Identification of Beta-Lactamase Enzymes and Prediction of Successful Beta-Lactam Therapy

Transcription

1 JOURNAL OF CLINICAL MICROBIOLOGY, May 1983, p /83/5791-8$2./ Copyright C 1983, American Society for Microbiology Vol. 17, No. 5 Relative Substrate Affinity Index Values: a Method for Identification of Beta-Lactamase Enzymes and Prediction of Successful Beta-Lactam Therapy RICHARD JAMES School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, Norfolk, England Received 26 July 1982/Accepted 21 January 1983 Using a nitrocefin competition assay, I determined the relative substrate affinity index (RSAI) values of nine clinically significant beta-lactamase enzymes against a range of beta-lactams. Using selected beta-lactam substrates, I observed large differences in the RSAI values of the nine enzymes that were sufficient in many cases to positively identify specific enzymes. I made use of the unique RSAI values of SHV-1, TEM-1, and TEM-2 beta lactamases with cefoxitin to screen for the presence of these enzymes in Klebsiella aerogenes clinical isolates. The RSAI values also allow for the prediction of the outcome of beta-lactam therapy against specific beta-lactamase-producing isolates. Resistance to beta-lactam antibiotics is now widespread among many bacterial genera. The most common mechanism of resistance is the production of beta-lactamases, enzymes which hydrolyze the beta-lactam ring and inactivate antibiotics. Although many gram-negative bacteria produce inducible, chromosomally coded beta-lactamases, in all but a few cases their role in resistance to beta-lactams appears to be open to doubt. In gram-negative bacteria, several constitutive, plasmid-coded beta-lactamases have been identified on the basis of their substrate profiles against a range of beta-lactams and their isoelectric points (5). A variety of methods is available to determine substrate profiles (6), but they suffer from a number of disadvantages. For example, hydrolysis of penicillins and cephalosporins may be monitored by a fall in UV absorption at a wavelength characteristic of each beta-lactam used, but it is readily apparent that relatively pure enzyme preparations have to be used in such assays to reduce background absorption. In addition to being laborious to perform, many of the assay methods are insensitive. Although capable of detecting gross differences in relative hydrolysis rates of beta-lactams, substrate profiles often fail to distinguish between a low rate of hydrolysis due to a low affinity of the enzyme for the substrate and one due to enzyme inhibition by the substrate. This distinction is becoming more important with the introduction of beta-lactamase inhibitors, such as clavulanic acid. Given the large differences in the hydrolysis rates of beta-lactams by different beta-lactamases, it is possible to predict that many betalactams are likely to be ineffective in vivo 791 against a pathogen producing specific beta-lactamases. Although a mass of evidence is available on the trends of antibiotic susceptibility of clinical isolates (1), little practical information is available on the contribution of specific betalactamases, largely because of the difficulty of identification. The problem cannot be ignored, however, because beta-lactam antibiotics constitute our principal defense against pathogenic bacteria, and a policy for sensible antibiotic use depends upon the avoidance of over-prescription of the newer cephalosporins when an older (and cheaper) beta-lactam would be effective. It is impractical to test every clinical isolate against every possible beta-lactam and all possible combinations of beta-lactamase inhibitors and beta-lactams; therefore, a data base of the sensitivity of specific beta-lactamase-producing clinical isolates to a range of beta lactams is needed. I have developed a very sensitive assay which allows the quantitation of the relative affinities of beta-lactamases for beta-lactam antibiotics. The assay is rapid, simple to perform, and amenable to automation. It yields a wealth of information on the interactions between betalactamases and their substrates which is useful in the prediction of successful beta-lactam therapy. Differences in relative affinities of a variety of clinically important beta-lactamases now also allow simple beta-lactamase identification and, in some cases, even bacterial identification. MATERIALS AND METHODS Bacterial strains and media. Escherichia coli C9 was obtained from the Coli Genetic Stock Center, New Haven, Conn. The transmissible plasmid RP4, which

