Cephalosporin, a New Diagnostic Chromogenic Reagent, and Comparison with Nitrocefin, Cephacetrile, and Other Beta-

Transcription

1 JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 1982, p /82/ $02.00/0 Vol. 15, No. 4 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 RONALD N. JONES,'* HAROLD W. WILSON,1 AND WILLIAM J. NOVICK, JR.2 Department of Pathology, Kaiser Foundation Laboratory (Oregon Region), Clackamas, Oregon 97015,1 and Hoechst-Roussel Pharmaceuticals, Inc., Somerville, New Jersey Received 13 August 1981/Accepted 20 October 1981 Pyridine-2-azo-p-dimethylanaline cephalosporin (PADAC), a chromogenic reagent which is purple and changes to yellow upon cleavage of its beta-lactam ring, was evaluated in comparison with other chromogenic cephalosporins. PADAC exhibited little antimicrobial activity against gram-negative bacteria, but did have good activity (minimum inhibitory concentration, 0.12 to 0.5,ug/ml) against Staphylococcus aureus, a quality comparable to nitrocefin. Nitrocefin, however, demonstrated an unexpected and uniquely potent activity against Streptococculs faecalis (minimum inhibitory concentration, to 0.12 p.g/ml). The relative hydrolysis rate of PADAC when subjected to six different beta-lactamases was substantially greater than that of cephacetrile, but less than that of nitrocefin. The relative hydrolysis rates of PADAC and nitrocefin were comparable with type Illa beta-lactamase and that derived from Bacillus cereus. The inhibition of betalactamase hydrolysis of the chromogenic cephalosporin substrates by six enzymestable inhibitors was generally greater with PADAC than with nitrocefin. Unlike nitrocefin, PADAC mixed with 50% human serum or various broth culture media showed no evidence of color change or degradation over several hours. The subsequent enzyme hydrolysis rates of such mixtures were the same as in phosphate buffer. Beta-lactamase-containing bacterial suspensions and clinical specimens containing such bacteria produced positive visual and spectrophotometric color changes when mixed with PADAC or nitrocefin. Although color changes occurred more slowly with PADAC than with nitrocefin, PADAC was not adversely influenced (non-enzyme-related color change) by the protein content of specimens. PADAC appears to be a promising alternative for betalactamase diagnostic testing in the clinical and research microbiology laboratory. Pyridine-2-azo-p-dimethylanaline chromophore 3-substituted cephalosporin (PADAC) is a new chromogenic beta-lactam developed by Hoechst AG (Frankfurt, West Germany) that has a distinct color change from purple to clear yellow concomitant with the cleaving of the beta-lactam ring (19) (Fig. 1). Other reported chromogenic cephalosporin reagents have included nitrocefin (formerly 87/312) and the parenteral therapeutic agent cephacetrile (14, 18). These latter compounds have a light-yellow color in the absence of beta-lactamase, but rapidly convert to red when the beta-lactam ring is broken (14, 18). Both have been applied to the monitoring of beta-lactamase enzyme kinetics in the research setting, along with cephaloridine, and also to the rapid screening of clinical isolates for enzyme production (6a-8). The limited availability of nitrocefin and the lower sensitivity of 677 the cephacetrile reagent have discouraged the wide use of these drugs as clinical diagnostics. In the mid-1970s, beta-lactamases were detected in heretofore beta-lactam-susceptible microorganisms (2, 15, 20). These genetic events stimulated the simultaneous development of beta-lactamase refractory antimicrobial agents (5-7, 12) and simple, easy to use beta-lactamase detection tests (1, 8, 9, 14, 17, 21). The quick and accurate identification of these enzyme-mediated resistances is paramount to successful treatment of Haemophilus meningitis or other infections and for treatment of gonococcal disease with associated epidemiological investigations. Penicillinase-producing Neisseria gonorrhoeae has now achieved near 1% endemic rates in some areas of the United States (3). Both of these organisms have well-defined plasmid-mediated type III-TEM beta-lactamases (16) that

2 678 JONES, WILSON, AND NOVICK lwj3-ch2-co-nht N, -Er 0 C0--CH2H-NL cooe / N beta - lactamase U7ICH2-CO- NH-7-V S 2Q-= - CH2 S coo e CH3 N CH3 + FIG. 1. Structural configuration of PAD) before posed standard M7-P (6a, 7, 10). A single lot of and after hydrolysis by a typical beta-lactam ase. Color microdilution trays (Prepared Media Laboratories, change was from purple to a clear yellow. Portland, Ore.) was stored at or below -20 C until utilized. Inocula (ca. 5 x 105 colony-forming units/ml) were delivered to the wells by a plastic replicate device said to deliver 5,ul to each well. The MIC was are commonly detected by most beta-l tests (1-3, 8, 9, 17-19, 21). In this report we compared the in vi broth (Difco Laboratories, Detroit, Mich.) supplemented to 50 mg of calcium and 25 mg of magnesium per liter as described in a published standard (10). Bacterial strains. One hundred seventy-four recent clinical isolates representing 17 bacterial species (Table 1) were collected from the routine cultures submitted to the Kaiser Foundation Laboratory (Oregon Region) and some others generously provided by A. L. Barry, University of California, Davis, Medical Center (Sacramento), P. C. Fuchs, St. Vincent Hospital and Medical Center (Portland, Ore.); T. L. Gavan, The Cleveland Clinic Foundation (Cleveland, Ohio); E. H. Gerlach, St. Francis Hospital (Wichita, Kans.) and H. M. Sommers, Northwestern Memorial Hospi- Nc3 tal (Chicago, Ill.). The latter strains were used in / previously reported studies of new beta-lactams (8; N Jones et al., in press). % N Six reference strains with known types of beta- / lactamase production were included for the enzyme inhibition and hydrolysis studies (5-7, 11-13). Antimicrobial susceptibility testing. The minimum inhibitory concentrations (MICs) were determined by,ns the reference broth microdilution methods described CH3 CH3 in detail in previous publications and in the National Committee for Clinical Laboratory Standards, pro- AC interpreted as the lowest cephalosporin concentration lactamase completely inhibiting bacterial growth in a well, as examined by the unaided eye, after 15 to 18 h of tro quali- incubation at 35 C. J. CLIN. MICROBIOL. ties of PADAC with those previously I published Beta-lactamase studies. The beta-lactamase hydrolycefin and sis and inhibition analyses were performed on a scan- for other chromogenic substrates (nitre cephacetrile). These investigations included: the ning UV spectrophotometer (model 552, The Perkinantimicrobial activity of each chrome )gen; the Elmer Corp., Norwalk, Conn.) at 37 C. The enzyme- labile chromogenic substrates used were nitrocefin beta-lactamase hydrolysis rates of the cchromobomo- (87/312), cephacetrile, and PADAC (14, 18, 19). Studnsu y se- ied drug mixtures were either in 0.5 ml of 0.05 M gens and other reference cephalospori lected beta-lactamase preparations; the utlity of phosphate buffer at ph 7 (for enzyme hydrolysis) or using PADAC and nitrocefin as enzyrme-labile with an added enzyme-stable inhibiting beta-lactam substrates in beta-lactamase hydrolys ;is inhibi- (for enzyme inhibition) also in the buffer. Enzyme tion analyses; and the interaction of PADAC hydrolysis inhibition tests were performed by replicate with commonly used microbiological nnedia and analysis in a centrifugal fast analyzer (CentrifiChem physiological fluids. 400, Union Carbide, Terrytown, N.Y.), using the PADAC and nitrocefin substrates, 550- or 482-nm peak rates, and four inhibitor (cefotaxime, ceftazidime, CP 45,899, dicloxicillin, HR221, SCH29482) MATERIALS AND METHODS Antimicrobial agents. PADAC, cefotaxim ie, and ce- concentrations ranging from 0.1 to ,uM. Crude fodizime (HR221) cephalosporin reagents wtere provid- enzyme preparations were made by methods de- scribed by Neu (11) from organisms known to produce ed by Hoechst-Roussel Pharmaceuticals, Ssomerville, N.J. The remaining reference antimicrob ial agents type I to IV beta-lactamases (16). Filtered enzymes were kindly provided by the following: cieftizoxime were then stabilized by the addition of a surfactant and from Smith Kline & French Laboratories Philadel- an antifoam agent. A commercially available penicilphia, Pa.; SCH29482 from Schering Cor,p., Kenil- linase (BBL Microbiological Systems, Cockeysville, worth, N.J.; nitrocefin and ceftazidime fr*om Glaxo