Vol. 17, No. 2JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 1983, p. 232-2390095-1137/83/020232-08$02.OO/0Copyright © 1983, American Society for Microbiology

Evaluation of the Cefonicid Disk Test Criteria, Including DiskQuality Control Guidelines

ARTHUR L. BARRY,lt* RONALD N. JONES,2 AND CLYDE THORNSBERRY3

Clinical Microbiology Laboratory, University of California, Davis, Medical Center, Sacramento, California958171; Department ofPathology, Kaiser Foundation Laboratory, Clackamas, Oregon 971052; and

Antimicrobics Investigations Section, Centers for Disease Control, Atlanta, Georgia 303333

Received 22 July 1982/Accepted 21 October 1982

Cefonicid (SKF 75073) is a second-generation cephalosporin which has a

spectrum of antimicrobial activity similar to that of cefamandole, but cefoxitin (acephamycin) and cephalothin have uniquely different spectra of activity. Thesecond-generation cephalosporins tested displayed comparable susceptibility toP-lactamases and inhibited type I P-lactamases. Although cefonicid has a longerserum half-life (3 to 4 h) compared with the currently used drugs, the same

minimal inhibitory concentration breakpoints separating susceptible and resistantcategories were applied to tests with cefonicid, cefamandole, and cephalothin.Regression analysis of the disk diffusion test results confirmed the use of identicalzone size breakpoints for 30-,ug cefonicid, cefamandole, and cephalothin disks: allthree produced similar parabolic regression lines. Further analysis of disk testdata confirmed the fact that cefonicid and cefamandole disks might be usedinterchangeably. But for routine tests, cefonicid disks might be preferred in orderto minimize the number of very major (false-susceptible) interpretive errors.

Suggested cefonicid 30-,ug disk interpretive criteria are: susceptible, -18 mm(<8.0 ,ug/ml), and resistant, <14 mm (>16 ,ug/ml). Quality control zone diameterlimits were calculated from data obtained in a multilaboratory collaborative study.

Cefonicid (SKF 75073) is a new cephalosporin(Fig. 1) with a prolonged serum half-life (17) anda spectrum of activity similar to that of thesecond-generation cephalosporins (2, 10, 14). In1980, Grappel et al. (10) recommended interpre-tive standards for tests with 30-,ug disks andpresented data that demonstrated the fact thatcephalothin disks could not be used to predictsusceptibility or resistance to cefonicid.

In the present report, we compare cefonicidwith other cephalosporins, in terms of their invitro spectra of antimicrobial activity, resistanceto P-lactamases, ability to inhibit the hydrolyticactivity of P-lactamases, and the penetrationinto bacterial cells. In addition, we computedregression statistics and calculated interpretivezone size standards for 30-,ug cefonicid, cefa-mandole, and cephalothin disks. The data wereanalyzed to determine whether cefonicid andother cephem disks might be used interchange-ably to accurately predict bacterial susceptibil-ity. Quality control limits for the disk diffusiontest were calculated from data obtained in anine-laboratory study using several lots of agarand disk products.

t Present address: The Clinical Microbiology Institute, Tua-latin, OR 97062.

MATERIALS AND METHODSAntimicrobial agents. Cefonicid (SKF 75073) and

cefazolin were provided by Smith Kline & FrenchLaboratories, Philadelphia, Pa. Cefoxitin was provid-ed by Merck Sharp & Dohme, West Point, Pa. Cepha-lothin, cephaloridine, and cefamandole were providedby Eli Lilly & Co., Indianapolis, Ind. Nitrocefin wasobtained from Glaxo Research Group, Greenford,Middlesex, United Kingdom. The cefoperazone camefrom Pfizer Inc., New York, N.Y., the ceforanide wasprovided by Bristol Laboratories, Syracuse, N.Y.,and the cefotaxime was provided by Hoechst-RousselPharmaceuticals Inc., Somerville, N.J. Antibiotic-containing disks were manufactured by BBL Microbi-ology Systems (Cockeysville, Md.) and Difco Labora-tories (Detroit, Mich.).

Bacterial strains. Recent clinical isolates were ob-tained from seven geographically separated institu-tions. In addition to the authors' laboratories, contrib-utors to the culture collection included P. C. Fuchs(St. Vincent Hospital, Portland, Oreg.), T. L. Gavan(The Cleveland Clinic Foundation, Cleveland, Ohio),E. H. Gerlach (St. Francis Hospital, Wichita, Kans.),and H. M. Sommers (Northwestern Memorial Hospi-tal, Chicago, Ill.). The strains and species representedare described in Table 1. The 422 isolates included 79Pseudomonas spp. and 15 Acinetobacter spp. whichwere generally resistant to the cephalosporins studiedand thus of little value in evaluating the disk test; theywere, however, included for comparative activity pur-poses. Six reference strains producing known types of

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CEFONICID DISK EVALUATION 233

3IJ.CHCONH NSN

OH Nf CH2S N'IC02H 1

CephalosporinCH3 Cefanandole

CH2S03H Cefonicid

FIG. 1. Structural similarity of cefonicid and cefa-mandole.

,B-lactamase were included for the enzyme inhibitionand hydrolysis studies (11-14, 18, 19).

Antimicrobial susceptibility tests. The microdilutionprocedure for determining minimal inhibitory concen-trations (MICs) was that defined by the NationalCommittee for Clinical Laboratory Standards(NCCLS) (16). Briefly, microdilution trays were pre-pared with doubling dilutions (0.12 to 64 ,ug/ml) ofcefonicid, cefamandole, cephalothin, and cefoxitin; allwere diluted in divalent-cation-supplemented Mueller-Hinton broth (16). As previously described (5, 6, 11-13), the test panels were frozen (-20°C or lower) andthen distributed to the three participating laboratories.Just before use, the test panels were allowed to thaw atroom temperature and then inoculated so as to obtainan inoculum of 1 x 105 to 5 x 105 CFU per ml. The

MIC was defined as the lowest concentration of drugwhich completely inhibited bacterial growth after 16 to18 h at 35°C. The testing procedure in the threelaboratories was controlled by repeatedly testing stan-dard control stains and by duplicate testing of selectedrepresentative strains.Disk diffusion tests were performed by the proce-

dure defined by the NCCLS (15) which is a slightmodification of the procedure described by Bauer etal. (7). Each isolate was tested with 30-,ug diskscontaining cefonicid, cefamandole, or cephalothin;cefoxitin disks were not included in this portion of thestudy.

P-Lactamase hydrolysis and inhibition studies. Hy-drolysis of eight 1-lactams was studied with a scanningUV spectrophotometer (Perkin-Elmer 552; The Per-kin-Elmer Corp., Norwalk, Conn.) at 37°C. Relativehydrolysis rates (RHRs) were calculated for eachstudy drug with cephaloridine as the reference sub-strate. Reaction mixtures were composed of l0' Msubstrate in 0.05 M phosphate buffer (pH 7.0). Crudeenzyme extracts were prepared by exposing cell sus-pensions to repeated freeze-thaw cycles, and if neces-sary, the enzymes were concentrated by precipitatingwith ammonium sulfate, as done in previous studies(11-13). Extracts were prepared from organismsknown to possess type I to IV enzymes as classified bythe scheme of Richmond and Sykes (19). A commer-cially prepared Bacillus cereus penicillinase prepara-tion (BBL Microbiology Systems) was also tested.The ability of cefonicid to inhibit P-lactamases was

determined by procedures described by Jones et al.

TABLE 1. Relative activity of cefonicid, cefamandole, cefoxitin, and cephalothinMIC50, (,ug/ml) MIC90b (Rg/Ml)

Species (no. tested) Cefoni- Cefa- Cefoxi- Cephalo- Cefoni- Cefa- Cefoxi- Cephalo-cid mandole tin thin cid mandole tin thin

Citrobacter diversus (9) 0.25 1.0 2.0 4.0 4.0 2.0 8.0 32Citrobacter freundii (6) 4.0 2.0 64 >64 64 4.0 >64 >64Enterobacter spp. (36)C 8.0 4.0 >64 >64 64 >64 >64 >64Escherichia coli (25) 0.5 0.5 2.0 8.0 64 16 8.0 >64Klebsiella spp. (25) 0.5 1.0 2.0 4.0 8.0 8.0 4.0 32Morganella morganii (4) >64 64 8.0 >64 >64 >64 16 >64Proteus mirabilis (25) s0.12 0.5 2.0 4.0 s0.12 1.0 4.0 8.0Proteus vulgaris (10) >64 >64 4.0 >64 >64 >64 4.0 >64Providencia spp. (18)d 8.0 8.0 2.0 >64 >64 >64 >64 >64Serratia marcescens (25) >64 >64 16 >64 >64 >64 64 >64Staphylococcus aureus

Methicillin sensitive (50) 2.0 0.5 2.0 0.25 8.0 1.0 4.0 0.5Methicillin resistant (6) >64 8.0 32 8.0 >64 8.0 64 32

Streptococcus faecalis (25) >64 32 >64 32 >64 64 >64 64Streptococcus pneumoniae (19) 0.5 s0.25 NTe -0.12 1.0 -0.25 NT <0.12Streptococcus pyogenes (20) 0.5 <0.25 NT <0.12 1.0 <0.25 NT 0.25Neisseria meningitidis (25) <0.12 s0.25 NT <0.12 <0.12 -0.25 NT 0.25Pseudomonas spp. (79f >64 >64 >64 >64 >64 >64 >64 >64Acinetobacter spp. (15) >64 64 64 >64 >64 >64 >64 >64

a MIC5o, Drug concentration inhibiting 50%o of the test strains within each species or subgroup.b MIC90, Drug concentration inhibiting 90%o of the test strains within each species or subgroup.Includes 16 Enterobacter aerogenes, 8 Enterobacter agglomerans and 12 E. cloacae.

d Includes 8 Providencia rettgeri and 10 Providencia stuartii.I NT, Not tested.f Includes 50 Pseudomonas aeruginosa, 11 P. putidalfluorescens group, 10 P. stutzeri, 4 P. cepacia, 3 P.

maltophilia, and 1 P. acidovorans.

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234 BARRY, JONES, AND THORNSBERRY

(11-13). The P-lactamase hydrolysis of nitrocefin wasfollowed on an UV spectrophotometer. A concentra-tion (1.0 x 10-4 M) of the nitrocefin substrate and fourconcentrations (2 x 10-4, 2 x 10-5, 2 x 10-6, 2 x 10-7M) of cefonicid were mixed in pH 7.0 phosphatebuffer. After adding the crude enzyme preparation, therate of nitrocefin hydrolysis was determined spectro-photometrically by replicate analysis in a centrifugalfast analyzer (CentrifiChem; Union Carbide Corp.,Tarrytown, N.Y.). Results were expressed as RHRs,compared to nitrocefin without the added inhibitor.Inhibition studies were also performed with pyridine-2-azo-p-dimethylanaline cephalosporin (PADAC) sub-strate combined with increasing concentrations ofcefonicid (that produced results (data not shown)identical to those found in Fig. 2).

Bacterial cell permeability study. The Escherichiacoli parent strain (DCO) and its sensitive cell mem-brane mutant (DC2) described by Richmond et al. (18)were obtained from Hoechst AG, Frankfurt, FederalRepublic of Germany. Both strains were tested inmicrodilution test panels (16), and the geometric meanMICs from at least 10 separate tests were calculated.A permeability index was obtained by dividing thegeometric mean MIC for the DCO parent strain by thegeometric mean MIC for the DC2 cell membranemutant. A permeability ratio of 1.0 would indicatemaximal permeability, and greater values would repre-sent diminished bacterial membrane permeability.

Statistical analysis. Scattergrams were first preparedby plotting the inhibitory zone diameters againstsimultaneously determined MIC values (log2 [,ug/ml]+9). To mathematically describe the relationship be-tween these two types of measurements, regressionanalysis was applied, using the method of leastsquares. Data with off-scale endpoints (MICs of s0.12or >64 ,ug/ml or no zone of inhibition) were excludedfrom these calculations. The method of least squaresassumes a straight-line relationship between the twovariables, but the scattergrams demonstrated a para-bolic relationship. Regression analysis was repeatedafter excluding all strains with MICs of .2.0 ,ug/ml;that eliminated the parabolic portion of the curve andbest described the relationship around the interpretivebreakpoints. Application of limited-range regressionanalysis has been used successfully in similar studies(4, 6, 11).

Regression analysis was also utilized to directlycompare MICs or zone diameters with two relateddrugs. Idealistically, a perfect correlation between thetwo parameters should provide a correlation coeffi-cient of 1.0, a y intercept of 0.0, and a slope of +1.0.Those three statistics express the degree of related-ness between the two values being compared. Inter-pretive agreement was also expressed as the percent-age of strains that was susceptible, intermediate, orresistant to both drugs being compared. For all fourdrugs included in this report, organisms were consid-ered to be susceptible if the MIC was <8.0 ,ug/ml,resistant if the MIC was >16 ,ug/ml, and intermediateif the MIC was 16 ,ug/ml. Discrepancies were consid-ered to be major differences if susceptible to one drug,but resistant to the other, and minor differences ifintermediate to one and susceptible or resistant to theother.

Quality control study. Two of the strains recom-mended by the NCCLS for quality control of the diskdiffusion test (15) were sent to nine participating

institutions. These strains were E. coli ATCC 25922and Staphylococcus aureus ATCC 25923. The partici-pating investigators in the quality control protocolwere A. L. Barry, S. Brown (Good Samaritan Hospi-tal, Portland, Oreg.), P. C. Fuchs (St. Vincent Hospi-tal, Portland, Oreg.), T. L. Gavan (The ClevelandClinic Foundation, Cleveland, Ohio), E. H. Gerlach(St. Francis Hospital, Wichita, Kans.), J. M. Matsen(University of Utah Medical Center, Salt Lake City,Utah), L. G. Reller (University of Colorado MedicalCenter, Denver, Colo.), H. M. Sommers (Northwest-ern Memorial Hospital, Chicago, Ill.), and C. Thorns-berry (Centers for Disease Control, Atlanta, Ga.).Each investigator performed 50 tests for each qualitycontrol organism on a lot of agar unique to that facility(10 total agar lots from four manufacturers) and 5 testson a Mueller-Hinton agar lot common to all investiga-tors. The total number of zones reported for each diskand control organism was 988. These data were statis-tically analyzed to detect variations in the disk lots,agar lots, or investigator by methods previously de-scribed (9).

RESULTS

The in vitro activity of cefonicid, cefaman-dole, cefoxitin, and cephalothin is shown inTable 1 as the drug concentration inhibiting 50and 90% of the test strains within each speciessubgroup (MIC50 and MIC90). Cefonicid andcefamandole had similar spectra of activity: bothwere more active than cephalothin against En-terobacter spp., Citrobacterfreundii, and Provi-dencia spp. Cefoxitin, however, had a uniquelydifferent spectrum of activity, i.e., more activeagainst indole-positive species of the Proteus-Providencia group and some strains of E. coli,Klebsiella pneumoniae, and Serratia marces-cens, but not active against Enterobacter spp.and C. freundii. All four drugs were activeagainst methicillin-susceptible strains of S. aure-us, but cephalothin was the most active drug.Although methicillin-resistant S. aureus are usu-ally considered clinically resistant to the cepha-losporins, cefamandole and cephalothin oftenproduced MICs of <8.0 1Lg/ml (susceptible),whereas cefonicid and cefoxitin had little, if any,activity in vitro. Strains of Streptococcus faeca-lis, Pseudomonas spp., and Acinetobacter spp.were resistant to the four drugs. Streptococcuspyogenes, Streptococcus pneumoniae, andNeisseria meningitidis isolates were extremelysusceptible to cefonicid, cefamandole, and ceph-alothin.

,B-Lactamase hydrolysis rates. Table 2 containsthe results of the spectrophotometric ,-lacta-mase hydrolysis studies with seven P-lactamasepreparations against eight drugs representing allthree generations of cephalosporins. Cefonicidwas determined most comparable to ceforanideand cefazolin in stability to most enzyme types.Cefoperazone, cefoxitin, and cefotaxime weregenerally more resistant to these P-lactamases.Cefonicid was very stable (RHR = 0.7%) to type

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CEFONICID DISK EVALUATION 235

TABLE 2. 3-Lactamase hydrolysis rates of cefonicid compared with those of five other cephemsNitrocefin RHR compared with cephaloridine

Omctminb RHRC Cefonicid Ceforanide Cefoperazone Cefoxitin Cefotaxime Cefazolin

Bacillus cereus (NT)d 8.5 159 3.6 0.9 8.9 0.3 0.3 6.9Enterobacter cloacae P99 (Ia) 16.0 260 0.7 19 0.8 0.4 0.9 49Escherichia coli TEM1 (Illa) 2.2 285 32 12 72 4.2 2.6 23Escherichia coli TEM2 (IlIb) 3.8 269 32 10 81 1.0 <1.0 25Klebsiella oxytoca Kl (IVc) 30.3 129 183 56 2.2 0.4 3.8 102Klebsiella pneumoniae (V) 1.7 519 29 19 24 <1.0 <1.0 NDePseudomonas aeruginosa (II) 1.9 578 19 ND <1.0 6.2 5.8 30

a Enzymes were classified by the schema of Richmond and Sykes (19).b ,uM/min per ml of partially purified P-lactamase preparation.c RHR = relative hydrolysis rate compared with that of cephaloridine ,-lactamase expressed as a value of

100%. The P-lactamase hydrolysis was determined by the UV spectrophotometric method using 260 to 482 nm at37°C. Reaction mixtures were at a volume of 1.0 ml of 1i-' M cephalosporin substrate in 0.05 M (pH 7)phosphate buffer.

d NT, Not typed (commercial penicillinase from B. cereus).e ND, Not determined.

I ,3-lactamases such as those found in Entero-bacter spp. This latter stability was similar tothat of third-generation cephalosporins. Com-monly found TEM plasmid-mediated P-lacta-mases hydrolyzed second-generation drugs atvery low rates, e.g., 1.0 to 32%. Cefonicid wasmost susceptible to the Klebsiella oxytoca Kl 1B-lactamase.

Inhibition of P-lactamases. The effects of fourconcentrations of cefonicid upon the 13-lacta-mase hydrolysis of enzyme-labile nitrocefin areshown in Fig. 2. Only type Ta (P99) ,B-lactamasewas significantly inhibited at cefonicid concen-trations below that of substrate. Nearly 80%

100\lI,V

III a80 III b

NTIV

60

~40

% of Substrate Conc.FIG. 2. Inhibition of seven beta-lactamases by ce-

fonicid used as an enzyme inhibitor. Labile substratewas nitrocefin at a concentration of 10-4 M.

inhibition was produced by a cefonicid concen-tration of 2 x 10-5 M (substrate, 1.0 x 10-4 M).Only trace enzyme inhibition was detected for ,B-lactamase types IlIa, IIIb, and IVc and for theuntyped B. cereus penicillinase at cefonicid lev-els twofold higher than the substrate.

Bacterial cell permeability. By using an E. colimodel developed by Richmond et al. (18), theability of ,B-lactam substances to gain access tothe bacterial periplasmic space can be evaluat-ed. Cefonicid has a permeability index of 1.3,slightly less than that of cephaloridine (1.0 in-dex) and comparable to cefazolin (1.4 index).This index was closest to that of cefoxitin (1.1index) among the second-generation cephems,but superior to cefamandole (5.0 index). Thecefonicid MIC for the two test strains was 0.25to 0.33 ,ug/ml in contrast to 0.25 to 1.2 ,ug/ml forcefamandole.

Cefonicid disk tests. Data correlating zonediameters with MIC values were accumulatedfor tests with 30-,ug cefonicid, cefamandole, andcephalothin disks. Regression formulae whichmathematically express the relationships arepresented in Table 3. All three drugs give similarregression lines (Fig. 3). Examination of the datasuggested a parabolic relationship for all threedrugs. To avoid the parabolic portion of theregression line, data with strains giving MICs of<2.0 ,ug/ml were excluded, and new regressionlines were calculated (Table 3 and line A in Fig.3). Zone size breakpoints for the resistant cate-gory would be <14 mm for all three drugs,regardless of the regression line utilized; i.e., thetwo lines intersect at or near the 14-mm break-point. A susceptible breakpoint of 218 mm isappropriate if the limited-range regression line isutilized.The interpretive errors that were encountered

when breakpoints of <14 and .18 mm were

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236 BARRY, JONES, AND THORNSBERRY

TABLE 3. Summary of regression statistics comparing zone diameters (30-1Lg disks) with microdilution MICvalues (jjg/ml) and interpretive discrepancies between the two types of in vitro tests

Antimicrobial agents compared All MICs >0.25 and -64 Fjg/ml Excluding MICs|2.0 or >64Interpretive agreement'~Lg/ml

No.ofCrRegression _ Regression % Discrepancies (%)

Disk tet MICs No. of orrelatioi No. o CorrelationDISk test MI s testsb coefficient Inter- Sb testsb coefficient Inter-Slop Agree- Verycet lp etlpe ment Minor Major mjry

Cefonicid Cefonicid 191 0.81 17.7 -0.32 88 0.69 16.4 -0.23 94.7 5.0 0 0.2dCefamandole Cefamandole 195 0.86 18.4 -0.35 102 0.86 17.4 -0.26 95.7 4.2 0 0Cephalothin Cephalothin 162 0.87 18.4 -0.32 121 0.70 17.2 -0.25 90.4 5.6 0 3*9e

Cefamandole Cefonicid 178 0.63 15.8 -0.21 83 0.74 16.8 -0.21 93.3 4.7 0 2.GfCefonicid Cefamandole 219 0.73 14.8 -0.24 48 0.82 16.2 -0.23 93.6 5.0 1.5g 0

a For all three drugs, susceptible = MIC of <8.0 ,ug/ml or zones of -18 mm, intermediate = MIC of 16 ,ug/mlor zones of 15 to 17 mm, and resistant = MIC >16 ,ug/ml or zones of <14 mm. Minor discrepancies =intermediate with one test but susceptible or resistant by the other, major discrepancies = resistant by the disktest but susceptible by MICs, and very major discrepancies = susceptible by the disk test but resistant by MICs.Data are expressed as the percentage of 422 strains tested.

b Number of tests included in regression analysis.I MIC expressed as log2 + 9 (pgIml).d Represents one E. cloacae.I Represents 15 Enterobacteriaceae, 2 methicillin-resistant S. aureus, and 1 C. faecalis.f Represents six methicillin-resistant S. aureus, one S. faecilis, and one C. freundii.8 Represents six methicillin-resistant S. aureus.

utilized are also listed in Table 3. Completeinterpretive agreement was observed with 90.4%of the strains (cephalothin) to 95.7% of thestrains (cefamandole). Minor discrepancies wereobserved with 4.2 to 5.6% of the strains. Onlyone Enterobacter cloacae was susceptible tocefonicid by disk tests, but resistant by MICs.No such very major errors were seen withcefamandole, but 18 (3.9%) strains were cepha-lothin susceptible by the disk test, but resistantby MICs. These data confirm the acceptableaccuracy of zone size breakpoints that havebeen previously applied to all three drugs, i.e.,<14 mm for resistant (MIC, >16 ,ug/ml) and .18mm for susceptible (MIC, -8.0 ,ug/ml).

Direct comparison of MICs. Because we wereinterested in determining whether susceptibilityor resistance to one drug can be predicted fromthe results of tests with another related drug, wefirst compared MIC values for cefamandole,cefoxitin, and cephalothin with those obtainedwith cefonicid. Table 4 presents the results ofregression analysis of such data. Near perfectcorrelation (r = 0.94) was observed betweencefamandole MICs and cefonicid MICs. Cepha-lothin and cefoxitin MICs, on the other hand,did not correlate as well with cefonicid MICs;i.e., correlation coefficients were 0.78 and 0.82,respectively. Similar analysis of disk test dataalso revealed a very acceptable correlation be-tween cefamandole and cefonicid zone diame-ters, i.e., correlation coefficient, 0.94; intercept,-0.79 mm; and slope, +0.93.

Interpretive categories (susceptible, interme-diate, and resistant) are also compared in Table

4. Complete interpretive agreement betweencefamandole and cefonicid was seen with 96% ofthe strains tested. Nine minor discrepancies (allintermediate to cefamandole, but resistant tocefonicid) included six S. faecalis isolates. Sev-en major discrepancies (susceptible to cefaman-dole, but resistant to cefonicid) included sixmethicillin-resistant S. aureus which are bestconsidered resistant to all cephalosporins. Whencefoxitin was compared with cefonicid, 48strains showed major discrepancies and 83.5%were in agreement. Cephalothin showed an86.9%o interpretive agreement with cefonicid,with 38 major discrepancies, 35 of which wereresistant to cephalothin but susceptible to cefo-nicid. Three methicillin-resistant S. aureus ap-peared to be susceptible to cephalothin, butwere clearly resistant to cefonicid. We conclud-ed that for the purposes of in vitro testing,cefamandole and cefonicid are essentially com-parable, especially if one excludes tests withenterococci and methicillin-resistant S. aureus.Both of the latter microorganisms are usuallyresistant to cefonicid, but may appear to besusceptible to cefamandole: clinically, neitherdrug is indicated for treating infections due tosuch microorganisms.Cefamandole or cefonicid disks. Zone diame-

ters obtained with the cefamandole disk testwere compared with cefonicid MICs, and zoneswith cefonicid disk tests were compared withcefamandole MICs, and the resulting data weresubjected to regression analysis (Table 2). Limit-ed-range regression analysis provided slightlydiminished correlation coefficients, but the lines

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CEFONICID DISK EVALUATION 237

Eh-..

4..W

0

*0

0

.0

C

0

64

16.

4.0-

1.0.0.25.

64

16-

4.0

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64-

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....... ...... .... ...

........ ...... . .. . .

Cefonicid:I~ ~ n

.: .. .. .. .. .. ..

Cefamandole\:: N~B

... .. . . . . . . .

. . RACephalothin\

B

6 10 14 1820 i5 30 35Zone Diameter (mm)

FIG. 3. Regression lines correlating MIC valueswith zone diameters with 30-p.g cephalothin, cefaman-dole, and cefonicid disks. Line B includes all on-scaledata, whereas line A excludes all strains with MICs of<2.0 ,ug/ml. Broken lines represent zone size break-points (14 and 18 mm) and MIC correlates evaluatedfrom regression line A.

were still similar in intercepts and slopes. Over-all interpretive agreement was excellent; mostmajor or very major discrepancies involved testswith methicillin-resistant S. aureus. If the latterare assumed to be clinically resistant to thecephalosporins, cefonicid disks or MICs provid-ed the correct in vitro response, and tests withcefamandole were generally in error. Intermedi-ate (equivocal) disk tests were observed 19 timeswith cefamandole disks and 23 times with cefo-nicid disks: the majority of strains producingsuch results were resistant to both drugs bymicrodilution tests. Most strains with intermedi-ate zone sizes represented species such as En-terobacter spp. that typically produce inducible,B-lactamases which may inactivate the twodrugs. It is difficult to decide whether suchstrains are truly resistant, but experience withcefamandole (8, 20) suggests that some treat-ment failures might be anticipated. We did notattempt to determine the number of strains withinducible P-lactamases that were categorized asbeing susceptible with the two disks, but use ofthe less active cefonicid disks should minimizesuch errors.

Quality control study. Table 5 shows the re-

sults of the nine-laboratory study of the 30-,ugcefonicid disks using the NCCLS M2-A2 meth-od (15). The study design has been reportedearlier by Gavan et al. (9) for studies of semisyn-thetic penicillins. Two lots of cefonicid diskswere used at each center to obtain 988 total zonediameters for each quality control organism,including 90 zones from the common agar lot.No significant differences were found betweenthe two lots of disks, and analysis of the zoneresults from a lot of agar, common to each

TABLE 4. Direct comparison of cefamandole, cefoxitin, and cephalothin MICs with cefonicid MICs with 422clinical isolates

Antimicrobial Regression analysis Interpretive agreementaagent compared Correlation % No. discrepantwith cefonicid coefficient Intercept Slope Agreemento. d a

Agreement Minor MajorCefamandole 0.94 1.23 0.94 96.0 gb 7CCefoxitin 0.82 -0.68 1.03 83.5 21 48dCephalothin 0.78 1.91 0.77 86.9 17 38ea For all four drugs, susceptible = MIC c8 ,ug/ml, intermediate = MIC 16 ,ug/ml, and resistant = MIC >16 ,ug/

ml. Major discrepancies = resistant to one drug, but susceptible to the other drug, and minor discrepanciesintermediate to one drug, resistant or susceptible to the other drug.

b Includes six S. faecalis, one Pseudomonas stutzeri, one K. pneumoniae, and one E. coli, all intermediate tocefamandole, but resistant to cefonicid.

c Included all six methicillin-resistant S. aureus and one C. freundii, all susceptible to cefamandole, butresistant to cefonicid.

d Includes 16 Proteus-Providencia spp., 15 Enterobacter spp., 5 Citrobacter spp., 4 E. coli, 2 K. penumoniae,2 S. marcesens, 2 Pseudomonas spp., 1 Acinetobacter sp., and 1 S. faecalis; 24 of 48 were susceptible tocefoxitin, resistant to cefonicid.

e Included 17 Enterobacter spp., 9 Proteus-Providencia spp., 6 Citrobacter spp., 2 E. coli, 1 K. pneumoniae,and 3 methicillin-resistant S. aureus; the latter appeared to be susceptible to cephalothin, but resistant tocefonicid; all others were resistant to cephalothin, but susceptible to cefonicid.

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238 BARRY, JONES, AND THORNSBERRY

TABLE 5. Recommended individual daily quality control limits and accuracy control parameters for the 30-,ug cefonicid disks and the two applicable NCCLS quality control organisms

Precision control rangebIndividual daily Accuracy control zone of five values

Organism Mean Median test control dcam (mm)"zi

zone diam (mm) diam (mm)" Maximum Avg(mm) (mm)

E. coli (ATCC 25922) 27.3 27 25-29 (24-31)d 25.7-28.4 (25.2-29.8) 5 (8) 2.6 (4.1)S. aureus (ATCC 25923) 24.7 25 22-28 (22-27) 23.0-27.0 (22.8-26.2) 7 (6) 3.5 (2.9)

' Mean of five values.b Maximum value minus minimum value obtained in a series of five consecutive tests should not exceed the

listed maximum limits; the mean should fall within the range under accuracy control.' In continuing series of ranges from consecutive groups of five tests each, the average range should

approximate the listed value.d Range as determined by the method described by Gaven et al. (9), with the second range calculated from the

mean + 2 standard deviations method given in parentheses.

participant, showed no evidence of significanttechnical variance. The individual daily test con-

trol zones (a range) were calculated by themethod of Gavan et al. (9) and by anothercommonly utilized procedure, mean ± 2 stan-dard deviations. The distribution of zones re-

ported for E. coli (ATCC 25922) was not normal-ly distributed, and the median statistic (9) seemspreferred, but the S. alureus (ATCC 25923) zone

population is essentially normal. Of all of thezones reported for E. coli, 93.3 and 98.3% wouldhave been within the control ranges calculatedby the median statistic and by the mean + 2standard deviations methods, respectively. TheS. aureus zones were 98.9 to 99.2% within eithercontrol range.

DISCUSSION

For all drugs included in this report, we haveapplied the same MIC breakpoints, i.e., <8.0pLg/ml for susceptible and >16 .Lg/ml for resist-ant. Those breakpoints are tentative and may bechanged if justified by further pharmacologicalor clinical experience with the new cephems.We also assume that routine testing of gram-

positive cocci with newer cephalosporins is notnecessary; such isolates were included in thecurrent study only for comparative purposes.Although non-enterococcal streptococci are pre-

dictably susceptible to the cephalosporins, theenterococci are considered resistant. Due totechnical problems in doing the in vitro tests,some enterococci might appear to be susceptibleto cefamandole or cephalothin. Methicillin-sus-ceptible S. aureus strains are also predictablysusceptible to the cephalosporins. Methicillin-resistant strains of S. aureus are relatively re-

sistant (3) and should be considered clinicallyresistant to all cephalosporins (1). In spite of thefact that some strains may have cefamandole or

cephalothin MICs of .8 ,ug/ml (susceptible), thelower MICs are not consistent or repeatable andwill be much higher if incubation time is pro-

longed, if the inoculum is increased, or if thetests are performed in a hypertonic medium (3).For these reasons, the practice of routinelytesting gram-positive cocci against cephalospo-rins is unlikely to yield unpredictable results, yetmight produce misleading information.

Against gram-negative bacilli, cefonicid andcefamandole demonstrated a similar spectrum ofactivity. The two were also nearly equal insusceptibility to the hydrolysis of most 3-lacta-mases, and both significantly inhibited (noncom-petitive) type I r-lactamases (14). The data ofMehta et al. (14) are supported by the resultspresented in Table 2 and Fig. 2.

Cefoxitin, cephalothin, and the third-genera-tion compounds tested in this study were signifi-cantly different. The RHR presented for cefo-perazone against E. coli TEM P-lactamaseswould be very similar to that of cefamandole,e.g., two to five times less stable than cefonicidor cefazolin (11, 12, 14).

Permeability studies showed that cefonicidreadily penetrates enteric bacilli, thus achievingaccess to the penicillin-binding-protein targetsites. This ability to enter bacteria is superior tothat reported for cefamandole and cefuroxime(18).The cefonicid 30-pLg disk quality control trial

produced consistent performance by all labora-tories participating. For this drug we prefer theranges and control parameters derived from themethod of Gavan et al. (9). These recommendedranges would predict that >90% of zone diame-ters would be routinely "in control." These willremain tentative pending official publication bythe NCCLS, adoption by the Food and DrugAdministration, and a review of the statisticsaccumulated by the College of American Pathol-ogists' programs after the release of cefonicid.The data in this report support recommenda-

tions of the following interpretive criteria fortests with 30-.ag cefonicid disks: .14 mm forresistant (MIC, >16 pLg/ml), 15 to 17 mm for

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CEFONICID DISK EVALUATION 239

intermediate (MIC = 16 ,ug/ml) and .18 mm forsusceptible (MIC, '8 p.g/ml). The same inter-pretive standards are also appropriate for testswith cefamandole and cephalothin disks (4, 6,15). The predictive value of all three disk testswas very acceptable, particularly if data withmethicillin-resistant strains of S. aureus andwith S. faecalis are first excluded because suchtests are deemed inappropriate.

Direct comparison of cefonicid MIC or zonesize data with cefamandole MICs or zone sizesconfirms the possibility of using the two disksinterchangeably. Because cefonicid is slightlyless active than cefamandole against those spe-cies likely to contribute the very major interpre-tive errors (Enterobacter spp., etc.), cefoniciddisks may be the best representative for thosesecond-generation cephalosporins tested here.That would minimize the number of potentiallyserious false-susceptible disk test results (8, 20).The possibility of using other second-generationcephalosporins to represent this spectrum groupof cephalosporins shall be considered separately(A. L. Barry, R. N. Jones, and C. Thornsberry,Am. J. Clin. Pathol., in press).

LITERATURE CITED

1. Acar, J. F., P. Courvalin, and Y. A. Chabbert. 1971.Methicillin-resistant staphylococci: bacteriological failureof treatment with cephalosporins. Antimicrob. AgentsChemother. 1970:280-285.

2. Actor, P., J. J. Uri, I. Aajac, J. R. Guarini, L. Phillips,D. H. Pitkin, D. A. Berges, G. L. Dunn, J. R. E. Hoover,and J. A. Weisbach. 1978. SK and F 75073, new parenteralbroad-spectrum cephalosporin with high and prolongedserum level. Antimicrob. Agents Chemother. 13:784-790.

3. Barry, A. L., and R. E. Badal. 1977. Reliability of themicrodilution technic for detection of methicillin-resistantStaphylococcus aureus. Am. J. Clin. Pathol. 67:489-495.

4. Barry, A. L., F. D. Schoenknecht, S. Shadomy, J. C.Sherris, C. Thornsberry, J. A. Washington II, and R. B.Kammer. 1979. Interpretive criteria for cefamandole andcephalothin disk diffusion susceptibility tests. Antimi-crob. Agents Chemother. 15:140-141.

5. Barry, A. L., C. Thornsberry, R. N. Jones, P. C. Fuchs,T. L. Gavan, and E. H. Gerlach. 1977. Cefuroxime, an invitro comparison to six other cephalosporins. Proc. R.Soc. Med. 70(Suppl. 9):63-70.

6. Barry, A. L., C. Thornsberry, R. N. Jones, P. C. Fuchs,T. L. Gavan, and E. H. Gerlach. 1978. Reassessment ofthe "class" concept of disc susceptibility testing: cephalo-thin disc versus mics with eleven cephalosporins. Am. J.Clin. Pathol. 70:909-913.

7. Bauer, A. W., W. M. M. Kirby, J. C. Sherris, and M.Turck. 1966. Antibiotic susceptibility testing by a stan-

dardized single disk method. Am. J. Clin. Pathol. 45:493-496.

8. Beckwith, D. G., and J. A. Jahre. 1980. Role of cefoxitin-inducible beta-lactamase in a case of breakthrough bacter-emia. J. Clin. Microbiol. 12:517-520.

9. Gavan, T. L., R. N. Jones, A. L. Barry, P. C. Fuchs,E. H. Gerlach, J. M. Matsen, L. B. Relier, C. Thorns-berry, and L. D. Thrupp. 1981. Quality control limits forampicillin, carbenicillin, mezlocillin, and piperacillin diskdiffusion susceptibility tests: a collaborative study. J.Clin. Microbiol. 14:67-72.

10. Grappel, S. F., F. R. Guarini, P. Actor, and J. A. Weis-bach. 1980. Correlation of agar disk diffusion tests withminimum inhibitory concentrations of cefonicid (SK andF 75073) and cephalothin. J. Antibiot. 33:85-87.

11. Jones, R. N., A. L. Barry, C. Thornsberry, E. H. Gerlach,P. C. Fuchs, T. L. Gavan, and H. M. Sommers. 1981.Ceftazidime (GR20263), a pseudomonas-active cephalo-sporin: in vitro antimicrobial activity evaluation includingrecommendations for disk diffusion susceptibility tests. J.Antimicrob. Chemother. 8(Suppl. B):187-211.

12. Jones, R. N., A. L. Barry, C. Thornsberry, and H. W.Wilson. 1981. In vitro antimicrobial activity evaluation ofcefodizime (HR221), a new semisynthetic cephalosporin.Antimicrob. Agents Chemother. 20:760-768.

13. Jones, R. N., H. W. Wilson, and W. J. Novick, Jr. 1982.In vitro evaluation of pyridine-2-azo-p-dimethylanalinecephalosporin, a new diagnostic chromogenic reagent,and comparison with nitrocefin, cephacetrile, and otherbeta-lactam compounds. J. Clin. Microbiol. 15:677-683.

14. Mebta, R. J., D. J. Newman, B. A. Bowie, C. H. Nash Ill,and P. Actor. 1981. Cefonicid: a stable beta-lactamaseinhibitor. J. Antibiot. 34:202-205.

15. National Committee for Clinical Laboratory Standards.1979, 1981, and 1982. Performance standards for antimi-crobic disc susceptibility tests. Approved standard M2-A2with supplements. National Committee for Clinical Labo-ratory Standards, Villanova, Pa.

16. National Committee for Clinical Laboratory Standards.1982. Standard methods for dilution antimicrobial suscep-tibility tests for bacteria which grow aerobically. Tenta-tive standard M7-T. National Committee for ClinicalLaboratory Standards, Villanova, Pa.

17. Pitkin, D. H., P. Actor, F. Alexander, J. Dubb, R. Stote,and J. A. Weisbach. 1980. Cefonicid (SK and F 75073)serum levels and urinary recovery after intramuscular andintravenous administration, p. 252-254. In J. D. Nelsonand C. Grassi (ed.), Current chemotherapy and infectiousdisease. Proceedings of the 11th ICC and the 19thICAAC. American Society for Microbiology, Washing-ton, D.C.

18. Richmond, M. H., D. C. Clark, and S. Wotton. 1976.Indirect method for assessing the penetration of beta-lactamase nonsusceptible penicillins and cephalosporinsin Escherichia coli strains. Antimicrob. Agents Chemo-ther. 10:215-218.

19. Richmond, M. H., and R. B. Sykes. 1973. The beta-lactamases of gram-negative bacteria and their possiblephysiological role. Textbook Adv. Enzymol. 9:31-88.

20. Sanders, C. C., and W. E. Sanders. 1979. Emergence ofresistance to cefamandole: possible role of cefoxitin-inducible beta-lactamases. Antimicrob. Agents Chemo-ther. 15:792-797.

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