2 792 JAMES codes for the TEM-2 beta-lactamase (2), was transferred into E. coli C9 by plate mating. Enterobacter cloacae was a laboratory strain which produces a constitutive, chromosomally coded beta-lactamase. E. coli strains 2136E, 28E, 1946E, 1527E, 2139E, and 214E, which produce TEM-1, SHV-1, HMS-1, OXA- 1, OXA-2, and OXA-3 beta-lactamases, respectively, were a generous gift from the Glaxo Research Laboratory, London, England. For the isolation of the reference beta-lactamase extracts, 3 ml of culture in a 1-liter flask was grown to stationary phase with shaking at 37 C in nutrient broth (Oxoid Ltd., London, England). For screening of beta-lactamases, 5 ml of culture was used to prepare the beta-lactamase. Preparation of beta-lactamase extracts. All procedures were carried out at 4 C. Cell pellets were obtained by centrifugation at 9, rpm for 15 min in a Sorvall RC2B centrifuge. The pellets were washed in 4 ml of 1 mm sodium phosphate buffer (ph 7.) and centrifuged again as described above. The washed cell pellets were suspended in 16 ml of the same buffer and sonicated in an Ultrasonics sealed-atmosphere type 753-5A treatment chamber for four periods of 3 s each with 6-s cooling periods. The sonicated extracts were centrifuged at 4, rpm for 4 min in a Beckman L2 centrifuge. The resulting supernatants were stored in portions at -2 C until required. Assay of beta-lactamase activity. The assay mixture (89,ul of 1 mm sodium phosphate buffer [ph 7. and prewarmed to 37 C], 1 p.l of the chromogenic substrate nitrocefin [7] made up in 1 mm phosphate buffer [ph 7.] at 5,ug/ml) was prepared in a 1-cm light path spectrophotometer cuvette. After the temperature was equilibrated to 37 C in a heated cuvette holder, the assay was started by the addition of 1,ul of the beta-lactamase extract (diluted if necessary in 1 mm phosphate buffer [ph 7.]). The change in optical density at a wavelength of 486 nm (OD486) was recorded over a 5-min period to follow nitrocefin cleavage. The reference cuvette contained buffer and nitrocefin but no enzyme. Data recording, graph plotting, and calculation of the percent inhibition of nitrocefin cleavage were performed by an Apple microcomputer interfaced to the spectrophotometer via an analog-digital converter. In all assays, I adjusted the amount of beta-lactamase extract to give an increase in the OD486 of the control of approximately.15 to.25 per min. Competition assays. For beta-lactam competition assays, up to 1 p.1 of individual beta-lactam antibiotics made up in 1 mm phosphate buffer (ph 7.) was substituted for an equivalent volume of buffer in the assay mixture. Concentrated stock solutions of the beta-lactams were stored at -2 C for up to 4 weeks without any significant loss of activity. Preincubation assays were carried out by incubation of buffer, enzyme, and competing beta-lactam in a cuvette at 37 C for various times before the addition of nitrocefin. The percent inhibition of nitrocefin cleavage was calculated for each concentration of competing beta-lactam used, and a graph was plotted of percent inhibition against concentration. Determination of MIC. For minimal inhibitory concentration (MIC) determinations, doubling dilutions of antibiotics were made in Oxoid nutrient broth in microtiter dishes. An inoculum of 16 cells of E. coli J. CLIN. MICROBIOL. C9 or E. coli C9RP4 was added and incubation was carried out at 37 C for 16 h. Isoelectric focusing. Volumes of 1 ml of the betalactamase extracts were loaded directly onto the surface of an Ampholine PAG plate (ph 3.5 to 