Md.) derived from Bacillus cereus was also studied. Research Ltd., Greenford, Middlesex, En gland; ce- Beta-lactamase induction was performed by grow- for Dis- ing cultures in broth microdilution tray wells in the phacetrile from Clyde Thormsberry, Centeirs ease Control, Atlanta, Ga.; cephaloridine from Eli presence of 0.12,ug of methicillin per ml in Mueller- Lilly Research Laboratory, Indianapolis, Ind.; CP Hinton media. After overnight growth, a chromogenic 45,899 from Pfizer Research, Groton, Conn.; dicloxa- cephalosporin (30- to 50-,ul drop) was added to the well cillin from Bristol Laboratories, Syracuse, N.Y.; and and examined for significant color change. cefoxitin from Merck Sharp & Dohme, AVest Point, PADAC-containing dipsticks (Behringwerke, West Pa. All compounds were diluted in Muelller-Hinton Germany) were used to detect beta-lactamase produc-

3 VOL. 15, 1982 tion among colonies grown on agar, as well as that present in clinical physiological fluids. PADAC IN VITRO EVALUATION 679 RESULTS The antimicrobial activities of PADAC, nitrocefin, cephacetrile, and cephaloridine were tested by reference microdilution methods in a 12- dilution schedule ranging from 0.06 to 128,ug/ml (Table 1). The 138 gram-negative isolates were generally resistant (median MICs, >128,ug/ml) to the inhibiting effects of PADAC and nitrocefin. Only rare strains of Providencia rettgeri had nitrocefin MICs of 8,g/ml. Cephacetrile and cephaloridine possess a spectrum similar to cephalothin, thus inhibiting only Escherichia coli, Klebsiella spp., and Proteus mirabilis among the Enterobacteriaceae and, also, Staphylococcus aureus. PADAC and nitrocefin were comparable to cephaloridine in anti-staphylococcal activity (median MICs, 0.25,ug/ml). Penicillinase-producing S. aureus strains had median MICs four- and.eightfold higher than penicillinase-negative strains for PADAC and nitrocefin, respectively. This indicates a partial susceptibility of these cephalosporins to the staphylococcal beta-lactamase. An unexpected finding was the very low nitrocefin MIC (range, s0.06 to 0.12,ug/ml) for enterococci. The other reference cephalosporin MICs for Streptococcus faecalis were.16,ug/ml, consistent with other cephalosporin drugs. Table 2 shows the results of spectrophotometric beta-lactam hydrolysis studies, using six beta-lactamases from reference bacteria or commercial sources. The color change for PADAC was from purple (lambda maximum, ca. 570 nm) to a clear yellow (lambda maximum, ca. 465 nm). This change was monitored at 570 nm for these studies, and the resulting scans before and after beta-lactamase (type la, Enterobacter cloacae) addition to the reaction mixture are shown in Fig. 2. Reaction hydrolysis rates were calculated in micromoles of substrate hydrolyzed per minute per milliliter of enzyme and compared with cephaloridine hydrolysis. The PADAC has a molecular weight of 621 and a molar extinction coefficient of ca. 5.7 x 104. The two cephalosporins used as parenteral drugs (cephacetrile and cefoxitin) had the lowest hydrolysis rates. Nitrocefin was most susceptible to hydrolysis by all enzymes, with PADAC also showing beta-lactamase lability, but at a reduced rate. PADAC was closest to nitrocefin in susceptibility to the type II-TEM enzymes commonly found in the Enterobacteriaceae and some Haemophilus spp. and N. gonorrhoeae. Table 3 and Fig. 3 demonstrate the results of using nitrocefin and PADAC as beta-lactamaselabile substrates in enzyme inhibition studies. Six beta-lactamase-stable inhibitors were selected (cefotaxime, ceftazidime, CP 45,899, dicloxacillin, HR221, and SCH29482) for their variability of inhibitory effect, particularly on type I beta-lactamases (5, 6, 16). At least one drug had a significant inhibitory effect (>50% of relative hydrolysis rate) for each enzyme (Table 3). For nearly all enzymes tested the inhibitor was more active against the PADAC hydrolysis than against that of nitrocefin. Only CP 45,899 versus type II and the B. cereus gram-positive enzyme slightly reversed this trend. Figure 3 graphically shows that beta-lactamase inhibitors achieve significant effects at lower percent concentrations for PADAC than for the nitrocefin substrate. Approximately a 10-fold difference is evident for HR221, SCH29482, and ceftazidime, and CP 45,891 produces a 50% inhibition of PADAC hydrolysis at an 8% concentration of substrate compared with a >200% concentration needed to inhibit nitrocefin hydrolysis. The comparisons for the very potent type I inhibitors dicloxacillin and cefotaxime were difficult to assess due to their extreme effects below 0.1 to 0.2% substrate concentrations. PADAC was mixed with 50% human serum in a spectrophotometer at 37 C. Little or no color change, compound degradation, or other adverse change was observed. Beta-lactamases added to PADAC mixed with serum resulted in hydrolysis rates comparable to those in phosphate buffer. Substrate (PADAC) degradation rates in phosphate buffer and serum were similar over several hours. Similar experiments were conducted mixing PADAC with various broth media, including Mueller-Hinton, brain heart infusion, Trypticase (BBL) soy, Schaedler, and Todd-Hewitt broths. Again, no significant adverse effects or reactions occurred. These data contrast markedly to the effects of media and serum proteins on nitrocefin (14). A pipette drop (30 to 50 p.l) of PADAC at a concentration of 128,ug/ml was mixed with Mueller-Hinton broth (100,ul) containing turbid growths of methicillin-induced S. aurtus cultures. Of 10 penicillinase-positive and -negative cultures, PADAC demonstrated the enzyme activity in all cases but only after >30 min of incubation at room temperature. Similar experiments were done on a cerebrospinal fluid from a clinical case known to be infected by betalactamase-producing Haemophilus influenzae. Again, in this case the mixture of PADAC or nitrocefin (1 drop) with 100,ul of cerebrospinal fluid produced positive color reactions in 10 to 30 min. The beta-lactamase-positive cerebrospinal fluid specimen nitrocefin results were very difficult to interpret due to a high cerebrospinal fluid protein level. The same reagents and dipsticks applied to patient urines containing betalactamase-positive bacteria (E. coli, resistant to

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5 VOL. 15, 1982 TABLE 2. PADAC IN VITRO EVALUATION 681 Substrate hydrolysis rates of six representative beta-lactamase preparations on chromogenic and enzyme-labile cephalosporinsa Organism (beta-lactamase type)b Cephaloridine RHR (%) compared with cephaloridine (glmol/min per ml) PADAC Nitrocefin Cephacetrile Cefoxitin Enterobacter cloacae (Ia) <0.1 Pseudomonas aeruginosa (II) <0.1 <0.1 Escherichia coli (IIIa) <0.1 Escherichia coli (IIIb) <0.1 Klebsiella spp. (IVc) <0.1 Bacillus cereus (NT) <0.1 a Beta-lactamase hydrolysis was determined by the UV spectrophotometric method, using 258 to 570 nm at 37 C. Reaction mixture was a 1.0-ml volume of a 10-4 M concentration of cephalosporin substrate (except PADAC at 5 x 10-5 M) in 0.05 M phosphate buffer, ph 7. RHR, Relative hydrolysis rate, compared with that of enzyme-cephaloridine given a value of 100%. b Enzyme classifications after the schema of Richmond and Sykes (16). NT, Not typed. > A(nm) FIG. 2. Absorption spectrum of PADAC (0.4 mm) before (-) and after (---) exposure to beta-lactamase. ampicillin and cephalothin) and a sample of infected urines with beta-lactamase-negative bacteria produced 100% accurate results within 30 min when compared with MIC results and beta-lactamase tests performed on resultant colonies. DISCUSSION Bacterial enzymes capable of destroying betalactam drugs have long been recognized in clinically relevant bacteria. Their frequency seems to have increased in recent years, and several previously beta-lactam-susceptible species have acquired beta-lactamase-mediated resistance through plasmids (2, 8, 15, 20). Among these, H. influenzae and N. gonorrhoeae (penicillinaseproducing strains) produce a large number of serious infections in children and adults, respectively. Early recognition of these beta-lactamase-producing strains allows the proper selection of antimicrobial agents (or deletion of unusable drugs) and a rapid follow-up of patient contacts, minimizing widespread circulation of penicillinase-producing N. gonorrhoeae in the community. The latter problem has been reemphasized in the recent report of a 0.6% incidence of penicillinase-producing N. gonorrhoeae in Florida (3). Rapid colorimetric diagnostic tests capable of detecting beta-lactamase have been developed and include an acidometric penicillinase test (17, 21), iodometric techniques (1, 9), and chromogenic reagents from basic cephalosporin structures (14). Varying enzyme detection susceptibilities for these tests are well known, and it is also known that they best correlate to the type of substrate beta-lactam used. Among the most sensitive tests has been the nitrocefin (87/312) chromogenic cephalosporin developed by Glaxo Research Group, Ltd., but its use has been generally limited to research laboratories due to inconsistent availability as a diagnostic. Thus, PADAC, a similar cephalosporin chromophore, appears to be a welcome addition to the diagnostic beta-lactamase reagents as a powder (Calbiochem-Behring) or on dipsticks (Behringwerke). PADAC has an easily seen color change, allowing application by most clinical microbiology laboratories. This reagent used as a test solution or on paper strips readily detected betalactamase in culture and in clinical specimens in our studies and those reported by others (C. Thornsberry and J. W. Biddle, personal communications). Their beta-lactamase-producing species were H. influenzae, N. gonorrhoeae, S. aureus, S. epidermidis, Bacteroides spp., and E. coli. PADAC's sensitivity to enzyme hydrolysis was less than that of nitrocefin, but comparable to cephaloridine, a compound commonly used as a cephalosporinase-labile substrate and monitored at 260 nm in the UV range. Like nitrocefin, PADAC's visual spectrum absorption peaks offer a wide application to non-uv-range spectrophotometers. Laboratories having a spectropho-

6 682 JONES, WILSON, AND NOVICK J. CLIN. MICROBIOL. TABLE 3. Comparative inhibition by six beta-lactam compounds, using nitrocefin and PADAC as betalactamase-labile substratesa RHR of substrate mixed with an equal Conc of the inhibitorb Organism (beta-lactamase type)' Substrate Dicloxacillin CP 45,899 SCH HR 221 Cefotaxime Ceftazidime Enterobacter cloacae (Ia) Nitrocefin < <1.0 < PADAC < <1.0 <0.1 5 Pseudomonas aeruginosa (II) Escherichia coli (Illa) Escherichia coli (IlIb) Klebsiella spp. (IVc) Bacillus cereus (NT) Nitrocefin PADAC Nitrocefin 23 7 PADAC 17 < Nitrocefin PADAC Nitrocefin PADAC Nitrocefin PADAC a Only results from equimolar concentration mixtures of inhibitor and substrate are shown. b RHR, Relative hydrolysis rate. Represents percent of base-line hydrolysis rate for substrate and enzyme mixture alone. c Enzymes classified by the method of Richmond and Sykes (16). NT, Not typed. tometer with a 550-nm filter could monitor enzyme hydrolysis, and those with a centrifugal t0 100 CC75- C)50- '425- _ Inhibitor Conc. (% of Substrate) FIG. 3. Type Ia (Enterobacter cloacae P99) betalactamase hydrolysis inhibition profiles, using PADAC and nitrocefin (87/312) as substrates monitored by a centrifugal fast analyzer at 550 and 482 nm, respectively. Symbols: Dicloxacillin (_-e); cefotaxime (o-); cefodizime (LI[); SCH29482 (e---eo); ceftazidime (o---o); sulbactam (U-U). RHR, Relative hydrolysis rate. fast analyzer can achieve great accuracy by rapid replicate analysis similar to that reported here and earlier by DeBell et al. (4). The latter investigators adapted the microiodometric method to a Rotochem and reported excellent enzyme kinetic results for a Vibrio parahaemolyticus beta-lactamase (4). PADAC can also be used as the beta-lactamlabile substrate for inhibition studies (6a, 7). Both nitrocefin and PADAC can be monitored in the non-uv range for those comparative analyses, in contrast to cephaloridine (5, 6, 13). Here PADAC may be a better substrate due to its decreased enzyme affinity compared with nitrocefin or cephaloridine (data not shown) when in combination with other beta-lactams used as inhibitors. Application to the direct examination of clinically infected fluids and other physiological materials seems practical due to the apparent lack of PADAC interaction with normal human serum constituents. Schindler and Huber describe the application of PADAC type I (Enterobacter cloacae P99) beta-lactamase hydrolysis to the quantitative measurement of those inhibiting beta-lactams down to the nanogram level on non-uv instruments (19). Similarly, the detection and quantitation of beta-lactamases in body fluids regardless of protein content seems both feasible and desirable (further studies in progress). In summary, PADAC is a new chromogenic cephalosporin developed by Schindler and Huber with absorption peaks at ca. 570 nm

7 VOL. 15, 1982 before beta-lactamase hydrolysis and ca. 465 nm after enzyme attack (19). Like nitrocefin, PADAC is a poor antimicrobial agent against gram-negative species, but has some activity against S. aureus. Representative beta-lactamase extracts and whole-cell suspensions (cultures and clinical fluids) produced a visual and spectrophotometric PADAC color change at a rate slower than nitrocefin. Both PADAC and nitrocefin were excellent substrates for beta-lactamase inhibition studies. PADAC should be a welcome addition to the beta-lactamase diagnostic tests that are urgently needed in the clinical microbiology laboratory and to the researcher as a reference beta-lactamase-labile chromogenic substrate. ACKNOWLEDGMENTS This study was supported in part by a grant from Hoechst- Roussel Pharmaceuticals, Inc. We thank Judy Bigelow for technical assistance and Barbara Beardsley for preparation of the manuscript. We also acknowledge the kind advice, help, and historical perspective offered by C. O'Callaghan. ADDENDUM IN PROOF Since this paper was accepted for publication, the cephalosporin HR221 has been named cefodizime, and the paper PADAC strips have been marketed by Calbiochem-Behring, La Jolla, Calif. LITERATURE CITED 1. Catlin, W. B Iodometric detection of Haemophilus influenzae beta-lactamase: rapid presumptive test for ampicillin resistance. Antimicrob. Agents Chemother. 7: Centers for Disease Control Penicillinase-producing N. gonorrhoeae. Morbid. Mortal. Weekly Rep. 25: Centers for Disease Control Infections due to penicillinase-producing Neisseria gonorrhoeae-florida. Morbid. Mortal. Weekly Rep. 30: DeBell, R. M., T. M. Hickey, and D. E. Uddin Partial characterization of a beta-lactamase from Vibrio parahaemolyticus by a new automated microiodometric technique. Antimicrob. Agents Chemother. 13: Fu, K. P., and H. C. Neu Beta-lactamase stability of HR 756, a novel cephalosporin, compared to that of cefuroxime and cefoxitin. Antimicrob. Agents Chemother. 14: Fu, K. P., and H. C. Neu The comparative betalactamase resistance and inhibitory activity of 1-oxa cephalosporin, cefoxitin and cefotaxime. J. Antibiot. 32: a.Jones, R. N., A. L. Barry, C. Thornsberry, E. H. Gerlach, P. C. Fuchs, T. L. Gavan, and H. M. Sommers PADAC IN VITRO EVALUATION 683 Ceftazidime (GR20263), a pseudomonas-active cephalospofin: 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., J. Slepack, and J. Bigelow Ampicillinresistant Haemophilus paraphrophilus laryngo-epiglottitis. J. Clin. Microbiol 4: Jorgensen, J. H., J. C. Lee, and G. A. Alexander Rapid penicillinase paper strip test for detection of betalactamase-producing Haemophilus influenzae and Neisseria gonorrhoeae. Antimicrob. Agents Chemother. 11: 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. 11. Neu, H. C Antibiotic inactivating enzymes of bacterial resistance. In V. Lorian (ed.), Antibiotics in laboratory medicine. The Williams & Wilkins Co., Baltimore. 12. Neu, H. C., K. P. Fu, N. Aswapokee, P. Aswapokee, and R. Kung Comparative activity and beta-lactamase stability of cefoperazone, a piperazine cephalosporin. Antimicrob. Agents Chemother. 16: 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: Percival, A., J. E. Corkill, and 0. P. Arya Penicillinase-producing gonococci in Liverpool. Lancet. 1i: Richmond, M. H., and R. B. Sykes The betalactamases of gram-negative bacteria and their possible physiological role. Textbook Adv. Enzymol. 9: Rosen, I. G., J. Jacobson, and R. Rudderman Rapid capillary tube method for detecting penicillin resistance in Staphylococcus aureus. Appl. Microbiol. 23: 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. 20. Thomas, W. J., J. W. McReynolds, C. R. Mock, and D. W. Bailey Ampicillin-resistant Haemophilus influenzae meningitis. Lancet i: Thornsberry, C., and L. A. Kirven Ampicillin resistance in Haemophilus influenzae as determined by rapid test for beta-lactamase production. Antimicrob. Agents Chemother. 6: