restriction pattern of pcff14 (CAZ- 1) was different. Mechanism of plasmid-mediated resistance to P-lactams. The three K. pneumoniae isolates had different P-lactamase profiles as revealed by isoelectric focusing. The chromosomally encoded SHV-1 penicillinase (pi 7.7) was present in the three isolates. As previously reported (12, 13), plasmidmediated P-lactamases CTX-1 (CF104) and CAZ-1 (CF504) focused, respectively, at pi 6.3 and In K. pneumoniae CF704 and its transconjugant, CF804, the CAZ-2 P-lactamase focused at a pi approximately 6.0 slightly higher than that of the TEM-4 enzyme (pl 5.9) (G. Paul, G. Gerbaud, A. Bure, A. Philippon, B. Pangon, and P. Courvalin, Program Abstr. 27th Intersci. Conf. Antimicrob. Agents Chemother., abstr. no. 520, 1987). In strains CF504 and CF704, there was also a TEM-1 penicillinase focusing at ph 5.4 (Fig. 3). In Table 4, the values of the kinetic constants for the three P-lactamases are reported. The three enzymes hydrolyzed penicillins with similar Vmax values for CTX-1 and CAZ-1, whereas the hydrolysis was very much higher with CAZ-2, particularly against amoxycillin (Vmax, 238) and piperacillin (Vmax, 440). Hydrolysis of cephalothin was higher with the CAZ-1 enzyme (Vmax, 380) than with the CTX-1 and CAZ-2,-lactamases (Vmax, 107 and 165, respectively). The three enzymes hydrolyzed broad-spectrum cephalosporins: CTX-1 and CAZ-2 showed much greater relative Vmax values for cefotaxime (445 and 640, respectively) than for ceftazidime (40 and 261, respectively). Conversely, with the CAZ-1 enzyme the greatest Vmax value was observed for ceftazidime (Vmax, 490) and was much higher than that for cefotaxime (Vmax, 150). Aztreonam was hydrolyzed by CAZ-1 (Vmax, 116) and much more by CAZ-2 (Vmax, 207). The hydrolysis of aztreonam with CTX-1 was 10-fold lower (Vmax, 8.5). The three enzymes lacked detectable activity against cephamycins and imipenem. DISCUSSION Since the outbreak of infections caused by CTX-1-producing K. pneumoniae and other species of the family FIG. 1. Agarose gel electrophoresis of plasmid DNA from K. pneumoniae CF704 (lane C), CF504 (lane E), and CF104 (lane 0) and their corresponding E. ccli K-12 transconjugants, CF804 (lane B, CF604 (lane D), and CF204 (lane F). Lane A, E. coli containing plasmids pip55 (150 kb), pip186 (111 kb), and pip135 (80 kb) used as molecular size standards (sizes indicated on the left). FIG. 2. Agarose gel electrophoresis of fragments produced by EcoRI digestion of plasmids extracted from E. coli transconjugants. Lanes: A, CF604(pCFF14); B, CF804(pCFF34); C, CF204(pCFF04).

5 1664 CHANAL ET AL. ANTIMICROB. AGENTS CHEMOTHER. FIG. 3. Analytical isoelectric focusing (ph range, S to 6.5) of 1-lactamases as follows. Lanes: A, E. coli transconjugant CF204 (CTX-1; pl 6.3); B, E. coli RP4 (TEM-2; pi 5.6); C, E. coli Rill (TEM-1; pi 5.4); D, E. coli 134K (TEM-4; pl 5.9); E, E. coli transconjugant CF804 (CAZ-2); F, K. pneumoniae CF704 (CAZ-2; TEM-1); G, E. coli transconjugant CF604 (CAZ-1; pl 5.55); H, K. pneumoniae CF504 (CAZ-1; TEM-1). The enzymes were located in the polyacrylamide gel by using a cephalosporin chromogen (CM32150) procedure. The SHV-1 penicillinases (p1 7.7) of K. pneumoniae strains are not visible because of the ph range (5 to 6.5). phs are shown in the left. Enterobacteriaceae (14), we have observed in the same hospital the advent of two novel expanded-spectrum,blactamases in K. pneumoniae isolates, CAZ-1 and, more recently, CAZ-2. Since the original CAZ-1- and CAZ-2- producing K. pneumoniae strains were isolated, 10 more CAZ-1-producing and 5 more CAZ-2-producing K. pneumoniae strains have been encountered in hospitals in Clermont- Ferrand, France. CTX-1-producing strains have spread to all intensive care units and to some other wards, whereas CAZ-1- and CAZ-2-producing strains were isolated separately in two different intensive care units. The first CAZ-1- and CAZ-2-producing strains were isolated from patients under treatment with ceftazidime alone or in combination with vancomycin. These isolates were more resistant to ceftazidime than to cefotaxime. Enzyme CAZ-1 can be considered as a ceftazidimase according to the resistance phenotype of producing strains and its hydrolytic activity. The Vm,g for ceftazidime was higher than that for cefotaxime; and aztreonam, which possesses the same side chain as ceftazidime, was also hydrolyzed. Likewise, the CAZ-2 enzyme appears to be a ceftazidimase as regards hydrolysis of ceftazidime and aztreonam (Vma,x 261 and 207, respectively). However, CAZ-2 differed from CAZ-1 by a rate of hydrolysis higher for cefotaxime (Vma,x 640) than for ceftazidime (Vmax, 261). CAZ-2 producers also had a lower level of resistance to cephalothin (MIC, 32,g/ml) than CAZ-1 producers (MIC, 1,024,ug/ml). The activities of the P-lactamase inhibitors against these two enzymes were different: sulbactam and clavulanic acid were equivalent against CAZ-2-producing strains, whereas sulbactam was less active than clavulanic acid against CAZ-1- producing strains. The enzymes differed also in their isoelectric points (CAZ-2, 6.0; CAZ-1, 5.55). Finally, these two enzymes were encoded by unrelated plasmids. On the other hand, some enzymatic characteristics bring CAZ-2 nearer to the CTX-1 enzyme, in particular the greatest Vmax values for cefotaxime and the comparable inhibitory activities of clavulanic acid and sulbactam. Most importantly, the plasmids encoding CAZ-2 (pcff34) and CTX-1 (pcff04) showed similar molecular weights (85 kb) and incompatibility groups (M or 7) and harbored the same associated resistance markers (Ak Km Nt Su Tc Tm). Moreover, the restriction patterns obtained after digestion by EcoRI endonuclease revealed that the pcff04 and pcff34 plasmids are closely related. Novel P-lactamase CAZ-2 also seems to be different from recently described ceftazidimases. The plasmid-mediated P-lactamase TEM-7 described by Gutmann et al. (7a) differs from CAZ-2 in its isoelectric point (5.41) and its kinetic TABLE 4. Kinetic constants of,b-lactamases from E. coli transconjugantsa PEN AMO CAR PIP CTN CTX CAZ IPM ATM Enzyme pi-_- Vmax K. V.. Km Vm. Km V.. Km V... Km Vm. Km Vmax Km Vm. K,b Vm. Km CTX < CAZ < CAZ < TEM <0.1 _c <0.1 - < <0.1 a PEN, Benzylpenicillin; AMO, amoxycillin; CAR, carbenicillin; PIP, piperacillin; CTN, cephalothin; CTX, cefotaxime; CAZ, ceftazidime; IPM, imipenem; ATM, aztreonam. VN.ax is expressed relative to penicillin G as loo1o. b Ki, Inhibitor constant determined when the antibiotic was not a substrate of the enzyme. c -, Poor affinity.

6 VOL. 32, 1988 EXPANDED-SPECTRUM P-LACTAMASES FROM K. PNEUMONIAE 1665 constant. Enzyme RHH-1 (17), which confers resistance to aminothiazolyl cephalosporins and aztreonam, differs from CAZ-2 in its isoelectric point (5.5) but like CAZ-2 confers a low level of resistance to first- and second-generation cephalosporins. This enzyme is nearer to the CAZ-1 enzyme by its isoelectric point (5.5) and its hydrolytic activity. The resistance markers cotransferred with RHH-1 differ from those observed with P-lactamases CAZ-1 and CAZ-2. The ceftazidime-hydrolyzing enzyme described by Bauernfeind and Horl (4) seems near to CAZ-2 by its isoelectric point (5.85). Data concerning kinetic constants and plasmid characterization were not given. Resistance markers associated with this enzyme differ from those observed with 1-lactamase CAZ-2. Recently, it has been demonstrated that the gene encoding the CTX-1 enzyme derives from the TEM-2 gene by two point mutations (16). DNA-DNA hybridization with a TEM probe showed that the CAZ-1 enzyme is also a TEM derivative (12). The same result was obtained with CAZ-2 (results not shown). Thus, these findings suggest a similar evolutionary process for the novel CAZ-2 ceftazidimase. ACKNOWLEDGMENTS We thank M. Jan and D. Rubio for their technical assistance. This work was supported by a grant from the University of Clermont-Ferrand. LITERATURE CITED 1. Acar, J., E. Bergogne-Berezin, Y. Chabbert, R. Cluzel, A. Courtieu, P. Courvalin, H. Dabernat, H. Drugeon, J. Duval, J. Fleurette, C. Morel, A. Philippon, J. Sirot, C. J. Soussy, A. Thabaut, and M. Veron Communique 1987 du Comitd de l'antibiogramme de la Socidtd Frangaise de Microbiologie. Pathol. Biol. 35: Bachmann, B. J Pedigrees of some mutant strains of Escherichia coli K-12. Bacteriol. Rev. 36: Barthelemy, M., J. Peduzzi, C. Verchere-Baur, H. Ben-Yaghlane, and R. Labia Purification and biochemical properties of the Pitton's type II P-lactamase (SHV-1). Ann. Microbiol. (Inst. Pasteur) 137B: Bauernfeind, A., and G. Horl Novel-R-factor borne 3-lactamase of Escherichia coli confering resistance to cephalosporins. Infection 15: Birnboim, H. C., and J. Doly A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7: Chabbert, Y. A., E. Derlot, and P. Courvalin A study by the National Reference Center for Antibiotics of inoculums for disc antibiotic sensitivity testing. Pathol. Biol. 34: Chabbert, Y. A., M. R. Scavizzi, J. L. Witchitz, G. R. Gerbaud, and D. H. Bouanchaud Incompatibility groups and the classification offi- resistance factors. J. Bacteriol. 112: a.Gutmann, L., M. D. Kitsis, D. Billot-Klein, F. Goldstein, G. Tran Van Nhieu, T. Lu, J. Carlet, E. Collatz, and R. Williamson Plasmid-medicated P-lactamase (T.E.M.-7) involved in resistance to ceftazidine and aztreonam. Rev. Infect. Dis. 10: Labia, R Comportement enzyme-substrat. Introduction de la notion de stabilitd enzymatique dans le cas des iplactamases. C. R. Acad. Sci. 279: Labia, R., J. Andrilion, and F. Le Goffic Computerized microacidimetric determination of P-lactamase Michaelis- Menten constants. FEBS Lett. 33: Lesage, D. D., G. R. Gerbaud, and Y. A. Chabbert Carte genetique et structure chez Escherichia coli K12 d'un plasmide de rdsistance isole de Salmonella ordonez. Ann. Microbiol. (Inst. Pasteur) 126A: Maniatis, T., E. F. Fritsch, and J. Sambrook Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 12. Petit, A., D. L. Sirot, C. M. Chanal, J. L. Sirot, R. Labia, G. Gerbaud, and R. A. Cluzel Novel plasmid-mediated,3- lactamase in clinical isolates of Klebsiella pneumoniae more resistant to ceftazidime than to other broad-spectrum cephalosporins. Antimicrob. Agents Chemother. 32: Sirot, D., J. Sirot, R. Labia, A. Morand, P. Courvalin, A. Darfeuille-Nichaud, R. Perroux, and R. Cluzel Transferable resistance to third-generation cephalosporins in clinical isolates of Klebsiella pneumoniae: identification of CTX-1, a novel P-lactamase. J. Antimicrob. Chemother. 20: Sirot, J., C. Chanal, A. Petit, D. Sirot, R. Labia, and G. Gerbaud Klebsiella pneumoniae and other enterobacteriaceae producing novel plasmid-mediated 13-lactamases markedly active against third-generation cephalosporins: epidemiologic studies. Rev. Infect. Dis. 10: Sirot, J., R. Labia, and A. Thabaut Klebsiella pneumoniae strains more resistant to ceftazidime than to other thirdgeneration cephalosporins. J. Antimicrob. Chemother. 20: Sougakoff, W., S. Goussard, G. Gerbaud, and P. Courvalin Plasmid-mediated resistance to third-generation cephalosporins caused by point-mutations in TEM-type penicillinase genes. Rev. Infect. Dis. 10: Spencer, R. C., P. F. Wheat, T. G. Winstanley, D. M. Cox, and S. J. Plested Novel P-lactamase in a clinical isolate of Klebsiella pneumoniae conferring unusual resistance to,blactam antibiotics. J. Antimicrob. Chemother. 20: