resistance to extended-spectrum cephalosporins. In this species, TEM-1 and TEM-2 1-lactamases confer additional
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1 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, May 1993, p /93/ $02.00/0 Copyright X 1993, American Society for Microbiology Vol. 37, No. 5 Characterization of a Novel Extended-Spectrum from Pseudomonas aeruginosa 1-Lactamase PATRICE NORDMANN,12t * ESTHEL RONCO,1 THIERRY NAAS,2 CATHERINE DUPORT,3 YVON MICHEL-BRIAND,3 AND ROGER LABIA4 Laboratoire de Microbiologie, H6pital Raymond Poincare, Faculte6 de Medecine Paris-Ouest, Garches, Laboratoire de Bacteriologie, Faculte6 de Me6decine, Besanqon,3 and Museum d'histoire Naturelle URA 401 Centre National de la Recherche Scientifique, Paris 05, 4 France, and Abteilung Mikrobiologie, Biozentrum der Universitat Basel, 4056 Basel, Switzerland2 Received 17 November 1992/Accepted 3 March 1993 A clinical isolate ofpseudomonas aeruginosa RNL-1 showed resistance to extended-spectrum cephalosporins which was inhibited by clavulanic acid. Although this strain contained three plasmids ca. 80, 20, and 4 kb long, the resistance could not be transferred by mating-out assays with P. aeruginosa or Escherichia coli. Cloning of a 2.1-kb Sau3A fragment from P. aeruginosa RNL-1 into plasmid pacyc184 produced ppz1, a recombinant plasmid that encodes a 1-lactamase. This f-lactamase (PER-1) had a relative molecular mass of 29 kda and a pl of 5.4 and was biosynthesized by P. aeruginosa RNL-1 along with a likely cephalosporinase with a pl of 8.7. PER-1 showed a broad substrate profile by hydrolyzing benzylpenicillin, amoxicillin, ticarcillin cephalothin, cefoperazone, cefuroxime, HR 221, ceftriaxone, ceftazidime, and (moderately) aztreonam but not oxacillin, imipenem, or cephamycins. Vmax values for extended-spectrum cephalosporins were uncommonly high, and the affinity of the enzyme for most compounds was relatively low (i.e., high Km)* PER-1 activity was inhibited by clavulanic acid, sulbactam, imipenem, and cephamycins but not by EDTA. A 1.1-kb SnaBI fragment from ppz1 failed to hybridize with plasmids that encode TEM-, SHV-, OXA-, or CARB/PSE-type 13-lactamase or with the ampc gene of P. aeruginosa. However, the same probe appeared to hybridize with chromosomal but not plasmid DNA from P. aeruginosa RNL-1. This study reports the properties of a novel extended-spectrum 13-lactamase in P. aeruginosa which may not be derived by point mutations from previously known enzymes of this species. More than 50 biochemically distinct P-lactamases responsible for resistance to,-lactams have been reported in gram-negative bacteria. The resistance of broad-spectrum cephalosporins to these,b-lactamases was a widely accepted concept in the beginning of the 1980s. However, overproduction of chromosomally mediated cephalosporinases has been described as responsible for failure of treatment of gram-negative bacterial infections with extended-spectrum cephalosporins (39). Since 1983, plasmid-mediated extended-spectrum 3-lactamases have been reported, primarily in Kiebsiella pneumoniae and then in numerous Enterobacteraceae species (16, 34). These enzymes hydrolyze extended-spectrum cephalosporins and aztreonam to various extents but usually neither cephamycins (cefoxitin and moxalactam) nor carbapenems (imipenem and meropenem). A common feature of these enzymes is inhibition of their activity by clavulanic acid. These enzymes are Ambler class A 1-lactamases, members of the TEM or SHV series that differ by a few point mutations in their structural genes (16, 34) Ṡuch enzymes from Pseudomonas aeruginosa have not been reported. In this species, induction or derepression of chromosomal cephalosporinases may lead to resistance to extended-spectrum cephalosporins but their activity is not inhibited by clavulanic acid (22). The other P-lactamases commonly found in P. aeruginosa do not confer clinical * Corresponding author. t Present address: Laboratoire de Microbiologie, H6pital Raymond Poincare, 104 Bd Raymond Poincare, Garches, France. 962 resistance to extended-spectrum cephalosporins. In this species, TEM-1 and TEM-2 1-lactamases confer additional resistance to ureidopenicillins (26). The OXA-type (oxacillin-hydrolyzing) enzymes possess high-level hydrolytic activity against cloxacillin, oxacillin, and methicillin (9, 10). Their activities are inhibited by clavulanic acid but to a lesser extent than TEM- or SHV-derivative P-lactamases. This group includes OXA-1 to OXA-7, as well as PSE-2, and possesses hydrolytic activities similar to and protein sequences related to those of oxacillin-hydrolyzing enzymes. CARB- and PSE-type j-lactamases (with the exception of PSE-2), also called carbenicillin-hydrolyzing enzymes, are found primarily in P. aeruginosa. They tend to hydrolyze cephalosporins much more slowly than penicillin and generally have slow rates of cloxacillin hydrolysis (9, 10). Both the oxacillin-hydrolyzing enzymes and carbenicillin-hydrolyzing enzymes described so far do not confer clinical resistance to extended-spectrum cephalosporins on P. aeruginosa. In this report, we describe a novel, likely chromosomally mediated, extended-spectrum,b-lactamase from a clinical isolate of P. aeruginosa. The purpose of this work was to (i) determine the enzymatic properties of the 1-lactamase, (ii) characterize its genetic determinant, and (iii) search for DNA homology with previously described TEM- and SHVderived 1-lactamases, as well as with oxacillin-hydrolyzing and carbenicillin-hydrolyzing enzymes. Special interest in this P-lactamase comes from its high level of hydrolytic activity against extended-spectrum cephalosporins and inhibition of its activity by clavulanic acid, sulbactam, imipenem, and cephamycins.
2 VOL. 37, 1993 EXTENDED-SPECTRUM j-lactamase FROM P. AERUGINOSA 963 TABLE 1. Strain or plasmid Strains Escherichia coli JM109 Pseudomonas aeruginosa RNL-1 P. aeruginosa PAO 2635 P. aeruginosa NTCT 8203 P. aeruginosa Pou P. aeruginosa Cil P. aeruginosa Da Xanthomonas maltophilia 6077T Plasmids pacyc184 phuc37 pkl9 ppzl prazl puc19 p124 p453 Rlll R151 Rmsl49 RP4 RGN238 R46 R57b Bacterial strains and plasmids used in this study Relevant genotype or phenotype end4l hsdr17gyra96 A(lac proa) recabi rel4 supe44 thi F' (laclq laczam15 proab+ trad36) PER-1 P-lactamase FP- trp-54 nf-3 fon-101 AmpC from P. aeruginosa blapse-l blacarb-3 blapse4 blal-l blal-2 cat tet Recombinant plasmid containing 450-bp PstI-NotI internal fragment of blashv-3 Kanamycin resistance 2.1-kb Sau3A fragment from P. aeruginosa RNL-1 cloned into pacyc kb SnaBI fragment from ppz1 cloned into SmaI site from digested pk19 Recombinant plasmid containing a 298-bp HincII-PstI internal fragment for blateml blashv-2 blashv-l blatem-l blapse-2 blapse-3 blatem-2 blaoxa-2 blaoxa-3 a IPSC, Institut Pasteur strain collection. MATERIALS AND METHODS Source or reference Bacterial strains and plasmids. The bacterial strains and plasmids used in this work are listed in Table 1. P. aeruginosa RNL-1 was isolated from the urinary tract of a hospitalized patient at Raymond Poincare Hospital, Garches, France, in Antimicrobial regimens before admission were not documented, and the patient did not receive any antibiotic treatment before isolation of the strain at the hospital. The strain was identified with API-20 NE (Biomerieux, La Balme-les-Grottes, France) and belonged to serogroup 0:2 (antisera from Diagnotics Pasteur, Mames- La-Coquette, France). On the basis of agar disc diffusion assay results, the isolate was resistant to extended-spectrum cephalosporins. A marked synergistic effect between clavulanic acid and extended-spectrum cephalosporins was observed that suggested the presence of an uncommon P-lactam resistance mechanism in P. aeruginosa. Analysis of resistance to,b-lactams is described below. P. aeruginosa RNL-1 was also resistant to amikacin, chloramphenicol, gentamicin, kanamycin, neomycin, netilmycin, sisomycin, streptomycin, sulfonamides, and tobramycin. Antimicrobial agents and MIC determinations. The antimicrobial agents used in this study were obtained from standard laboratory powders and were used immediately after solubilization. The agents and their sources were as follows: ampicillin, Bristol; amoxicillin, clavulanic acid, cloxacillin, and ticarcillin, Smith Kline French-Beecham; aztreonam, Squibb; cefamandole and moxalactam, Eli Lilly; cefpirome, ceftriaxone, and HR 221, Hoffmann-La Roche; cefuroxime, ceftazidime, and cephalothin, Glaxo; cefoxitin and imipenem, Merck Sharp & Dohme; cefotaxime, Hoechst-Roussel; meropenem, ICI; benzylpenicillin, Specia; sulbactam, Pfizer; piperacillin, Lederle; rifampin, Sigma. MICs were determined by an agar dilution technique on Mueller-Hinton agar (Diagnostics Pasteur) with a Steers multiple inoculator and an inoculum of 104 CFU per spot. All plates were incubated at 37 C for 18 h. MICs of 13-lactams were determined alone or in combination with 2,ug of clavulanic acid per ml or 4,ug of sulbactam per ml. Plasmid content and conjugation assays. Lysates of P. aeruginosa RNL-1 were prepared by the Kieser method (17). Plasmid DNA was detected by electrophoresis in a 0.7% agarose gel (95 V, 4 h) (27). The molecular sizes of plasmid DNAs were estimated by comparison with plasmids whose sizes are known (23, 43). Direct transfer of resistance into P. aeruginosa PAO 2635 and into in vitro-obtained rifampin-resistant Escherichia coli JM109 was attempted by liquid and solid mating-out assays at 30 and 37 C as previously described (36). Transconjugant selection was performed on Luria agar plates (Diagnostic Pasteur) containing rifampin (100 p,g/ml) and either ceftazidime (10,ug/ml) or ticarcillin (250,ug/ml). Cloning experiments and analysis of recombinant plasmids. Chromosomal DNA of P. aeruginosa RNL-1 was prepared as previously described (14). Fragments from partially Sau3A-digested genomic P. aeruginosa DNA (Boehringer, Mannheim, Germany) were ligated to the BamHI site of pacyc184 (11). BamHI-digested pacyc184 was dephosphorylated and electrophoresed in 0.8% low-melting-point agarose, and then the band was excised and purified with Genclean II (Bio 101, Inc., La Jolla, Calif.). Ligation was performed with a 2:1 vector-insert ratio at a final concentration of ca. 200 ng of DNA in a ligation mixture containing 1 U of T4 DNA ligase (Boehringer) at 15 C for 18 h. Recombinant plasmids were transformed by electroporation into E. coli JM109 (Bio-Rad, Richmond, Calif.). Antibiotic-resistant colonies were selected on Luria agar plates supplemented with 1 or 5,ug of ceftazidime per ml, 50 or 100,ug of ampicillin per ml, 100 or 200,ug of ticarcillin per ml. Recombinant DNA plasmids were obtained from 500-ml Luria broth cultures grown overnight at 37 C and isolated in accordance with the Quiagen protocol (Diagen). The reaction conditions used for restriction enzyme digestions were those suggested by the supplier (Boehringer). Restricted DNA fragments were separated on 0.8% agarose in 90 mm Tris-90 mm boric acid-3 mm EDTA (ph 8.0). The gels were stained in a solution of ethidium bromide for 15 min and washed for 15 min in distilled water. The standard molecular weight marker was a 1-kb DNA ladder (BRL, Basel, Switzerland). Estimation of fragment sizes and construction of physical maps were done after restriction enzyme double digestion.
3 964 NORDMANN ET AL. 13-Lactamase preparation. Cultures were grown overnight at 37 C in 100 ml of Trypticase soy broth (Diagnostics Pasteur). Bacterial suspensions were disrupted by sonification (4 x 20 s at 20 Hz) and centrifuged (30 min, 20,000 x g, 4 C). The supernatant contained the crude enzyme extracts. Isoelectric focusing. Crude,-lactamase extracts were submitted to analytical isoelectric focusing on a ph 3.5 to 9.5 ampholin polyacrylamide gel (Pharmacia, Uppsala, Sweden) for 36 h at 10 W of constant power on a flatbed apparatus (FBE-3000; Pharmacia). The 13-lactamases were visualized with an overlay of agar-iodine starch gel containing penicillin (0.01% [wt/vol]) in 0.1 M phosphate buffer (ph 7.0) (2). 13-Lactamase activity and determination of kinetic constants. The P-lactamase activity of crude extracts of E. coli JM109 harboring recombinant plasmid ppz1 (see Results) and the kinetic constants of preparations were determined by the one-line computerized microacidimetric method at ph 7.0 and 37 C as previously described (18). As assessed by isoelectric focusing, these crude extracts contained only a single f3-lactamase activity. The Km was expressed in micromolar concentrations, and Vma, was expressed relative to that of benzylpenicillin (Vm. = 100). In the case of substrates with low or undetectable Vm. values, enzymesubstrate affinity was measured as Ki (inhibition constant) rather than Km with benzylpenicillin as the substrate. Inhibition of 13-lactamase activity. Various concentrations of clavulanic acid, sulbactam, imipenem, and cephamycins were preincubated with the enzymes for 10 min at 37 C before testing of the rate of benzylpenicillin hydrolysis. Inhibition by cloxacillin was determined from the rate of benzylpenicillin hydrolysis in the presence up to 0.1 mm inhibitor. Ki values were consequently determined. Inhibition by EDTA (final concentration, 1 mm) (Sigma) or NaCl (final concentration, 100 mm) was tested by preincubation with the enzyme for 10 min at 37 C before testing of the rates of benzylpenicillin and ceftazidime hydrolysis. The rate of hydrolysis was also studied when the enzyme was preincubated with up to 10 mm ZnCl2. Determination of relative molecular mass. The relative molecular mass of the 3-lactamase obtained from E. coli JM109 harboring recombinant plasmid praz1 (see Results) was estimated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (19). Crude extracts and marker proteins (Pharmacia, Uppsala, Sweden) were treated with 1% SDS-3% P-mercaptoethanol at 100 C for 2 min and then subjected to electrophoresis in a 12.5% gel (20 ma, 5 h, room temperature). Renaturation of P-lactamase activity after denaturing electrophoresis was performed as previously described (25). Hybridization. DNA-DNA hybridizations were performed by the method of Southern (40). DNA was extracted from P. aenrginosa RNL-1, P. aeruginosa NTCT 8203, X. maltophilia 6077T (blal 1 blal 2), P. aeruginosa Pou (blapsel), P. aeruginosa Da (blapse4),p. aeruginosa Cil (blacarb-3), P. aeruginosa harboring plasmid Rmsl49 (blapse3) or R151 (blapse2), E. coli harboring plasmid RGN238 (blaoxa1), and Salmonella typhimurium harboring plasmids R46 (blaoxa2) and R57b (blaoxa-3). Approximately 10,ug of each of these DNAs was digested by SnaBI and run on a 0.7% agarose gel along with the following DNA fragments: the 298-bp HincII-PstI internal fragment of the blateml1 structural gene from puc19 (45), the 435-bp NotI-PstI internal fragment of the blashv-3 structural gene from phuc37 (30), and the 1.1-kb SnaBI fragment from ppz1 (as a positive control). DNAs were transferred onto and immobilized on a Biodyne A transfer membrane (PALL, PortspPZl ANTIMICROB. AGENTS CHEMOTHER. E Hc HcBE HcB H I I I LI LI L1 I I I * I I I Sc Pv Sc PV E I Pv Pv 1 kb FIG. 1. Restriction endonuclease map of recombinant plasmid ppz1, which codes for the PER-1,-lactamase. The thin lines indicate vector pacyc184, and the thick line represents the cloned insert from P. aeruginosa RNL-1. Restriction sites: B, BamHI; E, EcoRI; H, HindlIl; Hc, HincII; P, PstI; Pv, PvuI; S, SnaBI; Sc, Scal. The vector BamHI site marked with an asterisk was destroyed after cloning of the Sau3A insert from P. aeruginosa RNL-1. mouth, England). The probe consisted of the 1.1-kb SnaBI fragment from recombinant plasmid ppz1. The DNA probe was labeled with [32P]dATP with a random primer DNA labeling kit (Bio-Rad). Hybridization reactions were performed under high- and low-stringency conditions (24). RESULTS Cloning of the extended-spectrum 3-lactamase gene. Total DNA from P. aeruginosa RNL-1 was partially digested with restriction endonuclease Sau3A and ligated to BamHI-digested plasmid pacyc184. The ligated-dna mixture was transformed into E. coli JM109 with selection for ampicillin-, ticarcillin-, or ceftazidime-resistant colonies. Despite 12 attempts, only three colonies were obtained on ampicillin (50,ug/ml)-containing plates. These colonies were grown overnight, and their recombinant plasmids were extracted. The three recombinant plasmids contained inserts of the same size, i.e., 2.1 kb. One plasmid, ppz1, was retained for detailed restriction map analysis (Fig. 1). Subcloning of various restriction digests led to isolation of a 1.1-kb SnaBI fragment from ppz1, which was inserted into SmaI-digested multicopy vector pk19 (praz1), which expressed the same level of,b-lactam resistance as ppz1. It was used to determine the relative molecular mass of the cloned P-lactamase. Antibiotic susceptibility. MICs of,b-lactams showed the high resistance of P. aeruginosa RNL-1 to ticarcillin and extended-spectrum cephalosporins (Table 2). Susceptibility of P. aeruginosa RNL-1 to these antibiotics was partially restored with clavulanic acid. MICs of P-lactams for E. coli JM109 harboring recombinant plasmid ppz1 confirmed the extended-spectrum cephalosporin resistance conferred by the cloned 3-lactamase gene. In this strain, MICs of aztreonam and ceftazidime were markedly increased compared with those of cefotaxime and ceftriaxone. The MIC of moxalactam was slightly increased, while that of cefoxitin was not. All of the 13-lactams tested, except imipenem and cefoxitin, had decreased MICs in the presence of clavulanic acid or sulbactam, and these were more obvious in E. coli harboring ppz1 than in the original strain, P. aeruginosa I
4 VOL. 37, 1993 EXTENDED-SPECTRUM P-LACTAMASE FROM P. AERUGINOSA 965 TABLE 2. MICs of 3-lactams for P. aenrginosa RNL-1, E. coli JM109 harboring recombinant plasmid ppz1, and reference strain E. coli JM109 MIC (,ug/ml) for: Antibiotic(s) P. aeruginosa RNL-1 E. coli JM1O(pPZlr E. coi JM109 Amoxicillin >512 >512 <2 Amoxicillin-Clab > c Amoxicillin-Sued > Ticarcillin 512 >512 1 Ticarcillin-Cla Ticarcillin-Sul Piperacillin Piperacillin-Cla 32 1 Piperacillin-Sul 32 1 Cephalothin > Cephalothin-Cla >512 8 Cephalothin-Sul > Cefamandole > Cefamandole-Cla > Cefamandole-Sul >512 2 Moxalactam Moxalactam-Cla Moxalactam-Sul Cefoxitin > Cefoxitin-Cla >512 8 Cefoxitin-Sul >512 8 Ceftazidime Ceftazidime-Cla Ceftazidime-Sul Cefotaxime Cefotaxime-Cla Cefotaxime-Sul Ceftriaxone <0.03 Ceftriaxone-Cla 32 <0.03 Ceftriaxone-Sul Imipenem 0.5 <0.03 <0.03 Imipenem-Cla 0.5 <0.03 Imipenem-Sul 0.5 <0.03 Aztreonam Aztreonam-Cla Aztreonam-Sul a E. coli JM109 harboring recombinant plasmid ppz1 produced the PER-1 13-lactamase. b Cla, clavulanic acid at a fixed concentration of 2 pg/ml. The clavulanic acid MIC for E. coli JM109 was 16,ug/ml. c, not determined. d Sul, sulbactam at a fixed concentration of 4 pg/ml. The sulbactam MIC for E. coli JM109 was 64 pg/ml. RNL-1. Both P. aeruginosa RNL-1 and E. coli harboring ppz1 remained fully susceptible to imipenem. Isoelectric focusing and PER-1 I8-lactamase activity. Analytical isoelectric focusing revealed that P. aeruginosa RNL-1 had,b-lactamase activities at pis of 5.4 and 8.7. E. coli JM109 harboring recombinant plasmid ppz1 had P-lactamase activity only at pi 5.4 (Fig. 2). Therefore, P. aeruginosa RNL-1 possesses two 3-lactamases, one of a likely cephalosporinase and the other corresponding to the cloned extended-spectrum 3-lactamase. Kinetic parameters of the PER-1 13-lactamase obtained from a culture of E. coli JM109 harboring plasmid ppz1 were subsequently determined (Table 3). The enzyme had strong hydrolytic activity against amoxicillin, ticarcillin, cephalothin, and extended-spectrum cephalosporins. Surprisingly, both the Vm. and the Km values of extended-spectrum cephalosporins were high. Hydrolysis of aztreonam was low. Cloxacillin was not hydrolyzed and acted as a poor inhibitor. Interestingly, clavulanic e.w? qo d -W I I 1 a I I I I FIG. 2. Analytical isoelectric focusing of PER-1 and reference P-lactamases. Lanes: 1, TEM-1(R111), pi 5.4; 2, TEM-2(RP4), pl 5.6; 3, SHV2(p124), pl 7.1; 4, SHV1(p453), pi 7.7; 5, crude extracts from P. aeruginosa RNL-1, which produces the PER-1 P-lactamase, pi 5.4, and a likely cephalosporinase, pi 8.7; 6, crude extracts from P. aeruginosa NTCT 8203, which produces the AmpC cephalosporinase, pi 8.7; 7, crude extracts from E. coli JM109 harboring ppz1, which produces the PER-1 1-lactamase, pi 5.4. The numbers on the left are pis. acid, sulbactam, imipenem, and cephamycins were inhibitors. Clavulanic acid and sulbactam acted as time-dependent, irreversible inhibitors. Activity of the enzyme was inhibited by neither EDTA nor NaCl (100 mm) and was not enhanced upon addition of ZnCl2. Plasmid DNA, transfer of resistance, and location of the 13-lactamase gene. Three plasmids of approximately 80, 20, and 4 kb (barely visible) were detected in P. aeruginosa TABLE 3. Kinetic parameters of various 3-lactam antibiotics for PER-1,B-lactamase Substrate Vma Km (pm) ratio Amoxicillin Aztreonam Benzylpenicillin Cefamandole Cefotaxime 1, Cefoxitinb <0.5 4 x 10-2 NAC Cefpirome Ceftazidime 2, Ceftriaxone Cefuroxime Cephaloridine Cephalothin Clavulanic acid' < x 10-2 NA Cloxacillinb <0.5 2 x 10-1 NA HR 221 1, Imipenemb <0.5 7 x 10-2 NA Moxalactamb <0.5 3 x 10-3 NA Sulbactamb <0.5 4 x 10-2 NA Ticarcillin a Vin, and the Vin,,x/Km ratio are expressed relative to those of benzylpenicillin, which were set at 100. b Affinity constants as measured by Ki. c NA, not applicable. i
5 966 NORDMANN ET AL. ANTIMICROB. AGENTS CHEMOTHER. FIG. 3. Plasmid DNA analysis of P. aeruginosa RNL-1. Plasmid DNAs were extracted from P. aeruginosa RNL-1, run on a 0.7% agarose gel (lane B), and compared to standard plasmid molecular sizes (lane A). Corresponding hybridization of the same DNAs were performed with the 1.1-kb SnaBI fragment from ppz1 (lanes B and C). Sizes of reference plasmids are indicated on the left, and P. aeruginosa RNL-1 plasmid sizes are shown in the middle, along with the position of chromosomal DNA (Chr.) migration. RNL-1 (Fig. 3, lane B). Direct mating-out experiments failed to transfer the I-lactam resistance marker into P. aeruginosa or E. coli JM109. A probe consisting of the 1.1-kb SnaBI fragment from plasmid ppz1 failed to hybridize to plasmid DNA isolated from P. aeruginosa RNL-1. However, this probe did hybridize significantly with residual chromosomal DNA which was present as contamination of plasmid DNA preparations (Fig. 3, lane D). Molecular mass. The relative molecular mass of the cloned 13-lactamase was estimated from cultures of E. coli JM109 harboring prazi, which codes for the cloned 3-lactamase. Like E. coli JM109 harboring ppz1, E. coli harboring prazi (SnaBI fragment from ppz1 inserted into pkl9) showed the same 1-lactamase activity at pi 5.4. SDS-gel electrophoresis revealed a protein with a relative molecular mass of 29 kda (Fig. 4). After renaturation of the gel, only this 29-kDa protein band showed,b-lactamase activity. DNA-DNA homologies. Hybridization performed with the 1.1-kb SnaBI probe from ppz1 containing the 3-lactamase gene revealed a strong positive signal in response to a 1.1-kb fragment of SnaBI-digested P. aeruginosa RNL-1 DNA, thus confirming the origin of the cloned,-lactamase gene. Hybridization with the same probe failed to show any homology with the blatem-l, blasrv-3, blapse-l, blapse-2, blapse-3, blapse-4, blaoxa-1, blaoxa-2, blaoxa-3, or ampc gene from P. aeruginosa or the blal l or blal 2 gene from X. maltophilia. 1.- FIG. 4. SDS-polyacrylamide gel electrophoresis of PER-i 3-lactamase. Crude extracts of E. coli JM109 harboring pkl9 (lane A) and prazi (SnaBI fragment from ppz1 cloned into the pkl9 SmnaI site), which produces PER-i 13-lactamase (lane B). The additional band in lane B (compared with the lane A protein profile) corresponding to the PER-i P3-lactamase is indicated by the arrow. Protein molecular mass reference markers are shown on the left (sizes are in kilodaltons). DISCUSSION P. aeruginosa RNL-i, isolated from the urinary tract of an infected patient, encodes two 13-lactamases: a likely cephalosporinase with a pi of 8.7 (ranking within the pi range of cephalosporinases of P. aeruginosa (38]) and a novel 13-lactamase, PER-i, with a pi of 5.4. The PER-i enzyme is an extended-spectrum cephalosporin-hydrolyzing 13-lactamase sensitive to inhibition by clavulanic acid, imipenem, and cephamycins, and its gene appears to be chromosomally encoded. It is particularly interesting that this 13-lactamase is not derived from previously known extended-spectrum 13-lactamases, according to the results of hybridization experiments. Extended-spectrum cephalosporin-hydrolyzing 13-lactamases sensitive to clavulanic acid are mainly derivatives from plasmid-mediated TEM and SHV enzymes (16). They have been extensively described in Enterobacteriaceae species. The ceftazidime-cefotaxime MIC ratio of >8 indicates that the PER-i enzyme is a ceftazidime-hydrolyzing enzyme, like the TEM-5, TEM-6, and TEM-7 P-lactamases, with which it shares an acidic pi. However, the high Vm.,
6 VOL. 37, 1993 EXTENDED-SPECTRUM I-LACTAMASE FROM P. AERUGINOSA 967 combined with high Km values for extended-spectrum cephalosporins, with PER-1,3-lactamase is not present in any known TEM or SHV derivatives. PER-1,B-lactamase did not result from point mutations of TEM or SHV derivatives, as hybridization experiments failed to report any homology under high- or low-stringency conditions. According to the Bush 3-lactamase classification, one may classify PER-1,-lactamase within group 2b', although it differs from TEM and SHV derivatives (9). Recently, plasmid-mediated non- TEM, non-shv, extended-spectrum,-lactamases have been reported in Kiebsiella oxytoca and E. coli (4, 44). These enzymes, named MEN-1 and KH, differ from PER-1 P-lactamase by their pls of 8.4 and 5.2, respectively, and their lower hydrolytic properties regarding extended-spectrum cephalosporins. PER-1 differs particularly by its pi and its strong inhibition of activity by clavul4dic acid from rare plasmid-mediated extended-spectrum --lactamases which hydrolyze cephamycins, such as CMY-1, CMY-2, and MIR-1 (6, 31). These enzymes have hydrolytic properties that resemble those of chromosomal AmpC cephalosporinase. In fact, protein sequence analysis of CMY-2 isolated from K pneumoniae revealed that it isi97% similar to AmpC from Citrobacter freundii (7). Similarly, BIL-1 may derive from an AmpC-type origin (33). The PER-1 enzyme shares some similarities with the Bush; group 2e enzymes (10). These enzymes, such as FEC-1 from E. coli, Form II from Citrobacter diversus ULA-27, and L-2 from X. maltophilia, are active predominantly against cephalosporins and are inhibited by clavulanic acid. However, relatively low rates of hydrolysis of extended-spectrum cephalosporins and differences in pis (alkaline pi) indicate that these enzymes differ from the PER-1,-lactamase. Moreover, in the case of the L-2 j-lactamase from X. maltophilia, chrdmosomal DNA of this species did not hybridize with a probe containing the PER-1 13-lactamase gene. Hybridizations with the oxacillin-hydrolyzing and carbenicillin-hydrolyzing 3-lactamase genes tested failed to reveal any homology with the PER-1,-lactamase gene probe. Although genetic diversity has been reported among the oxacillin-hydrolyzing enzymes and all available oxacillinhydrolyzing enzyme genes were not tested in our hybridization study, the absence of oxacillin hydrolysis for the PER-1 P-lactamase excludes this enzyme from this group. Moreover, as opposed to the strong inhibition property of clavulanic acid described here, oxacillin-hydrolyzing enzyme activities are usually poorly inhibited by this inhibitor. In addition, oxacillin-hydrolyzing enzymes are inhibited by NaCl (100 mm) (10), which was not the case for PER-1 1-lactamase. The published sequences of carbenicillinases indicate that they are closely related at both the DNA and protein levels. Therefore, negative hybridization performed with blapse and blacarb genes excludes assignment of the PER-1 enzyme to this group of,-lactamases. By in vitro mutagenesis, it has been shown that mutation in the blapsea gene, as well as in the blacarb-4 gene may lead to extended-spectrum derivatives (8). Although the extended-spectrum properties of the PER-1 1-lactamase may suggest the occurrence of a similar event, the hybridization experiments we performed do not argue in favor of the isolation of such a carbenicillin-hydrolyzing 3-lactamase derivative. Recently, two novel,b-lactamases were reported in P. aeruginosa which are also different from PER-1 3-lactamase (21, 29). The first novel chromosomally mediated,b-lactamase hydrolyzes oxacillin strongly and extended-spectrum cephalosporins weakly (29). It has a pi of 8.0, and its activity is not inhibited by clavulanic acid. Similar to the PER-1 P-lactamase, its activity is inhibited by imipenem. The property of imipenem acting as an inhibitor has been already described for the Ambler class A P-lactamase of Bacillus cereus (28). In this case, imipenem reversibly reacted with the enzyme with transient formation of an altered,b-lactamase. Concomitant induction of the chromosomal cephalosporinase by imipenem and inhibition of the pi 8.0 enzyme gave an image of simultaneous synergy and antagonism when a disc of imipenem was placed in front of a disc of ceftazidime in an agar diffusion assay. Such an image could not be found with the P. aeruginosa RNL-1 strain, since the PER-1 f-lactamase strongly hydrolyzes extended-spectrum cephalosporins. The second recently reported,3-lactamase is a non-tem-, non-shv-derived 3-lactamase (21). It is plasmid mediated, has a pi of 6.4, and like the PER-1 enzyme, confers resistance to extended-spectrum cephalosporins. However, the Vm. values of extended-spectrum cephalosporins are much lower than that of the PER-1 P-lactamase, which hydrolyzes oxacillin and whose activity is not inhibited by clavulanic acid. As suggested by its biochemical properties, this pi 6.4 enzyme is an oxacillin-hydrolyzing enzyme. Indeed, sequence analysis revealed that it is a derivative of the blapse2 gene obtained by point mutations which confer resistance to extended-spectrum cephalosporins (21; oral communication). Interestingly, two discrepancies were noted between the MICs obtained for E. coli possessing recombinant plasmid ppz1 and the hydrolytic properties of the PER-1 enzyme. (i) Concerning aztreonam, its Vm., and VmaIjKm ratio were particularly low compared with the corresponding MIC. Such a high MIC may have resulted from poor penetration of aztreonam through the outer membrane barrier. Such a property has also been reported for the TEM-22 f-lactamase (1). (ii) It is intriguing how moxalactam could behave as an efficient inhibitor (Ki, 3 x 10-3 PM) and had a slight but reproducible increase in its MIC as measured in E. coli possessing recombinant plasmid ppz1, which codes for PER-1. As resistance to aminoglycosides and to sulfonamides was also found in P. aeruginosa RNL-1, and as such antibioticresistant genes are present within Tn2l and Tn3 transposon derivatives of this species (20), further work will evaluate the potential presence of a transposon containing these antibiotic resistance genes along with the PER-1 3-lactamase gene. Sequence analysis of the PER-i-encoding gene will help to compare it with other extended-spectrum,b-lactamases and will likely cotifirm its serine-enzymatic nature, as strongly suggested by its biochemical properties. Absence of inhibition of its activity by EDTA makes it different from the reported plasmid-mediated carbapenem-hydrolyzing 3-lactamase in P. aeruginosa, which also strongly hydrolyzes extended-spectrum cephalosporins (42). Finally, from a therapeutic point of view, this report emphasizes the fact that with P. aeruginosa, 3-lactamases other than the commonly found cephalosporinases may lead to failure of therapeutic regimens which include extended-spectrum cephalosporins. ACKNOVLEDGMENTS We thank C. Nauciel, in whose laboratory we isolated P. aeruginosa RNL-1; W. Arber, in whose laboratory part of this work was performed; and M. Barthelemy, for precious advice. We are also grateful to C. Bailly, A. Morand, and M. Thouverez for technical assistance. This study was supported by Merck Sharp & Dohme Chibret, the
7 968 NORDMANN ET AL. Fondation pour la Recherche Medicale, and the Ministere de l'education Nationale, France. REFERENCES 1. Arlet, G., M. Rouveau, G. Fournier, P. H. Lagrange, and A. Philippon Novel extended-spectrum 3-lactamase (TEM- 22) in Klebsiella pneumoniae conferring higher resistance to aztreonam than to extended-spectrum cephalosporins. Program Abstr. 32nd Intersci. Conf. Antimicrob. Agents Chemother., abstr Barthelemy, M., M. Guionie, and R. Labia Beta-lactamases: determination of their isoelectric points. Antimicrob. Agents Chemother. 13: Barthelemy, M., J. Peduzzi, H. Ben Yaghlane, and R. Labia Single amino-acid substitution between SHV-1 P-lactamase and cefotaxime-hydrolysing SHV-2 enzyme. FEBS Lett. 231: Barthelemy, M., J. Pduzzi, H. Bernard, C. Tancrede, and R. Labia Close amino-acid sequence relationship between the new plasmid-mediated extended-spectrum,b-lactamase MEN-1 and chromosomally encoded enzymes of Klebsiella oxytoca. Biochim. Biophys. Acta 1122: Barthelemy, M., J. PWduzzi, C. Verchere-Beaur, H. Ben Yaghlane, and R. Labia Purification and biochemical properties of Pitton's 2,-lactamase (SHV-1). Ann. Inst. Pasteur/ Microbiol. (Paris) 137B: Bauernfeind, A., Y. Chong, and S. Schweinghart Extended broad spectrum 1-lactamase in Klebsiella pneumoniae including resistance to cephamycins. Infection 17: Bauernfeind, A., P. Mangold, S. Schweighart, G. H. Miller, K. J. Shaw, H. Giamarellou, and K. D. Dornbush Molecular analysis of a transferable cephamycinase in Kiebsiella pneumoniae. Program Abstr. 32nd Intersci. Conf. Antimicrob. Agents Chemother., abstr Bejaoui, N., and R. C. Levesque Structure-function analysis of critical amino acids for extended-spectrum ceftazidimase activity in Pseudomonas aeruginosa carbenicillinases. Program Abstr. 32nd Intersci. Conf. Antimicrob. Agents Chemother., abstr Bush, K Classification of P-lactamases: groups 1, 2a, 2b, and 2b'. Antimicrob. Agents Chemother. 33: Bush, K Classification of P-lactamases: groups 2c, 2d, 2e, 3, and 4. Antimicrob. Agents Chemother. 33: Chang, A. C. Y., and S. N. Cohen Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J. Bacteriol. 134: Datta, N., R. W. Hedges, E. J. Shaw, R. B. Sykes, and M. H. Richmond Properties of an R factor from Pseudomonas aeruginosa. J. Bacteriol. 108: Hedges, R. W., and G. A. Jacoby Compatibility and molecular properties of plasmid Rms 149 in Pseudomonas aeruginosa and Eschenichia coli. Plasmid 3: Honore, N., M. H. Nicolas, and S. T. Cole Inducible cephalosporinase production in clinical isolates of Enterobacter cloacae is controlled by a regulatory gene that has been deleted from Escherichia coli. EMBO J. 5: Jacob, A. A., J. A. Shapiro, L. Yamamoto, D. I. Smith, S. N. Cohen, and D. Berg Plasmids studied in Escherichia coli and other enteric bacteria, p In A. I. Bukhari, J. A. Shapiro, and S. L. Adhya (ed.), DNA insertion elements, plasmids and episomes. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 16. Jacoby, G. A., and A. A. Medeiros More extendedspectrum,-lactamases. Antimicrob. Agents Chemother. 35: Kieser, T Factors affecting the isolation of CCCDNA from Streptomyces lividans and Eschenchia coli. Plasmid 12: Labia, R., M. Guionie, M. Barthelemy, and A. Philippon Properties -of three carbenicillin hydrolysing 3-lactamases (CARB) from Pseudomonas aeruginosa: identification of a new enzyme. J. Antimicrob. Chemother. 7: ANTIMICROB. AGENTS CHEMOTHER. 19. Laemmli, U. K., and M. Farve Maturation of the head of bacteriophage T4. I. DNA packaging events. J. Mol. Biol. 80: Levesque, R. C., and G. A. Jacoby Molecular structure and interrelationships of multiresistance P-lactamase transposons. Plasmid 19: Livermore, D. M., L. M. C. Hall, D. Gur, M. Akova, and H. E. Akalin Non-TEM, non-shv ceftazidimase, transferable from a P. aeruginosa isolate. Program Abstr. 32nd Intersci. Conf. Antimicrob. Agents Chemother., abstr Livermore, D. M., and Y. J. Yang Lactamase lability and inducer power of newer P-lactam antibiotics in relation to their activity against 3-lactamase-inducibility mutants of Pseudomonas aeruginosa. J. Infect. Dis. 155: Macrina, F. L., D. J. Kopecko, K. R. Jones, D. J. Ayers, and S. M. McCowen A multiple plasmid containing Escherichia coli strain: convenient source of size reference for plasmid molecules. Plasmid 1: Maniatis, T., E. F. Fritsch, and J. Sambrook Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 25. Massidda, O., G. M. Rossolini, and G. Satta The Aeromonas hydrophila cpha gene: molecular heterogeneity among class B metallo-p-lactamases. J. Bacteriol. 173: Medeiros, A. A., and G. A. Jacoby Beta-lactamase mediated resistance, p In S. F. Queener, J. A. Webler, and S. W. Queener (ed.), Beta-lactam antibiotics for clinical use. Marcel Dekker, Inc., New York. 27. Michel-Briand, Y., M. J. Dupont, I. Chardon-Loriaux, and M. Jouvenot Isolation of an antibiotic multiresistance plasmid from Pseudomonas aeruginosa. J. Antimicrob. Chemother. 7: Monks, J., and S. G. Waley Imipenem as substrate and inhibitor of,b-lactamases. Biochem. J. 253: Mugnier, P., M. D. Kitzis, A. Philippon, L. Gutmann, and E. Collatz Simultaneous synergy and antagonism between imipenem (Imi) and other P-lactam antibiotics in a clinical isolate of Pseudomonas aeruginosa (PAe). Program Abstr. 32nd Intersci. Conf. Antimicrob. Agents Chemother., abstr Nicolas, M.-H., V. Jarlier, N. Honore, A. Philippon, and S. T. Cole Molecular characterization of the gene encoding SHV-3 P-lactamase responsible for transferable cefotaxime resistance in clinical isolates of Klebsiella pneumoniae. Antimicrob. Agents Chemother. 33: Papanicolaou, G. A., A. A. Medeiros, and G. A. Jacoby Novel plasmid-mediated,b-lactamase (MIR-1) conferring resistance to oxyimino- and a-methoxy,-lactams in clinical isolates of Klebsiella pneumoniae. Antimicrob. Agents Chemother. 34: Paul, G., M. Barthelemy, A. Philippon, J. Peduzzi, L. Gilley, R. Labia, and P. Nevot Immunological comparison of constitutive P-lactamases of gram-negative bacteria by neutralization in zymogram gels: properties of anti-tem-1 and anti- TEM-2 sera. Ann. Inst. Pasteur/Microbiol. (Paris) 139: Payne, D. J., N. Woodford, and S. G. B. Amyes Characterization of the plasmid mediated 3-lactamase BIL-1. J. Antimicrob. Chemother. 30: Philippon, A., R. Labia, and G. Jacoby Extended-spectrum 3-lactamases. Antimicrob. Agents Chemother. 33: Philippon, A. M., G. Paul, and G. A. Jacoby Properties of PSE-2 13-lactamase and genetic basis for its production in Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 24: Plesiat, P., B. Alkhalaf, and Y. Michel-Briand Prevalence and profile of plasmids in Pseudomonas aeruginosa. Eur. J. Clin. Microbiol. Infect. Dis. 7: Pridmore, R. D New and versatile cloning vectors with kanamycin resistance marker. Gene 56: Sanders, C. C., M. L. Gates, and W. E. Sanders, Jr Heterogeneity of class I r-lactamase expression in clinical isolates of Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 32:
8 VOL. 37, 1993 EXTENDED-SPECTRUM,-LACTAMASE FROM P. AERUGINOSA Sanders, W. E., Jr., and C. C. Sanders Inducible P-lactamases: clinical and epidemiological implications for use of newer cephalosporins. Rev. Infect. Dis. 10: Southern, E. M Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98: Stanisich, V., and J. M. Ortiz Similarities between plasmids of the P incompatibility group derived from different bacterial genera. J. Gen. Microbiol. 94: Watanabe, M., S. lyobe, M. Inoue, and S. Mitsuhashi Transferable imipenem resistance in Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 35: Witchitz, J. L., and Y. A. Chabbert Resistance transferable a la gentamicine. Expression du caractere de resistance. Ann. Inst. Pasteur (Paris) 121: Wu, S. W., K. Dornbusch, M. Norgren, and G. Kronvall Extended-spectrum 13-lactamase from Klebsiella oxytoca, not belonging to the TEM or SHV family. J. Antimicrob. Chemother. 30: Yanisch-Perron, C., J. Vieira, and J. Messing Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mpl8 and puc19 vectors. Gene 33:
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