9.5; LKB Instruments, Inc., Rockville, Md.), and isoelectric focusing was performed according to the instructions of the manufacturer. Beta-lactamase bands were visualized by staining with nitrocefin; pink bands appeared within a few minutes and were photographed with a Polaroid MP4 camera, Polaroid 665 film, and a Kodak Wratten 44 green filter. Antibiotics. Cefoxitin was a generous gift from Merck Sharp & Dohme Inc., West Point, Pa.; cefotaxime was obtained from Hoechst-Roussel Pharmaceuticals Inc., Somerville, N.J.; ceftizoxime was obtained from Fujisawa Pharmaceutical Co., Osaka, Japan; moxalactam was obtained from Eli Lilly & Co., Indianapolis, Ind.; clavulanic acid, cefuroxime, ceftazidime, and nitrocefin were obtained from Glaxo Research Laboratory; SQ 14,359 was obtained from E. R. Squibb & Sons, Princeton, N.J.; Ro was obtained from Hoffmann-La Roche Inc., Nutley, N.J.; and CP 45,899, a beta-lactamase inhibitor, was obtained from Pfizer Inc., New York. All other antibiotics were purchased commercially. RESULTS Determination of RSAI values. The chromogenic substrate nitrocefin is readily cleaved by all of the beta-lactamases I used. Beta-lactam antibiotics which are substrates of a beta-lactamase enzyme readily compete with nitrocefin in my assay. The effect of various concentrations of ampicillin on the cleavage of nitrocefin by TEM-2 is shown in Fig. 1A. From a plot of percent nitrocefin cleavage against ampicillin concentration, the ampicillin concentration which results in 5% inhibition of the activity of TEM-2 (Fig. 1B) can be determined. This ampicillin concentration is a direct estimate of the relative affinity of TEM-2 for ampicillin and nitrocefin. For ease of comparison of the affinity values of a specific beta-lactamase against different beta-lactam antibiotics, I propose the term relative substrate affinity index (RSAI), which is the ratio of the beta-lactam concentration giving 5% inhibition of nitrocefin cleavage to the nitrocefin concentration used in the assay. In the case of ampicillin, the RSAI value is 1.3. The effect of competition by beta-lactams on nitrocefin cleavage kinetics also yields information on the mechanism of interaction between an antibiotic and a beta-lactamase. For example, most of the beta-lactams tested against TEM-2 show a pattern of inhibition similar to that of ampicillin (Fig. 1A); i.e., increasing the betalactam concentration reduces the rate of nitrocefin cleavage (shown as a reduced slope of the increase in OD486 value), but the plots are still linear. This is characteristic of competitive inhibition between ampicillin and nitrocefin. In con-

3 VOL. 17, 1983 RSAI VALUES FOR,-LACTAMASE IDENTIFICATION 793.8L QD -t.4.3.2h.1 1 CD8 W 6 z ii 4 w LI- '2 z.6r L TIME (min) B I I AMPICILLIN CONCENTRATION (pg /mi) FIG. 1. (A) Effect of various ampicillin concentrations on nitrocefin cleavage kinetics by TEM-2. a, Control. The ampicillin concentrations used were 5 (b), 1 (c), 2 (d), and 3 (e),ug/ml. (B) Percent nitrocefin cleavage at 4.5 min. Values were taken from Fig. 1A (control, 1%) and plotted against the ampicillin concentrations. C TIME hin) FIG. 2. Effect of various SQ 14,359 concentrations on nitrocefin (NCF) cleavage kinetics by TEM-2. (A) Results from my conventional assay, in which TEM-2 d was incubated with SQ 14,359 plus nitrocefin at con- 9. centrations of 5 (a), 1 (b), and 2 (c) mg/ml. (B) Effect of preincubation of TEM-2 and SQ 14,359 at concentrations of 25 (d), 5 (e), 2 (f), and 5 (g) mg/ml before the addition of 5,ug of nitrocefin at 5 min, as shown by the arrow. trast, SQ 14,359 at high concentrations exhibits 4 5 nonlinear kinetics of nitrocefin cleavage inhibition (Fig. 2). The same pattern of nitrocefin cleavage inhibition by TEM-2 was also observed with the beta-lactamase inhibitors clavulanic acid and CP 45,899 (data not shown) and with moxalactam and cefoxitin. Preincubation of TEM-2 with SQ 14,359 for 5 min before the addition of nitrocefin resulted in a significant reduction in nitrocefin cleavage (Fig. 2) typical of a beta-lactamase inhibitor. The RSAI values of nine clinically significant beta-lactamase enzymes against beta-lactams are shown in Table 1. It is perhaps not surprising that the chromosomal beta-lactamases of E. cloacae and Pseudomonas aeruginosa, which are classified as cephalosporinases, should exhibit very low RSAI values against most of the beta-lactams, in contrast to penicillinase enzymes like TEM-1 and SHV-1. However, the large differences in the RSAI values of the penicillinase enzymes with a single competing substrate were remarkable. Beta-lactamase screening. The large differences in RSAI values of beta-lactamases against a single beta-lactam suggests that simple betalactamase screening may be feasible. I tested this with SHV-1, which has a uniquely high RSAI value of 6 against cefoxitin (Table 1). I screened Klebsiella aerogenes isolates (National

4 794 JAMES J. CLIN. MICROBIOL. TABLE 1. RSAI values of nine clinically significant beta-lactamases RSAI value of following beta-lactamasea: Beta-lactam P. aeruginosa E. cloacae OXA-3 OXA-2 OXA-1 HMS-1 SHV-1 TEM-2 TEM-1 Penicillin Ampicillin Cloxacillin Cephalothin Cephalozolin Cephaloridine Cefoxitin * 6* Cefuroxime Cefotaxime SQ 14, *.7* 5* 1.5* 45* 15* 1* Moxalactam.6*.1* 1* 3* 4*.3* 28 6* 4* Cefoperazone Carbenicillin.8.3* Cefamandole Cephradine Mecillinam * Ceftizoxime , 1, Ceftazidime.46*.1* >4 9 >2, 1 1,14 1,5 >4 a *, Nonlinear inhibition kinetics. Collection of Type Cultures, Central Public Health Laboratory, London NW9 5HT, England) for the presence of SHV-1 by using a concentration of 3 mg of cefoxitin per ml in the standard nitrocefin competition assay. All but one of the K. aerogenes isolates contained a beta-lactamase, as judged by nitrocefin cleavage, The percent inhibition of nitrocefin cleavage by cefoxitin in all other isolates was 5 + 8%, with the exception of NCTC 173, which was inhibited by only 23%. Isoelectric focusing of the extracts of each isolate confirmed the presence of a single major band in each, which had the same isoelectric point as the SHV-1 control enzyme (pl = 7.6), except in the case of NCTC 173 (pl = 6.5). The RSAI values of the NCTC 173 beta-lactamase are shown in Table 2. It is of interest that this enzyme does not exhibit nonlinear inhibition kinetics with any of the beta-lactams tested, in contrast to SHV-1. Cefoxitin at a concentration of 3 mg/ml is also useful to screen for the presence of TEM-1 or TEM-2, owing to the characteristic nonlinear inhibition kinetics of nitrocefin cleavage (Fig. 3). I screened a large number of recent clinical isolates of K. aerogenes (Public Health Laboratory, Norwich, Norfolk, England) with cefoxitin. Over 6% exhibited the characteristic nonlinear inhibition kinetics of nitrocefin cleavage with cefoxitin. Isoelectric focusing with control SHV-1, TEM-1, and TEM-2 confirmed the presence of TEM-1 or TEM-2 in those isolates predicted from the above test. In all of the isolates which produced a TEM-type enzyme, we also detected SHV-1 on the isoelectric focusing gels when they were overloaded with respect to TEM-1 or TEM-2. TABLE 2. Comparison of RSAI values of SHV-1 and K173 RSAI value of following beta- Beta-lactam lactamasea: SHV-1 K173 Ampicillin Cephazolin Cephaloridine Cefoxitin 6 1,2 SQ 14,359 45* 6 Ceftizoxime 1,4 72 CP 45,899.18*.18 a*, Nonlinear inhibition kinetics. A third example of the use of RSAI values in beta-lactamase identification involves an ampicillin-resistant isolate obtained from a water sample from the River Wensum next to the University of East Anglia, Norwich, Norfolk, England. After confirming the presence of a beta-lactamase which had an RSAI value of.2 against cefoxitin, I suspected the E. cloacae beta-lactamase. This suspicion was confirmed by determining the RSAI value of the extract against cephaloridine (RSAI value, 15.6) and that against clavulanic acid (RSAI value, 48). My identification of this river isolate as E. cloacae was confirmed by a blind bacterial identification test kindly performed for me by the Public Health Laboratory. RSAI values and MICs. A high RSAI value implies that the beta-lactam is a poor substrate for that beta-lactamase. Therefore, in the absence of any permeability barrier to their penetration, beta-lactams should be clinically effec-

5 VOL. 17, 1983 D o -9F O*61.51 a L TIME (min) FIG. 3. Effect of various cefoxitin concentrations on nitrocefin cleavage kinetics by TEM-2. a, Control. The cefoxitin concentrations used were 1 (b), 2 (c), 5 (d), 1 (e), 2 (f), and 3 (g) mg/ml. tive against bacteria producing a beta-lactamase against which they exhibit high RSAI values in vitro. I tested this hypothesis by determining the MICs of a range of beta-lactams for E. coli C9RP4 and E. coli C9 (Table 3). The penicillins which exhibited low RSAI values against TEM-2 in vitro were ineffective in vivo against E. coli C9RP4. In contrast, the new cephalosporins, such as ceftazidime, exhibited very high RSAI values and very low MICs. The use of isogenic strains, differing only in the presence of the plasmid coding for a beta-lactamase, is essential for interpretation of this experiment. Using antibiotic sensitivity test disks, I tested my collection of recent clinical isolates of K. aerogenes, which produce TEM-1 or TEM-2, against cefoxitin and cefotaxime. All isolates were extremely sensitive to both of these antibiotics. Many E. cloacae isolates are resistant to most beta-lactams, including the new cephalosporins (1). This resistance is presumably the result of the permeability barrier to beta-lactams in the E. cloacae outer membrane, together with the presence of a beta-lactamase enzyme. A comparison of the RSAI values of the E. cloacae enzyme with the MICs of the same beta-lactams for E. cloacae (Table 4) reveals that the lowest MIC is shown by moxalactam. Although the RSAI value of the E. cloacae enzyme against moxalactam is very low, implying that it would be readily destroyed by this enzyme, the nonlinear inhibition kinetics suggest that moxalactam could act RSAI VALUES FOR P-LACTAMASE IDENTIFICATION 795 as a combined beta-lactamase inhibitor and killing agent. I therefore predicted that a combination of moxalactam and a beta-lactam having a high RSAI value against this enzyme should be synergistic. This synergism is illustrated in Fig. 4 with cephaloridine and moxalactam. A similar but less effective synergy was observed between moxalactam and carbenicillin, mecillinam, cephazolin, cefoperazone, or cefoxitin. The absence of synergy with all other beta-lactams is illustrated by the results obtained with ampicillin and cefuroxime (Fig. 5). The synergistic action of moxalactam in combination with carbenicillin or cephaloridine was prolonged; no regrowth of E. cloacae was observed for at least 5 h after addition (Fig. 6). E. cloacae laboratory strains which have an inducible cephalosporinase are readily killed by many cephalosporins at concentrations of less than 1,ug/ml (data not shown). We are currently searching for further E. cloacae isolates which are constitutive cephalosporinase producers to test for synergy. DISCUSSION Determination of the RSAI values of betalactamase enzymes is relatively simple and requires neither a purified enzyme nor any detailed analysis of enzyme-substrate interactions. The nonlinear inhibition kinetics of nitrocefin cleavage are also very easy to detect. My findings with cefoxitin and the TEM-type enzymes are in agreement with those of a recent report (3), whereas the similar properties of SQ 14,359 have not been reported previously. The very large differences in RSAI values between penicillinase and cephalosporinase enzymes were not unexpected; however, the ap- TABLE 3. Comparison of RSAI values against TEM-2 and MICs for E. coli C9 and E. coli C9RP4 in vitro MIC (slg/ml) for E. coli RSAI Beta-lactam value with strain: nitrocefina C9 C9RP4 Ampicillin >1, Carbenicillin.4 16 >1, Cephalothin >1, Cephradine >1, Cephaloridine 4 16 >1, Cefamandole >1, Cefoxitin 6* 8 16 Cefuroxime Moxalactam 4* 4 8 Ro Cefotaxime 6 <.125 <.125 Ceftizoxime 1, <.125 <.125 Ceftazidime 1,5 <.125 <.125 a *, Nonlinear inhibition kinetics.

6 796 JAMES TABLE 4. MICs and RSAI values for E. cloacae Beta-lactam val RSAI MIC value' (,ug/ml) mi) Ampicillin.8 >1, Cephalothin Cephazolin Cephaloridine Cefoxitin Cefuroxime Cefotaxime Moxalactam.1* 64 Cefoperazone Carbenicillin.3* 512 Mecillinam 1.3* 512 Ceftizoxime Ceftazidime.1* 128 a, Nonlinear inhibition kinetics TIME (minl FIG. 4. Synergistic combinations of moxalactam plus cefoxitin or cephaloridine against E. cloacae. Fifty milliliters of E. cloacae was grown to an OD55 of approximately.2 before transfer of 5-ml samples to 25-ml flasks containing appropriate amounts of the combination to be tested. Controls () were performed with the single beta-lactams alone. A, Cephaloridine (1,ug/ml); A, cefoxitin (1 FLg/ml); V, cephaloridine (1 pg/ml) plus cefoxitin (1,ug/ml);, moxalactam (5,g/ml); O, moxalactam (5,ug/ml) plus cefoxitin (1,ug/ml); *, moxalactam (5,ug/ml) plus cephaloridine (1,ug/ml). The time of antibiotic addition is indicated by the arrow. J. CLIN. MICROBIOL TIME (min) FIG. 5. Absence of synergy between moxalactam plus ampicillin or cefuroxime against E. cloacae., Control;, moxalactam (5,ug/ml); O, moxalactam (5,ug/ml) plus ampicillin (1,ug/ml); A, moxalactam (5,ug/ml) plus cefuroxime (1,ug/ml); *, moxalactam (5,ug/ml) plus cephaloridine (1,ug/ml). preciable differences in RSAI values of enzymes classified within the same group, i.e., OXA-1, OXA-2, and OXA-3, were surprising. I exploited these differences in a beta-lactamase screening system to detect the presence of SHV-1 or a TEM-type enzyme in a large number of K. aerogenes clinical isolates with a single competing beta-lactam. My predictions on the betalactamase present in these strains were confirmed by isoelectric focusing. The amount of a TEM-type enzyme present in a K. aerogenes isolate "swamps" the much smaller amount of SHV-1, thus allowing its identification on the basis of the observed nonlinear inhibition kinetics of nitrocefin cleavage. The presence of a novel enzyme such as that produced by NCTC 173 is readily detected by its response to inhibition by cefoxitin alone. I am currently studying the immunological relatedness between this enzyme and SHV-1. In a search for natural Klebsiella isolates isolated from a local river, I isolated a betalactamase enzyme which had an RSAI value of

7 VOL. 17, 1983 RSAI VALUES FOR 13-LACTAMASE IDENTIFICATION 797 1tO 4 4 I XU 9 1U9 S LU TIME nin cl I a 2V 2 M FIG. 6. Prolonged nature of synergy between 5 Fg of moxalactam per ml plus 1,ug of either carbenicillin (O) or cephaloridine () against E. cloacae., Control..2 against cefoxitin. The beta-lactamase was confirmed as the E. cloacae chromosomal betalactamase by further RSAI determinations, and my identification of the producing organism was subsequently proven independently. Such a bacterial identification is obviously possible only for chromosomally coded beta-lactamases, which are constitutively produced or have been induced by pregrowth with a beta-lactamase inducer. RSAI values are a direct measure of the relative affinity of a beta-lactamase for nitrocefin and the competing beta-lactam. The affinity of beta-lactamase for a specific beta-lactam in vitro is, of course, only one component in the interaction of a number of factors which determine the clinical usefulness of individual beta-lactam antibiotics against beta-lactamase-producing bacteria. In addition to (i) the permeability properties of the outer cell membrane of gram-negative bacteria, (ii) the Km and Vm. values of the enzyme for that specific beta-lactam, and (iii) the amount of enzyme produced (9), one must also consider the affinity of the beta-lactam for its target, one or more of the penicillin-binding proteins (8). It would thus be surprising if high RSAI values of beta-lactams in my assay against, e.g., TEM-2 correlated exactly with low MICs of the same antibiotics. It is encouraging, therefore, that the new beta-lactamase-resistant cephalosporins exhibit high RSAI values and low MICs against TEM-2 producing E. coli. In this regard, ceftizoxime and ceftazidime appear to be major breakthroughs; their RSAI values of 1, and 1,5, respectively, are the highest I have recorded against a range of 12 clinically significant beta-lactamases. These very high RSAI values also match the very low MICs of the same antibiotics for E. coli C9RP4. I am currently testing a series of isogenic E. coli strains which differ only by the presence of a plasmid-coded beta-lactamase to study further the correlation between RSAI values and MICs. It is also encouraging that none of the K. aerogenes clinical isolates producing a TEMtype beta-lactamase show resistance to cefoxitin or cefotaxime. Inspection of the RSAI values of a range of beta-lactams against the E. cloacae beta-lactamase suggested likely synergistic beta-lactams to use in combination in vivo against this constitutive, beta-lactamase-producing isolate. In vivo tests have supported the predictive value of the RSAI data. It would be surprising if the predictions of synergistic combinations effective against E. cloacae were absolutely reliable, given the impermeability of its outer membrane to many beta-lactam antibiotics (4); however, I am encouraged that the E. cloacae isolate recovered from the River Wensum was also susceptible to the same synergistic combinations (data not shown). I am currently developing a panel of competing beta-lactams with the aim of providing a rapid, automated beta-lactamase identification assay. Such an assay would be invaluable in the further development of rational beta-lactam therapy. ACKNOWLEDGMENTS I am grateful to Jill Debbage, Jacky Heath, and Lynne James for their excellent technical assistance and to Bill Shepherd for stimulating discussions of this work. This research was supported initially by a project grant from the Medical Research Council and subsequently by the National Research Development Corporation. Part of this work is the subject of patent applications. LITERATURE CITED 1. Atkinson, B. A Species incidence, trends of susceptibility to antibiotics in the United States, and minimum inhibitory concentrations, p In V. Lorian (ed.), Antibiotics in laboratory medicine. The Williams & Wilkins Co., Baltimore. 2. 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. 18: Fisher, J., J. G. Belasco, S. Khosba, and J. R. Knowles. 198.,B-Lactamase proceeds via an acyl-enzyme intermediate. Interaction of the Escherichia coli RTEM enzyme with cefoxitin. Biochemistry 19: Kojo, H., Y. Shigi, and M. Nishida Enterobacter cloacae outer membrane permeability to ceftizoxime (FK 749) and five other new cephalosporin derivatives. J. Antibiot. 33:

8 798 JAMES J. CLIN. MICROBIOL. 5. Matthew, M., R. W. Hedges, and J. T. Smith Types of beta-lactamase determined by plasmids in gram-negative bacteria. J. Bacteriol. 138: Neu, H. C Antibiotic inactivating enzymes and bacterial resistance, p In V. Lorian (ed.), Antibiotics in laboratory medicine. The Williams & Wilkins Co., Baltimore. 7. O'Callaghan, C. H., A. Morris, S. M. Kirby, and A. N. Shingler Novel method for detection of beta-lactamase by using a chromogenic cephalosporin substrate. Antimicrob. Agents Chemother. 1: Spratt, B. G Distinct penicillin binding proteins involved in the division, elongation and shape of Escherichia coli K12. Proc. Natl. Acad. Sci. U.S.A. 72: Sykes, R. B., and M. Matthew The beta-lactamases of Gram-negative bacteria and their role in resistance to beta-lactam antibiotics. J. Antimicrob. Chemother. 2: