Susceptible Chromogenic Cephalosporin Reagent

Transcription

1 JOURNAL OF CLINICAL MICROBIOLOGY, May 1982, p /82/ $02.00/0 Vol. 15, No. 5 In Vitro Evaluation of CENTA, a New Beta-Lactamase- Susceptible Chromogenic Cephalosporin Reagent RONALD N. JONES,'* HAROLD W. WILSON,1 WILLIAM J. NOVICK, JR.,2 ARTHUR L. BARRY,3 AND CLYDE THORNSBERRY4 Department of Pathology, Kaiser Foundation Laboratories (Oregon Region), Clackamas, Oregon ; Hoechst-Roussel Pharmaceuticals, Inc., Somerville, New Jersey ; Department of Clinical Microbiology, University of California, Davis, Medical Center, Sacramento, California ; and Antimicrobics Investigation Section, Centers for Disease Control, Atlanta, Georgia Received 8 October 1981/Accepted 9 December 1981 CENTA is a newly synthesized, beta-lactamase-labile, chromogenic cephalosporin reagent which changes color from light yellow (K maximum ca. 340 nm) to chrome yellow (A maximum ca. 405 nm) concomitant with hydrolysis of the betalactam ring. This compound offers promise as a diagnostic reagent comparable to other chromogens (PADAC and nitrocefin) for the early detection of betalactamase-producing clinical isolates, while retaining some antimicrobial effect against Escherichia coli, Klebsiella spp., Proteus mirabilis, Staphylococcus aureus, and non-enterococcal Streptococcus spp. CENTA is relatively unaffected by commonly used microbiological media and human serum. CENTA is a newly synthesized chromogenic cephalosporin structurally resembling nitrocefin, cephaloridine, and cephalothin (9; Fig. 1). Schindler and colleagues at Hoechst AG have described its physicochemical properties and possible applications to the studies of bacterial beta-lactamases and enzyme inhibitors and measurements of cephalosporin serum concentrations (13, 14; P. Schindler, G. Huber, J. Blumbach, and H. G. Berscheid, Program Abstr. Intersci. Conf. Antimicrob. Agents Chemother. 21st, Chicago, Ill., abstr. no. 434, 1981). Like PADAC, cephacetrile, and nitrocefin, CENTA seems to offer promise as a diagnostic reagent in the clinical microbiology laboratory, particularly in the early rapid recognition of beta-lactamase production by clinical bacterial isolates (5, 9, 11). Research applications of such substances have also been accelerated in the quest for betalactamase-stable antimicrobial agents (3, 4, 13). In this report we present the in vitro characteristics of CENTA as compared to those previously published for other chromogenic substrates, PADAC and nitrocefin. These investigations included: a comparison of the antimicrobial activity with the two other chromogens; a beta-lactamase hydrolysis rate study of the chromogens and two other reference cephalosporins against eight selected enzyme preparations; the utility of using CENTA, PADAC, or nitrocefin as an enzymelabile substrate in beta-lactamase hydrolysis inhibition studies; and the interaction of CENTA with commonly used microbiological liquid media and with human serum. The CENTA, PADAC, cefotaxime, and cefodizime (HR221) cephalosporin reagents were provided by Hoechst-Roussel Pharmaceuticals Inc., Somerville, N.J. The other reference antimicrobial agents were contributed by the following pharmaceutical companies: SCH from Schering Corp., Kenilworth, N.J.; nitrocefin and ceftazidime from Glaxo Group Research Ltd., Greenford, Middlesex, U.K.; cephalothin from Eli Lilly Research Laboratory, Indianapolis, Ind.; CP 45,899 from Pfizer Research, Groton, Conn.; and dicloxacillin from Bristol Laboratories, Syracuse, N.Y. The CENTA, PADAC, and nitrocefin chromogenic compounds were diluted in Mueller-Hinton broth (Difco Laboratories, Detroit, Mich.) supplemented with calcium to 50 mg/liter and magnesium to 25 mg/liter as described in the National Committee for Clinical Laboratory Standards standard for dilution susceptibility testing (Standard Methods; 6). A single lot of microdilution trays (Prepared Media Laboratories, Portland, Oreg.) was prepared for this study and was stored at or below -20 C until utilized. Inocula (ca. 5 x 105 colonyforming units per ml) were delivered to the wells by a plastic replicating device at a volume of 5,ul to each well. The minimal inhibitory concentration (MIC) was interpreted as the lowest cephalosporin concentration completely inhibiting bacterial growth in a well as examined by the 954 unaided eye, after 15 to 18 h of incubation at 350C. A total of 519 recent clinical isolates representing 27 bacterial species were collected from

2 VOL. 15, 1982 CH2CONH co2 FIG. 1. The structural configuration of CENTA. the routine cultures submitted to the Kaiser Foundation Laboratory (Oregon Region), and others were kindly contributed by A. L. Barry (University of California-Davis Medical Center, Sacramento, Calif.), P. C. Fuchs (St. Vincent Hospital and Medical Center, Portland, Oreg.), T. L. Gavan (The Cleveland Clinic Foundation, Cleveland, Ohio), E. Hugh Gerlach (St. Francis Hospital, Wichita, Kans.), and H. M. Sommers (Northwestern Memorial Hospital, Chicago, Ill.). The latter strains were used in earlier reports of new beta-lactams and chromogenic cephalosporins (3-5). Seven reference strains with known types of beta-lactamase production were included for the enzyme inhibition and hydrolysis studies (2-5, 7-11). The enzyme hydrolysis analyses were performed on a scanning UV spectrophotometer at 37 C in 1.0 ml of 0.05 M phosphate buffer at ph 7.0. The enzymelabile chromogenic substrates used were CENTA, PADAC, and nitrocefin and, in addition, the nonchromogenic reference therapeutic cephalosporins cephalothin and cefotaxime (2-5, 9, 13). For the beta-lactamase inhibition tests, the drugs were mixed in 0.5 ml of 0.05 M phosphate buffer at ph 7. Enzyme-stable inhibiting beta-lactams were added to the labile substrate-enzyme mixture and tested in a centrifugal-fast analyzer (CentrifiChem 400, Union Carbide, Tarrytown, N.Y.) using CENTA, PA- DAC, and nitrocefin as substrates, with 405-nm, 550-nm, or 482-nm peak rates and four inhibitor concentrations (cefotaxime, ceftazidime, CP 45899, dicloxacillin, cefodizime [HR221], SCH 29482) ranging from 0.1 to 100 pum (1-5, 8, 13, 14). Crude enzyme preparations were made by methods described by Neu (7) from organisms known to produce types I, II, III, and IV and "new" beta-lactamases (10, 11). Some enzymes were partially purified by ammonium sulfate fractionation by a technique reported by DeBell et al. (1) and then stabilized as described before (3-5). A commercially available penicillinase (BBL Microbiology Systems, Cockeysville, Md.) derived from Bacillus cereus was also studied. The antimicrobial activity of CENTA, PA- DAC, and nitrocefin was tested by the Standard Methods reference microdilution method (6) with a 10-log2 dilution schedule ranging from 0.25 to 128,ug/ml (Table 1). Results indicated NOTES 955 CENTA to be the most active antimicrobial among these chromogens. Against the 256 Enterobacteriaceae isolates tested, only the Proteus mirabilis, Klebsiella spp., Escherichia coli, and Citrobacter diversus strains were significantly inhibited by CENTA (MIC for 50% of isolates [MIC50] = s32 ij.g/ml). In contrast, none of these species was inhibited by PADAC, and only P. mirabilis was inhibited by nitrocefin (MIC50 = 32,ug/ml). All three chromogenic cephalosporins were active against Staphylococcus aureus strains (MIC50 = <0.25,ug/ml). PA- DAC demonstrated an anti-staphylococcal activity comparable to the most active of the cephalosporins, e.g., cephalothin and cefamandole (3, 4). CENTA showed little effect (MIC50 = 128,ug/ml) against Streptococcus faecalis as compared to the marginal inhibition demonstrated by PADAC (MIC50 = 16,ug/ml) and the excellent activity of nitrocefin (highest MIC = 2.0,ug/ml). This latter feature for nitrocefin seems unique among the cephalosporins and was consistent with data reported earlier (5). All three drugs were active against Streptococcus pneumoniae and Streptococcus pyogenes (all MICs, <32,ug/ml). The highest MIC for each chromogen was for an S. pneumoniae isolate having a penicillin G MIC of 0.5,ug/ml. A marked variation in chromogen MICs was seen between the beta-lactamase-producing Haemophilus influenzae and those strains not exhibiting detectable enzyme. CENTA showed the best anti-haemophilus activity, generally 16- to 128- fold more potent than either PADAC or nitrocefin. None of these drugs was active against Acinetobacter spp. or the pseudomonads tested. The results of the cephalosporin substrate relative hydrolysis rate (RHR) studies on eight beta-lactamase preparations are summarized in Table 2. CENTA undergoes a detectable color change from faint yellow (A maximum 345 nm) to chrome yellow (A maximum 410 nm) concomitant with enzymatic hydrolysis of the beta-lactam ring (Fig. 2). CENTA appears to be susceptible to all the beta-lactamases tested and comparable to the previously described chromogenic cephalosporins (5, 12, 13; M. H. Richmond, personal communication). The RHR of CENTA was generally 5 to 20% of the nitrocefin reference rates, with the exception of type IV enzyme preparations, for which CENTA exhibited rates close to that of nitrocefin (79 to 81%). The RHR profile of CENTA was thus different from either of the other tested chromogenic reagents and of the compounds tested was most similar to cephalothin. PADAC had more rapid hydrolysis rates than CENTA for enzyme types II, III, and "new" and the new penicillinase derived from B. cereus. CENTA may yield faster positive results than PADAC with enzyme

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4 VOL. 15, 1982 TABLE 2. Substrate hydrolysis rates of eight representative beta-lactamase preparations on chromogenic and other cephalosporinsa Nitrocefin RHR as % compared to nitrocefin Organism (beta-lactamase type)" hydrolysis rate (>M/min) CENTA PADAC Cephalothin Cefotaxime Enterobacter cloacae (Ia) <1.0 Pseudomonas aeruginosa (II) <1.0 Escherichia coli (Illa) <1.0 Escherichia coli (IlIb) <1.0 Klebsiella oxytoca (IV) Klebsiella oxytoca (IVc) Klebsiella pneumoniae (New) <1.0 Bacillus cereus (NT) <1.0 a Beta-lactamase hydrolysis was determined by the UV-spectrophotometric method using 258 to 570 nm at 37 C. Reaction mixture was 1.0 ml of a 10-4 M concentration of the cephalosporin substrate (except PADAC, which was at 5 x 10-5 M) in 0.05 M phosphate buffer (ph 7). RHR was compared to that of enzyme-nitrocefin preparations, which was given a value of 100%o. b Enzyme classifications after references 10 and 11. NT, Not typable. types I and IV, both commonly found in gramnegative clinical isolates. CENTA, PADAC, and cephacetrile (5, 12) all had slower relative hydrolysis rates than nitrocefin. The RHR inhibition profiles (Fig. 3) show CENTA to be somewhat less effective than nitrocefin or PADAC at demonstrating the inhibiting action of beta-lactam drugs on type Ia (P99) beta-lactamase. The differential affinity of these chromogens for the type I enzymes appears to rank in the order: CENTA > nitrocefin > PA- DAC. As an enzyme-labile substrate, CENTA failed to demonstrate any significant inhibitory effect for CP 45,899 against the type I enzyme, 1.0 I~~~~~~~~~~~~~01 M A(nm) FIG. 2. Absorption spectrum of CENTA (10-4 M) before (-) and after (--) exposure to type I (E. cloacae P99) beta-lactamase. NOTES 957 and its hydrolysis was only marginally inhibited by ceftazidime and SCH Nitrocefin, when used as the enzyme-labile substrate, showed minimal inhibition for CP 45,899, a finding expected for this inhibitor from previous analyses using cephaloridine as the beta-lactamase-labile substrate. In contrast, PADAC seemed more sensitive to the inhibitory qualities of all tested beta-lactam compounds. All six inhibitors showed -75% RHR reduction at concentrations of no more than 20% of the PADAC substrate, confirming a previous report (5). When CENTA was added to commonly used microbiological broth media (Mueller-Hinton, brain heart infusion, Schaedler, and Trypticase soy [BBL Microbiology Systems]) no significant nonspecific (beta-lactamase-like) color change occurred as monitored by a spectrophotometer. Human serum (50%) also did not adversely affect the color reaction, as it does with nitrocefin (9). The positive chrome-yellow reaction obtained with 25- to 50-pug/ml CENTA solutions was difficult to interpret with the unaided eye, especially in the broths that are yellowish and in human body fluids which contain yellow-colored constituents such as bilirubin. Thus, this chromogen seems most applicable to spectrophotometric analyses of beta-lactamase action. We recommend that these analyses be performed at 340 or 405 nm, preferably the latter. In conclusion, CENTA is seen as a useful tool for detecting and measuring beta-lactamases. It is also applicable to the classification of these enzymes through the development of hydrolysis and inhibition profiles. Like its congener PA- DAC, CENTA was found to be relatively unaffected by human serum (13, 14) and was sufficiently labile to common beta-lactamases for use as an indicator reagent, particularly in instrumental analyses by non-uv range equipment.

5 958 NOTES 100) Z, < of substrate concentration FIG. 3. Type Ia (E. cloacae P99) beta-lactamase hydrolysis inhibition patterns on three chromogenic cephalosporin substrates, monitored by a centrifugalfast analyzer. Six inhibitor beta-lactams were used: dicloxacillin (0), cefotaxime (0), cefodizime (A), SCH (A\), ceftazidime (i), and CP 45,899 (O). Thus, CENTA is another addition to the armamentarium of reagents capable of detecting betalactamases that can be used in the clinical microbiology laboratory. LITERATURE CITED 1. DeBell, R. M., T. M. Hickey, and D. E. Uddin Partial characterization of beta-lactamase from Vibrio parahaemolyticus by a new automated micro-iodometric technique. Antimicrob. Agents Chemother. 13: J. CLIN. MICROBIOL. 2. Fu, K. P., and H. C. Neu The comparative betalactamase resistance and inhibitory activity of 1-oxa cephalosporin, cefoxitin and cefotaxime. J. Antibiot. 32: Jones, R. N., A. L. Barry, C. Thornsberry, E. H. Gerlach, P. C. Fuchs, T. L. Gavan, and H. M. Sommers Ceftazidime, a pseudomonas-active cephalosporin: in vitro antimicrobial activity evaluation including recommendations for disk diffusion susceptibility tests. J. Antimicrob. Chemother. 8(Suppl. B): Jones, R. N., A. L. Barry, C. Thornsberry, and H. W. Wilson In vitro antimicrobial activity evaluation of cefodizime (HR221), a new semisynthetic cephalosporin. Antimicrob. Agents Chemother. 20: Jones, R. N., H. W. Wilson, and W. J. Novick, Jr In vitro evaluation of pyridine-2-azo-p-dimethylaniline cephalosporin, a new diagnostic chromogenic reagent, and comparison with nitrocefin, cephacetrile, and other beta-lactam compounds. J. Clin. Microbiol. 15: National Committee for Clinical Laboratory Standards Standard methods for dilution antimicrobial susceptibility tests for bacteria which grow aerobically. Proposed Standard, M7-P. National Committee for Clinical Laboratory Standards, Villanova, Pa. 7. Neu, H. C Antibiotic inactivating enzymes of bacterial resistance, p In V. Lorian (ed.), Antibiotics in laboratory medicine. The Williams & Wilkins Co., Baltimore. 8. O'Callaghan, C. H., and A. Morris Inhibition of beta-lactamases by beta-lactam antibiotics. Antimicrob. Agents Chemother. 2: O'Callaghan, C. H., A. Morris, S. M. Kirby, and A. H. Singler Novel method for detection of beta-lactamases by using a chromogenic cephalosporin substrate. Antimicrob. Agents Chemother. 1: Petrocheilou, V., R. B. Sykes, and M. H. Richmond Novel R-plasmid-mediated beta-lactamase from Klebsiella aerogenes. Antimicrob. Agents Chemother. 12: Richmond, M. H., and R. B. Sykes The betalactamases of gram-negative bacteria and their possible physiological role. Textbook Adv. Enzymol. 9: Russell, A. D Interaction of a new cephalosporin, 7- cyanacetamidocephalosporanic acid, with some gramnegative and gram-positive beta-lactamase-producing bacteria. Antimicrob. Agents Chemother. 2: Schindler, P., and G. Huber Use of PADAC, a novel chromogenic beta-lactamase substrate, for the detection of beta-lactamase producing organisms and assay of beta-lactamase inhibitors/inactivators, p In U. Brodbeck (ed.), Enzyme inhibitors. Verlag Chemie, Weinheim, West Germany. 14. Siebert, G., and A. Biebach Eine enzymatische Methode zur Bestimmung von Cefotaxim in Serum- und Harnproben. J. Clin. Chem. Clin. Biochem. 19: