Quality Control of Moxalactam Susceptibility Disks

Transcription

1 JOURNAL OF CLINICAL MICROBIOLOGY, June 1983, p Vol. 17, No /83/ $02.00/0 Copyright 1983, American Society for Microbiology Quality Control of Moxalactam Susceptibility Disks ARTHUR L. BARRY,'* DAVID A. PRESTON,2 RONALD N. JONES,3 THOMAS L. GAVAN,4 AND CLYDE THORNSBERRY5 University of California, Davis, Medical Center, Sacramento, California ; Lilly Research Laboratories, Indianapolis, Indiana ; Kaiser Foundation Laboratories, Clackamas, Oregon ; The Cleveland Clinic Foundation, Cleveland, Ohio ; and Centers for Disease Control, Atlanta, Georgia Received 14 January 1983/Accepted 2 March 1983 In vitro evaluation of two types of moxalactam disks revealed significant performance differences when Staphylococcus aureus was being tested. The differences were traced to the amount of decarboxylated moxalactam present in the disks. The decarboxylated analog was much more active than the parent compound against S. aureus, not active against Pseudomonas aeruginosa, and approximately as active as the parent compound against Escherichia coli. A ninelaboratory coordinated study was performed to establish quality control parameters for 30-,ug moxalactam disks. Problems with the establishment of interpretive standards for moxalactam disk tests were evaluated in the light of differences between disks utilized in earlier studies and those that are now commercially available. The type of disk greatly influences standards for tests with S. aureus but has insignificant influence on testing gram-negative bacilli. Moxalactam (MOX) is a 1-oxa-p-lactam compound with a broad spectrum of antibacterial activity resembling that of the third-generation cephalosporins (2, 4, 5, 8, 9, 12, 13, 15-17). In 1980, Barry et al. (3) made recommendations for the interpretation of disk diffusion tests. Other interpretive standards have been recommended by Preston et al. (14) and by Hall and Opfer (7). The present report describes the influence that technical problems involving the disk manufacturing process have on the selection of interpretive zone size standards and on the establishment of quality control (QC) parameters. During the development of the MOX susceptibility test disk, the methods for manufacturing susceptibility disks were altered. The earliest disks were prepared with the disodium salt of MOX in an amorphous form (3). Later, it was found that the MOX in the disks manufactured in this way was more vulnerable to a decarboxylation reaction which occurs in the dry state and appears to be enhanced by some undefined component in the paper disks. Decarboxylation converts MOX to another antibiotic, the decarboxylated analog of MOX, D-MOX (Fig. 1). Because D-MOX has antibiotic properties differing from those of MOX, the presence of D-MOX in MOX susceptibility test disks can alter the performance of the disks against certain microorganisms. Further developments provided a manufacturing process which uses a crystalline t Present address: Clinical Microbiology Institute, P.O. Box 947, Tualatin, OR form of the diammonium salt of MOX and results in MOX disks which are stable to drystate decarboxylation. Studies have been performed to evaluate two types of MOX disks containing different amounts of D-MOX. The two types of disks differed in their performance when tested against Staphylococcus aureus, but not when tested against other bacterial species. These differences dramatically influenced the establishment of interpretive breakpoints. Data collected during the current study are contrasted to those obtained from our previous evaluation to determine whether interpretive zone size standards need to be modified because of improvements in disk manufacturing procedures. Three separate batches of 30-,ug MOX disks containing different initial amounts of D-MOX were evaluated in a nine-laboratory coordinated study designed to establish QC limits (6). MATERIALS AND METHODS Bacteria. To evaluate disk diffusion tests, 388 isolates were studied. These included 15 Acinetobacter spp., 50 Pseudomonas aeruginosa, 9 Pseudomonas stutzeri, 6 Pseudomonas fluorescens, 5 Pseudomonas putida, 3 Pseudomonas maltophilia, 3 Pseudomonas cepacia, 3 Pseudomonas acidovorans, 25 Escherichia coli, 26 Klebsiella pneumoniae, 10 Citrobacter diversus, 11 Citrobacter freundii, 19 Enterobacter aerogenes, 14 Enterobacter cloacae, 9 Enterobacter agglomerans, 2 Enterobacter gergoviae, 2 Serratia marcescens, 25 Proteus mirabilis, 10 Morganella morganii, 19 Providencia stuartii, 10 Providencia rettgeri, 48 S. aureus, 25 Streptococcus faecalis, 20 Strepto- 1032

2 VOL. 17, 1983 OCH3 HO- CH-CONH1 N-N R Moxalactam - COOH D-MOX - H ~~~CH2 I.K COOH CH3 FIG. 1. Structure of MOX and its decarboxylated analog, D-MOX. coccus pyogenes, and 19 Streptococcus pneumoniae. Many of these isolates were included in the culture collection which we tested in 1979 and reported in 1980 (3) Ṫhree strains for QC were derived from the following ATCC strains: E. coli ATCC 25922, S. aureus ATCC 25923, and P. aeruginosa ATCC Additional stock cultures were used to evaluate the activity of D-MOX relative to that of MOX. Antimicrobial susceptibility tests. Microdilution susceptibility tests were performed with MOX kindly provided by The Lilly Research Laboratories (Indianapolis, Ind.). Twofold dilutions of MOX (0.008 to 256,ug/ml) were prepared in cation-supplemented Mueller- Hinton broth and then were dispensed into the wells of microdilution test panels. The trays were stored at -20 C or lower until ready for use. Microdilution susceptibility tests and disk diffusion susceptibility tests were performed according to the most recent recommendations of the National Committee for Clinical Laboratory Standards, Villanova, Pa. (10, 11). These studies included two types of disks, both prepared at The Lilly Research Laboratories by different manufacturing procedures. A third lot of disks was prepared by Difco Laboratories (Detroit, Mich.) and used for the multilaboratory QC phase of this study. All lots of disks contained c2.5% D-MOX and.97.5% MOX, as determined by high-performance liquid chromatography of solutions extracted from the disks. One lot of disks contained only 0.25% D-MOX. Activity of the decarboxylated analog. Using an agar dilution procedure (11), one of the authors (D.A.P.) compared the in vitro activity of D-MOX with that of a pure preparation of MOX (<0.5% D-MOX). Additional disk tests (10) were performed with disks prepared to contain 30,ug of drug per disk, but with mixtures of MOX and D-MOX in various ratios. QC parameters. To establish tentative QC limits for MOX disk tests, a nine-laboratory coordinated study was performed following a protocol described in detail elsewhere (1, 6). Participants in this study included four of the authors (A.L.B., R.N.J., T.L.G., and C.T.) as well as J. M. Matsen (University of Utah Medical MOXALACTAM QUALITY CONTROL 1033 Center, Salt Lake City, Utah), P. C. Fuchs (St. Vincent Hospital, Portland, Ore.), L. B. Reller (University of Colorado Medical Center, Denver, Colo.), S. Brown (Good Samaritan Hospital, Portland, Ore.), and E. H. Gerlach (St. Francis Hospital, Wichita, Kans.). All nine participants tested three lots of disks, two prepared by The Lilly Research Laboratories. The three disk lots contained 0.25, 2.0, and 2.5% D-MOX. Three standard control strains were provided in the form of desiccated disks and were tested after reconstitution and serial transfer. Each participant utilized a different lot of Mueller-Hinton agar, representing products from four different media manufacturers (BBL Microbiology Systems, Cockeysville, Md.; Difco Laboratories; GIBCO Diagnostics, Madison, Wis.; and Oxoid Ltd., London, England). Each laboratory performed 50 separate tests with each of the three control strains. To provide further internal protocol control, a common lot of Mueller-Hinton agar was also sent to each participant, and five additional tests were performed with each of the control strains. The resulting zone diameters were compared to detect significant variations in the performance of the different lots of Mueller-Hinton agar. Zone diameters observed with the three different MOX disks were analyzed separately, and the data were then pooled to establish the overall range of variability. Tentative QC limits were established according to the criteria of Gavan et al. (6). The more traditional method of estimating control limits as the mean ± 2 standard deviations was also utilized but was not applicable because the zone measurements were not normally distributed. Performance of the three different types of disks was compared by applying an analysis of variance. When significant differences among disk types were observed, the Newman-Keuls multiple range test was used to compare each type of disk with each of the others. RESULTS Performance of MOX disks. The two types of MOX disks (<0.25% and.2.0% D-MOX) were evaluated separately by plotting zone diameters against minimum inhibitory concentrations (MICs). The method of least squares was used to calculate regression formulae, excluding data with off-scale endpoints (Table 1). As noted previously (3), the regression was not a straightline relationship. The very susceptible Enterobacteriaceae all produced zones of >23 mm in diameter, but the zones were not as large as the regression line would have predicted. To avoid TABLE 1. Regression analysis of 30-,ug MOX disk test data Range Ragf Regression CorreofAmt of No. off formula lation MICs tests D-MOXa in- -coe (pkg/mi) cludedb Inter- Slope cient cept Slp cin High Low High Low High Low a High, 2.0%o D-MOX; low, 0.25% D-MOX. b Data with isolates giving no zones and those with MICs outside the indicated ranges were excluded to avoid the parabolic portion of the curve.

3 1034 BARRY ET AL. the parabolic portion of the line, regression analysis was applied after excluding data with MICs of <2.0,g/ml and again by including only those strains for which MICs were in the critical range of 32 to 2.0,ug/ml (susceptibility breakpoint of 8.0,ug/ml ± 2 log2 dilution intervals). The slopes and intercepts of the lines calculated for the two types of disks were essentially identical (Table 1). The strains tested in the current study were also included in the larger collection of strains which we tested in 1979 in a study published in 1980 (3). The intercepts displayed in Table 1 are 1 to 2 log2 dilution intervals lower than those which we previously described, presumably because a larger number of moderately susceptible and resistant strains of P. aeruginosa and Acinetobacter spp. were included in the 1979 study. More very susceptible Enterobacteriaceae were also tested in the 1979 study. Careful review of the data revealed that the most significant differences between the current data and those of the previous study involved the zone sizes produced by S. aureus strains. Figure 2 compares data obtained from the 1979 series with those observed with the two types of disks used in the present series. Data obtained with Pseudomonas spp., Acinetobacter spp., and S. aureus isolates are displayed in Fig. 2. The enteric bacilli were essentially the same in both series of tests. In the previous study (1979 series), S. aureus isolates generally required MICs of.8.0,g/ml and produced zones of.23 mm, and P. aeruginosa and Acinetobacter spp. with moderately susceptible MICs of 16 or 32,ug/ml generally produced zones 15 to 22 mm in diameter. Those observations led us to recommend tentative zone standards of <14 mm for resistant, 15 to 22 mm for moderately susceptible, and.23 mm for susceptible (3). However, in the current study, the majority of S. aureus isolates gave zones in the moderately susceptible category, although the MICs for them were.8.0,ug/ml. Most S. aureus isolates yielded zones of.20 mm, which is the breakpoint for susceptible currently recommended in the package insert. Disks containing.0.25% D-MOX categorized 12 moderately susceptible Pseudomonas spp. (MIC, 16 to 32,ug/ml) as being resistant (.14 mm), whereas disks containing 2.0% D-MOX provided only 8 such misclassifications. On the other hand, the latter disks misclassified eight resistant isolates (MIC, >32 Fig/ml) as being moderately susceptible (zones of 15 to 19 mm), and the former disks provided only three such misclassifications. Less than half of the pseudomonads for which MICs were 16,ug/ml appeared to be susceptible if a breakpoint of.20 mm was used, but very few strains gave zones of -23 mm. E - 0 UJ ;z C-,) 0 C-) cc 0 -J m si :u I:I:s I I---- f SERIES *.1 1i I 0 o I tii *1 2.0% D-MOX IN - *: t S I:$S 30upg DISKS t 0% 0 - *o~~~;~ J. CLIN. MICROBIOL. * I c So o og 8 I ll I ~~~I 0.25% D-MOX IN > <4- > <4- > * f.sf.<.. I 30jig DISKS --Yi[i -t- _. _ -* ' -.1 P '.i *I -T-** U i 10 < >30 ZONE DIAMETER (mm) FIG. 2. Correlation between microdilution MICs and zone diameters with 30-,ug MOX disks with Pseudomonas spp. plus Acinetobacter spp. (0) and with S. aureus (0). Data with the two types of disks included in the current study (2.0 and 0.25% D-MOX) are compared with similar data (1979 series) previously published (3). In vitro activity of D-MOX. The in vitro activity of D-MOX was compared with that of a relatively pure (.99.5%) solution of MOX by one of the authors (D.A.P.). Against some Enterobacteriaceae, MOX was more active than D-MOX (Table 2). However, against the grampositive cocci, D-MOX was much more active. For S. aureus isolates, the geometric mean MICs were 9.75,ug/ml (MOX) and 0.35,ug/ml (D-MOX). Methicillin-resistant strains of S. aureus demonstrated similar differences, but both compounds were less active. P. aeruginosa was essentially unaffected by D-MOX but was susceptible to moderate concentrations of MOX. Since all MOX susceptibility testing disks contain trace amounts of D-MOX, we designed a study to determine the effect of varying the amount of D-MOX in 30-,ug disks. Figure 3 displays the results of such tests performed with the three standard control strians. With S. aureus, there was a direct linear relationship between the zone size and amount of D-MOX in the disk. With the E. coli, zone sizes were not _

4 VOL. 17, 1983 TABLE 2. Enterobacter cloacae (7) Enterobacter aerogenes (4) Pseudomonas aeruginosa (30) Morganella morganii (4) Proteus mirabilis (15) Haemophilus influenzae, I-lactam+ (6) Providencia stuartii (8) Providencia rettgeri (8) Proteus vulgaris (14) Salmonella spp. (7) Serratia marcescens (8) Haemophilus influenzae, P-lactam- (7) Klebsiella pneumoniae (6) Shigella spp. (8) Escherichia coli (6) Streptococcus pneumoniae (11) Staphylococcus aureus, methicillin susceptible (14) Staphylococcus aureus, methicillin resistant (17) Streptococcus spp., not group D (8) Streptococcus pyogenes (11) Neisseria gonorrhoeae (5) Streptococcus spp., group D (14) MOXALACTAM QUALITY CONTROL 1035 In vitro activity of D-MOX compared with that of MOX, against selected pathogens MIC (p.g/ml) No. on scale Geometric means' Ratio of D-MOX MOX D-MOX MOX D-MOX MOX means 8.0-> > < > < < < s >32 < < >32 > > > < > > > > > c <0.06 < >32 < <0.02 a Number in parentheses shows number of strains tested. b Geometric means were calculated after excluding data that were not on scale, except in groups for which the mode endpoint was off scale. In these cases, the off-scale mode is shown. affected. P. aeruginosa, on the other hand, was not affected until the amount of D-MOX exceeded 20%; i.e., when the concentration of active MOX significantly decreased, zones with P. aeruginosa became smaller. If disks were controlled to maintain 0 to 2.0% D-MOX, some variability in zones sizes would be expected with S. aureus but not with E. coli or P. aeruginosa. QC parameters. The nine-laboratory coordinated study provided data for comparing the two types of MOX disks prepared by The Lilly Research Laboratories (Fig. 4). With E. coli, the performances of both of disk types were essentially identical. With P. aeruginosa, the disks containing 2.0% D-MOX tended to produce more small zones, but the modes and medians were the same. The differences were much more dramatic when the tests were performed with the S. aureus strain. The disks containing 2% D- MOX produced zones much larger than those seen with the other disks containing only 0.25% D-MOX. The disks prepared by Difco Laboratories provided zones that were essentially the same as those observed with disks prepared by The Lilly Research Laboratories to contain 2.0% D-MOX. Table 3 summarizes the overall results of the QC studies with the two types of disks and the commercially prepared 30-,ug MOX disks. With S. aureus, the latter disks produced zones significantly (P < 0.01) larger than those obtained with either of the other two lots of disks. Disks that contained only 0.25% D-MOX produced significantly (P < 0.01) smaller zones of inhibition with S. aureus than the other types of disks did. Tests with P. aeruginosa revealed signifi-

5 1036 BARRY ET AL. I 30 z cn U_\ 20 o \ % D-MOX IN 30 pg DISKS FIG. 3. Effect of varying the amount of D-MOX in disks containing a total of 30,ug of drug. The concentration of MOX decreased as the amount of D-MOX increased. Replicate tests were performed with the standard QC strains for E. coli (O), S. aureus (A), and P. aeruginosa (0). cantly (P < 0.01) larger zones with the disks containing the greatest amounts of active MOX (<0.25% D-MOX), but the differences between means were only 0.5 or 0.6 mm (Table 3). Performances of the three types of disk did not differ significantly when tested with E. coli. The zone size measurements were not normally distributed, and consequently the traditional method for establishing QC limits (mean ± 2 standard deviations) may not be applicable. The method of Gavan et al. (6) establishes control limits as the median + one-half of the alllaboratory median range. This approach better describes control limits for data that are not normally distributed. Both types of control lim- CD c o E coli P. aeruginosa ATCC ATCC Ī5.1 2 its are defined in Table 3, along with the control limits currently recommended by the Food and Drug Administration (FDA), Washington, D.C. A fairly broad tolerance limit is needed for tests with S. aureus because the results appear to be very method dependent. One might expect significant lot-to-lot variation in the performance of commercially prepared disks owing to variability in the trace amount of D-MOX. Tests with E. coli and P. aeruginosa are most reproducible (overall range of 9 mm, compared with a 15-mm range for tests with S. aureus). Control limits for the S. aureus should anticipate the amount of D- MOX that might be present in commercially prepared disks. The upper limit would represent zones expected with disks containing the maximum amount of D-MOX that could be tolerated (2.0%), and the lower limit would reflect zones expected with disks containing only trace amounts of D-MOX and amounts of MOX acceptable to the FDA. DISCUSSION Some decarboxylation of MOX occurs during the preparation of susceptibility testing disks. An analogous situation occurs during the preparation of carbenicillin disks, with the production of trace amounts of benzylpenicillin. MOX prepared for parenteral use in humans should contain less than 2% D-MOX, and similar standards should, therefore, be applied to susceptibility disks. However, variation in disk lots (0 to 2% decarboxylated analog) produces significant variations in zone sizes with the control strain of S. aureus, but has little effect with those of E. coli or P. aeruginosa. Zones with the control CJ 44 S. aureus ATCC J. CLIN. MICROBIOL. z U.)C cm;z 20 C.)U LU DIAMETER (mm) ZONES OF INHIBITION FIG. 4. Distribution of zone diameters recorded during a nine-laboratory coordinated study. Two types of 30-,ug MOX disks were tested 450 separate times.

6 VOL. 17, 1983 TABLE 3. Summary of nine-laboratory coordinated study with three control strainsa Zone site (mm)' D-MOX amt and control limits E. coli ATCC 5 S. aureus ginosa ATCC ATCC Amt of D-MOX in MOX disks 2.5% 31.6 (28-36) 19.7 ( (19-30) 2.0% 31.6 (27-36) 19.6 ( (17-28) 0.25% 31.7 (27-36) 20.2 ( (15-24) Combined (all 31.6 (27-36) 19.8 ( (15-30) MOX disks) Control limits Median ± 1/ range Mean ± SD FDA recom mendation a Three types of MOX disks, each tested 450 separate times, were used. b Means and (in parentheses) ranges are shown. For control limits, only ranges are shown. strain of P. aeruginosa should be reduced only if a large excess (>20%) of the MOX in the disk has been converted to D-MOX, which is essentially inactive against P. aeruginosa. Since the decarboxylated analog is much more active than the parent compound against S. aureus, zones with the control strain of S. aureus will be markedly increased in size when decarboxylation occurs. For QC purposes, it is important to emphasize the need for testing MOX disks against the control strain of S. aureus as well as against those of P. aeruginosa and E. coli. Since E. coli is equally susceptible to both forms of the drug, zones will not be affected if the drug is decarboxylated: tests with the control strain of E. coli best serve to monitor the total potency of the MOX disks. Control limits calculated from data obtained during our nine-laboratory study are similar to those currently recommended by the FDA (Table 3). The latter control limits seem to be acceptable. The technical problem with MOX susceptibility testing disks also greatly influences the establishment of interpretive zone standards. Clearly, the zone standards for interpreting tests with S. aureus will be influenced by the method of preparing MOX disks. In the 1979 series, the MIC was 16 p.g/ml for only 2% of the methicillinsusceptible S. aureus strains; all the other strains were inhibited by a concentration of -8.0,ug/ml. In the current study, the MICs for all S. MOXALACTAM QUALITY CONTROL 1037 aureus isolates were s8 jig/ml. Although MOX is not a primary anti-staphylococcal drug, it is highly effective against methicillin-susceptible S. aureus at obtainable serum concentrations. Clinical experience with MOX therapy in patients found to have staphylococcal infections has been encouraging, with results of >90% bacteriological and clinical responses (D.A.P., unpublished data). With the disks utilized in the current study, most isolates of S. aureus gave zones of 20 to 22 mm in diameter, but in the 1979 study, most S. aureus isolates gave zones of >23 mm in diameter. The current FDA recommendations for MOX disks define susceptible isolates as those with for which MICs of <16,ug/ml and zones of.20 mm are observed; resistant isolates are defined as those for which MICs of.64 j,g/ml and zones of <14 mm are found. If those zone size breakpoints had been used in the current study, fewer S. aureus isolates would have been misclassified in the moderately susceptible category (15 to 19 mm). However, most pseudomonads for which MICs were 16,ug/ml (susceptible by FDA standards) were not categorized as being susceptible since they had zones of <19 mm. Preston et al. (14) recommended a MOX breakpoint of -18 mm for the susceptible category, and that breakpoint would be more appropriate for the S. aureus data included in the current study. If the susceptible category is defined as an MIC of s16 jig/ml, many P. aeruginosa isolates would be miscategorized, even with the breakpoint of -18 mm. Regression statistics for data accumulated in the 1979 study suggested breakpoints of.23 mm for MICs of -8.0 jlg/ml and.18 mm for MICs of <16 j,g/ml. Data accumulated in the current study provided regression statistics which gave MIC correlates of 4.9 and 5.2 jig/ml for a 23-mm zone with the two types of disk, and an 18-mm zone correlated with MICs of 11.8 and 12.3,ig/ml for the two types of disk. Clearly, the present report documents the need for zone size breakpoints that are smaller than those we previously recommended for interpreting tests with S. aureus. For tests with gram-negative bacilli, the position of the interpretive zone standards depends upon the MIC breakpoint to be used for definition of the susceptible category ('8 or s16,ug/ml). Since methicillin-susceptible S. aureus strains have a very predictable level of susceptibility to MOX, and since MOX is not a primary antistaphylococcal drug, one can logically argue that in vitro susceptibility tests are not routinely required. Although methicillin-resistant strains of S. aureus might occasionally appear to be susceptible to MOX, they should be considered resistant. For these reasons, routine tests with 30-j,g MOX disks should be limited to tests with

7 1038 BARRY ET AL. gram-negative bacilli. In that case, variability in the amount of the decarboxylated analog would have minimal effects on the zone size standards as long as less than 20% of the drug is decarboxylated. If data with S. aureus are excluded, a zone size breakpoint of.23 mm for the susceptible category would be appropriate for an MIC breakpoint of -8.0,ug/ml for the susceptible category. Such breakpoints would appropriately categorize most P. aeruginosa in the moderately susceptible category (3). If isolates were considered susceptible for which the MICs were s16 jig/ml, zone size breakpoints of.18 mm (14) would be more appropriate, but many P. aeruginosa isolates with susceptible MICs would give zones in the moderately susceptible or resistant categories. The former MIC breakpoints (s8.0 jig/ml) are preferred because they separate clearly different organism populations and do not cut through the center of a major population of MICs. Although not required routinely, occasional S. aureus isolates might require in vitro tests with MOX disks, e.g., when recovered from a patient receiving MOX therapy for treatment of other infections. When such S. aureus isolates are tested with MOX disks, methicillin-resistant strains should be considered resistant to MOX, regardless of the results of the in vitro tests. Methicillin-susceptible strains of S. aureus with MOX zones of.15 mm (intermediate or susceptible) can be reported as susceptible, and those with zones of <14 mm should be reevaluated; they may be methicillin resistant or may be an organism other than S. aureus. Even if S. aureus isolates are not being tested routinely with MOX disks, monitoring the performance of MOX disks with the control strain of S. aureus is recommended. If unusually large zones (.28 mm) are observed, decarboxylation should be suspected. If sufficient decarboxylation occurs, zones with the P. aeruginosa control strain will decrease in size because the amount of active MOX has been reduced. The control strain of E. coli is equally susceptible to both forms of the drug, and so zones will not be altered if the drug is decarboxylated; consequently, it is useful for monitoring the overall potency of the disks. LITERATURE CITED 1. Barry, A. L., R. N. Jones, C. Thornsberry, and T. L. Gavan Azlocillin, a ureido penicillin active against Pseudomonas aeruginosa: interpretive zone standards and quality control parameters for tests with 75-,ug disks. J. Clin. Microbiol. 16: Barry, A. L., C. Thornsberry, and R. N. Jones In vitro evaluation of LY (6059-S) compared with J. CLIN. MICROBIOL. cefotaxime, eight P-lactams and two aminoglycosides. J. Antimicrob. Chemother. 6: Barry, A. L., C. Thornsberry, R. N. Jones, and E. H. Gerlach Tentative interpretive standards for disk susceptibility tests with moxalactam (LY127935). Antimicrob. Agents Chemother. 18: Barza, M., F. P. Tally, N. V. Jacobus, and S. L. Gorbach In vitro activity of LY Antimicrob. Agents Chemother. 16: Fu, K. P., and H. C. Neu The comparative,blactamase resistance and inhibitory activity of 1-oxa cephalosporin, cefoxitin and cefotaxime. J. Antibiot. (Tokyo) 32: Gavan, T. L., R. N. Jones, A. L. Barry, P. C. Fuchs, E. H. Gerlach, J. M. Matsen, L. B. Reller, C. Thornsberry, and L. D. Thrupp Quality control limits for ampicillin, carbenicillin, mezlocillin, and piperacillin disk diffusion susceptibility tests: a collaborative study. J. Clin. Microbiol. 14: Hall, W. H., and B. J. Opfer Correlation of moxalactam (LY127935) susceptibility tests by disk diffusion and agar dilution methods. Antimicrob. Agents Chemother. 19: Jones, R. N., P. C. Fuchs, H. M. Sommers, T. L. Gavan, A. L. Barry, and E. H. Gerlach Moxalactam (LY127935), a new semisynthetic 1-oxa- -lactam antibiotic with remarkable antimicrobial activity: in vitro comparison with cefamandole and tobramycin. Antimicrob. Agents Chemother. 17: Komatsu, Y., and T. Nishikawa Moxalactam (6059- S), a new 1-oxa-1-lactam: binding affinity for penicillinbinding proteins of Escherichia coli K-12. Antimicrob. Agents Chemother. 17: National Committee for Clinical Laboratory Standards Approved standard M2-A2. Performance standards for antimicrobic disc susceptibility tests. National Committee for Clinical Laboratory Standards, Villanova, Pa. 11. National Committee for Clinical Laboratory Standards Tentative standard M7-T. Standard methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. National Committee for Clinical Laboratory Standards, Villanova, Pa. 12. Neu, H. C., N. Aswapokee, K. P. Fu, and P. Aswapokee Antibacterial activity of a new 1-oxa cephalosporin compared with that of other 3-lactam compounds. Antimicrob. Agents Chemother. 16: Parsons, J. N., J. M. Romano, and M. E. Levison Pharmacology of a new 1-oxa-1-lactam (LY127935) in normal volunteers. Antimicrob. Agents Chemother. 17: Preston, D. A., M. A. Surprenant, and L. C. Hawley Susceptibility testing with LY (Shionogi 6059-S): proposals for disk content and interpretive criteria, p In J. D. Nelson and C. Grassi (ed.), Current chemotherapy and infectious disease. American Society for Microbiology, Washington, D.C. 15. Reimer, L. G., S. Mirrett, and L. B. Reiler Comparison of in vitro activity of moxalactam (LY127935) with cefazolin, amikacin, tobramycin, carbenicillin, piperacillin, and ticarcillin against 420 blood culture isolates. Antimicrob. Agents Chemother. 17: Wise, R., J. M. Andrews, and K. A. Bedford LY127935, a novel oxa-1-lactam: an in vitro comparison with other,b-lactam antibiotics. Antimicrob. Agents Chemother. 16: Yu, V. L., R. M. Vickers, and J. J. Zuravleff Comparative susceptibilities of Pseudomonas aeruginosa to 1-oxacephalosporin (LY127935) and eight other antipseudomonal antimicrobial agents (old and new). Antimicrob. Agents Chemother. 17:96-98.