In Vitro Studies with Cefotaxime: Disk Diffusion Susceptibility Tests

0095-1137/82/080213-05$02.00/0

In

Vitro

Studies with Cefotaxime:

Disk

Diffusion

Susceptibility Tests

SMITH SHADOMY* ANDEDWARD L. CHAN

Departmentof Medicine, Medical College of Virginia, Virginia Commonwealth University, Richmond,

Virginia23298

Received 22February 1982/Accepted 29April1982

Until recently two sets ofconflicting interpretive criteria existed foruse with thestandardized 30-,ug cefotaxime diffusion disk [National Committee for Clinical Laboratory Standards (NCCLS) M2-A2S, supplement 1; product information packageinsertfor Claforan (cefotaxime sodium),edition

10/81.]

The lattercriteria recently weresuperseded byathirdsetwhich differsradically fromthefirsttwoin both minimalinhibitoryconcentration (MIC)andzonesizebreakpoints. The first two sets of criteria differed mainly in the zone of inhibition diameters used to predict resistantandintermediate organisms. Theaccuracies of thesetwosetsof criteria were evaluated by paired agar dilution (World Health Organization-International Collaborative Study) and disk diffusion tests (Food and Drug Administration) with 347 clinical isolates of aerobic bacteria. A total of 274 isolates (79%) wereclinically susceptible by agardilutionasdefined by NCCLS (MIC

.8

,ug/ml) and 61, including 48 Pseudomonas spp., were of intermediate susceptibility (MIC of16or32

p,g/ml).

Twelve were resistant(MIC .64 ,ug/ml). The original product information package insert criteria proved to be most unreliable in identification of the intermediate organisms (zone of inhibition diameters of18 to 22mm);only5werecorrectlypredictedasbeing intermediate, whereas 54 were predicted as being resistant, and 2 were predicted as being susceptible. In contrast, the NCCLS criteria(zone of inhibition diameter of 15to 22mm)predicted41 asbeing intermediateand 18asbeing resistant; again,2were susceptible. The 12isolatesresistanttocefotaximebyagardilutionwerecorrectly predictedtobe resistant byeithersetofbreakpoints(zoneof inhibitiondiameter of .14 or .17 mm). These results suggest that the package insert criteria previously recommended for use with the 30-,ug cefotaxime disk were overly conservative and required revision to bring them into agreement with NCCLS performance standards. Unfortunately however, therecent revision of the pack-age insert criteria has furtherconfused this issue [product information package insert forClaforan (cefotaxime sodium), edition 2/82].

Cefotaxime (HR 756; CTX) is a broad-spec-trumcephalosporin antibiotic possessing ahigh degree ofresistanceto ,-lactamases (4,7). Itis intended for parenteraluse in treatmentof seri-ousinfectionscaused bysusceptible organisms, both gram-negative and gram-positive. The in vitro spectrum of CTXincludesmanyorganisms resistanttoothercephalosporinsandpenicillins (9). Because ofthis extended spectrum, a sepa-rate30-,ug CTX disk has beendeveloped for use in disk diffusion susceptibility tests. Until re-cently, two sets of interpretive "breakpoint" criteria had been promulgated for use with the 30-,ug CTX disk. One set comprises those in-cluded in thestandardsrecently proposedby the National Committee for Clinical Laboratory Standards (NCCLS; 8). These standards agree with and appear to be derived from those pro-posed by Fuchs et al. (3). The second set of

criteriacomprises thosepreviously containedin theproduct information package insert for Cla-foran (CTXsodium; 5). These two setsof crite-ria differed primarily in the zone ofinhibition diameters usedtodefine intermediate and resist-antorganisms (Table1). Intermsof correspond-ing minimal inhibitory concentration (MIC) val-ues,bothsetsofcriteriaregarded 8,ug/mlasthe upper limitof probable clinical susceptibility. In termsof clinicalresistance, NCCLS defines this as an MIC

.64

p.g/ml,

whereas the package insert usedanMICof >16

jig/ml.

Left undefined bythesecriteria is the intermediate, or indeter-minate, category as suggested by Fuchs et al. (3),which, by default, appearstobe 16or32,ug/ ml. The study reported here had two specific goals: evaluation of the performance character-istics of the 30-,ig CTX disk and evaluation of the reliability ofeachof thetwo sets of criteria

213

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TABLE 1. Interpretive criteria for use in disk

diffusion susceptibility tests with the

30-Rg

CTX disk

Zonediam (mm)breakpoints

Category Claforan NCCLS

packageinsert interpretive criteria(10/81 standards

edition)

Susceptible .23 (.8)" 23 (s8)

Intermediate 18-22 15-22

Resistant .17(>16) <14(.64)

aCorrespondingMIC

(pg/ml).

previously promulgated forusein interpretation of resultsobtained with this diskaswellasthose now contained in the revised Claforan product

information package insert (6).

MATERIALS AND METHODS

Organisms. A total of 347 isolates of gram-negative

andgram-positive aerobic bacteriaweretested. These

included 78 organisms collected from several different

medical centers inthis country, as wellas a diverse

population of gram-negative organisms of Asiatic

ori-gin. Identifications of all isolates were confirmed by

eitheraccepted laboratory criteria (2, 7) or the API

20E system (Analytab Products) as required. Stock

cultureswere maintained incystine Trypticase (BBL

Microbiology Systems)agarmedium (CTA medium).

Susceptibility tests. Paired agar dilution and disk

diffusion tests were performed with all 347 isolates.

The agar dilution tests employed the World Health

Organization-InternationalCollaborativeStudy

proce-dureasdescribedby Washingtonand Sutter(10);final

testconcentrationsof CTX(HR 756,Hoechst-Roussel

Pharmaceuticals, Inc.;lot17011) rangedfrom 0.13 to

64 ,ug/ml. Themedium employed intheagardilution

tests was a commercial dehydrated Mueller-Hinton

agar (MH; BBL). The disk diffusion tests were

per-formed by thestandardized Food and Drug

Adminis-tration procedureas described by Barryand

Thorns-berry (1). A standardized 30-p.g CTX disk(BBL; lot

104026, assayed contentof 30 pg) and commercially

prepared 150-mm-diameter MH plates (MH-11; BBL)

wereused in the diskdiffusiontests.

Inocula for the disk diffusion tests were prepared

from overnight broth cultures and then adjusted to

match the turbidity of a 0.5 MacFarland standard.

Inocula for the agar dilution tests were prepared to

contain approximately 2 x 106colony-forming units

permlor5 x 103colony-forming units perspot. Both

agar dilution and disk diffusion tests then were

per-formed.Inoculatedplateswereincubatedovernightat

35°C. The MIC was defined as being the lowest

concentration ofdrug yielding no more than one or

two colonies; barely discernable or hazy responses

wereignored. Zones of inhibition in the disk diffusion

testsweremeasured intriplicatetothenearest0.1mm

andaveraged.

Dataanalyses.Originaldatawereevaluatedinterms

of numbers oforganisms inhibited ateachgiven

con-centration of CTX and their corresponding zones of

inhibition. Allmeasurablezonesofinhibition foreach

given concentration were then averaged. Results of

the paired tests then were analyzed in terms of each

testeddrug concentration (log2 value) and the

corre-sponding average zone of inhibition using standard

regression analysis (least-squares method). Statistics

from this analysis were then used to derive disk

diffusion zone size correlates for MIC values of 8

(susceptible)and 64 (resistant)p.g/ml.Finally,

individ-ualpaired zone size andMIC responses were analyzed

in terms of agreement between actualMIC values and

susceptibility categories as predicted by the

corre-sponding zone size responses. Three sets of criteria

were used in this analysis: the two previously pub-lished sets of zone size criteria described above (5, 8)

andthe zone size criteria now contained in the current

edition of the Claforan product information package

insert (6).

RESULTS

In vitro susceptibility data. A total of 274 isolateshadCTX MIC values of 8

jig/ml

or less. Thus, by NCCLS MIC standards, these were susceptible organisms (Table 2). A total of 61 isolates had MIC values of 16 or 32 pLg/ml and thuswereof intermediate orindeterminant sus-ceptibility.Ten isolates wereclinically resistant, with MIC values of 64 pLg/ml, and 2 more were totally resistant (MIC >64 ptg/ml). Isolates of Pseudomonas spp. accounted for 68 of the 73 organisms inthe latter two categories.

Regression analysis.The average zonesof inhi-bition for all isolatesinhibitedateachgivendrug concentration were calculated (Table 2). Data for several isolates were rejected from these calculations becauseof either apparent noncon-gruency, two values, or nonmeasurable values (nomeasurablezoneof inhibitionorMIC values of >64 ,ug/ml, seven values). Regression analy-siswasthenperformed, matchingthelog2 value of each concentration against the average zone

TABLE 2. Invitro susceptibilitytoCTX as

determinedby pairedagardilutionand diskdiffusion

tests

Drug No. Avg zone of Excluded

(pg/mi) inhibited inhibition data"

0.13 204 31.10 2

0.25 19 30.05 0

0.5 21 28.83 0

1 6 28.52 0

2 6 26.35 0

4 7 26.55 0

8 11 20.49 1

16 45 16.00 1

32 16 14.61 1

64 10 10.96 2

>64 2 6.9 2

a Data rejected from further regression analysis:

contamination, two values; no measurable zones or

MICs >64,g/ml, sevenvalues.

MICROBIOL.

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ofinhibition for that concentration. Results of this analysis indicated excellent correlation (r = 0.9551,P< 0.001; Fig. 1). Regression statistics a(26.78) and r(-2.2872) then were used to cal-culate disk diffusion zone size correlates for MIC values of 8

(Y,

= 19.92 mm) and 64(YI. = 13.06 mm) p.g/ml.Thesevalues were rounded to 20and 14 mm, respectively, leaving an interme-diate region of15 to19 mm.

Analysis of disk diffusion test results. Paired MIC and disk diffusion test data were analyzed for all 347 isolates. According to MIC data, using NCCLS standards, there were three groupsof organisms:resistant(MIC.64

,ug/ml),

12; intermediate (MIC of16 or 32 ,ug/ml), 61; susceptible(MIC s8,ug/ml), 274.Application of the zone size breakpoints generated by these samedatacorrectlypredicted the probable cate-gory of susceptibility for 315 isolates (91%). Incorrect predictions involved 22 isolates that were intermediate by MIC and 10 isolates that weresusceptible by MIC. Of the 22 intermediate isolates,4 werepredicted to be susceptible, and 18 were predicted to be resistant. Of the 10 isolatessusceptibleby MIC, 3 werepredictedto beresistant,and 7 werepredicted to be interme-diate.

Results similar to thoseabove were obtained when the NCCLS interpretive zone size stan-dards were applied (Table 3). Both sets of data werein agreement for 309(89%) isolates. There were38 instancesof disagreement between

mea-27

-6

-E

11- 5

-4

-0

2

3(8)-2 2

-<1I

O O

( -I_

-2

-3i

sured MICvaluesandinterpretationofzonesize responses. The isolates for which data were inconsistent involved 20 intermediate by MIC and 18 susceptible by MIC. Of the intermediate organisms, two were predicted by the disk test asbeing susceptible, and 18 were predicted as being resistant. The latter were the same orga-nisms predicted as being resistant with the re-gression statistics correlates. Two isolates each ofPseudomonas spp. and Escherichia coli and one isolateeach of Enterobacter spp. and Pro-teusmirabilis,susceptible byMIC andpredicted as being susceptible by the regression statistic correlates, now wereplacedin the intermediate category.

Application oftheoriginal package insert cri-teria to the results of the disk diffusion tests resulted in 74 (21%) instances ofdisagreement betweenpairedMICand diskdiffusiontestdata. Three isolates of Pseudomonas susceptible by MICandpredictedasbeing intermediatebythe NCCLS standards now were predicted to be resistant. Moreimportantly, 38 isolates interme-diate by MIC nowwere placed in the resistant category. Only5

(8%)

ofthe 61 isolates interme-diatebyMICwerepredictedasbeing intermedi-atebythese criteria.

Recently and subsequent to the performance ofthese studies,thepackage insert criteriawere revised (6). The revision suggests use ofMIC values of -16 and

.64

jig/ml,

respectively, to define clinical susceptibility and resistance.

Or-I

411 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~1

3 2 2 4

2 2 1 3 3 12 3 12 2 2

1 12 1 1 1

1\ 1 2

I

1 2 3

1 1 51 1 32

1 4 I1 1 2

I I 1 2 1 2 1 6 13 1727 3634K 5 1 5

I I I I I I I I I 1 I I I I I 1 I I I I I I

slO

15 20 25 30 35 >38

ZONE OF INHIBITION (AVG. Z.D.,ea MIC),mm, 30-pg CTX DISC

FIG. 1. Correlation between CTX agar dilution MIC values and zones of inhibition obtained with a 30-,ug CTX diffusion disk. Numbers reflect distribution of individual zone size responses (rounded to nearest

millimeter). Z.D., Zonediameter.

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TABLE 3. Analysis of disk diffusion test results-correlation of MIC responses with resultsof diskdiffusion tests using three different sets of interpretive criteria

No.incategoryby:a

CTX MIC(p.g/ NCCLSzonesize standards with Original package insert criteria, Revised package insert criteria,

ml)(no. zonesizediam(mm)of: 10/81edition,withzonesize diam 2/82edition,withzonesize diam

inhibited)

Res. Intermed. Suscept. Res. Intermed. Suscept. Res. Intermed. Suscept.

.14 15-22 -23 .17 18-22 -23 .14 15-19 >20

264(12) 12 0 0 12 0 0 12 0 0

32(16) 7 9 0 16 0 0 7 9 0

16 (45) 11 32 2 38 5 2 11 30 4

8(11) 1 7 3 3 5 3 1 5 5

c4(263) 4 8 253 3 7 253 2 2 259

aRes, Resistant;

Intermed.,

intermediate;suscept., susceptible.

ganisms having MICs between 16 and 64 ,ug/ml are defined as being ". . . susceptible if high

dosage is used orif the infection is confined to

tissues ... in which high antibiotic levels are

attained (6)." The correspondingzone size

cor-relates are as follows: susceptible, .20 mm;

intermediate, 15 to 19 mm; resistant, <14 mm.

These new zone size and MIC criteria were appliedtothepreviously described data with the following results (Table 3). Of 274 organisms having MIC values of 8

jig/ml

or less, the new zone size correlates predicted 264 (96%) to be susceptible and 10to be resistantor

intermedi-ate. However, of 45 organisms having MIC values of 16 ,ug/ml and now considered to be susceptible by MIC, only4werepredictedtobe susceptible by the disk test, whereas 30 were

predicted to be intermediate, and 11 were

pre-dictedtobe resistant.Of the latter 41 organisms, 39wereisolates of Pseudomonas.

Mostinstances ofdisagreement between

mea-sured MIC values and categories of susceptibil-ity predicted by the 30-,ug CTX disk diffusion

testinvolved isolates of Pseudomonasspp. (Ta-ble 4). Paired data for 78 such isolates were

analyzed. Ten isolates were resistant by MIC;

all 10werecorrectly predictedasbeingresistant

by the NCCLS standards and bothsetsof

pack-ageinsert criteria. Tenweresusceptibleto8,ug/ mlorless; onlytwowerecorrectlypredictedas

being susceptible by either the NCCLS criteria

orthe original package insert criteria. The

origi-nal packageinsert criteria predicted resistant for two and intermediate for six of the remaining eight, whereas the NCCLS standards predicted intermediate for all eight. A total of 58

Pseudo-monasisolates had intermediate CTX MIC val-ues asdefinedby NCCLS, 15at32 p.g/ml and 43

at 16 ,ug/ml. Application of the NCCLS

stan-dardstothe diskdiffusiontestdata for these 58 isolates correctly predicted 40 (70%) as being

intermediate;twowerepredictedtobe suscepti-ble, and 16 were predicted to be resistant. In

contrast, when theoriginal package insert

crite-riawereapplied, 52 (90%) werepredictedto be resistant, with only 4asbeing intermediate and

twoasbeing susceptible. Only confusion result-ed when the revisresult-edpackage insert criteriawere

applied to the data for Pseudomonas spp. Ac-cording to the revised MIC criteria, 53 isolates could be considered tobesusceptible. The disk

test data predicted the following for these 53 isolates: susceptible, 8; intermediate, 34;

resist-ant, 11.Of 15 isolatesnowintermediateby MIC, the disktestdatapredicted 10tobe intermediate and 5 tobe resistant.

TABLE 4. Performance ofthe30-p.gCTXdisk indiskdiffusiontestswith 78isolates of Pseudomonas spp.

No. in categoryby:a

CTXMIC(,ug/ NCCLSzonesize standards with Original packageinsertcriteria, Revisedpackageinsertcriteria,

C

mlC(n.g/

NCCSzonesize

stan

s w 10/81edition,with zonesize diam 2/82 edition,with zonesizediam

m')(no. zonesize diam (mm) of: (mm) of: '(mm)of:

inhibited)

Res. Intermed. Suscept. Res. Intermed. Suscept. Res. Intermed. Suscept.

.14 15-22 .23 .17 18-22 .23 .14 15-19 >20

264(10) 10 0 0 10 0 0 10 0 0

32(15) 5 10 0 15 0 0 5 10 0

16(43) 11 30 2 37 4 2 11 28 4

8(8) 0 7 1 2 5 1 0 5 3

'4(2) 0 1 1 0 1 1 0 1 1

aSeeTable 3, footnoteaforabbreviations.

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DISCUSSION

Two conclusions can be drawn from the re-sults of this study.

First,

the zone size criteria previously recommended by the Claforan prod-uct information package insert (5) for use with the 30-,ug CTX disk diffusion test were overly conservative. This applied primarily to orga-nisms of intermediate orindeterminate suscepti-bility (MICs of 16 or 32

jig/ml).

Many such organisms will be predicted by disk diffusion testing to beresistant when these older criteria areapplied. Revision of the package insertzone size criteria, bringing them in line with current NCCLS standards for the 30-,ug CTX disk, would have been desirable. Unfortunately, the recentrevision ofthesecriterianowdiffers from NCCLS standards in both MIC and zone size criteria. Thisonlyaddsconfusion totheissue.

Second, diskdiffusion susceptibilitytestswith

the

30-,Ig

CTX disk and isolates of

Pseudomo-nas spp. appear to be a mostunreliable way of predicting probable clinical susceptibility or resistance regardless of which criteriaareused. More importantly, and as already cautioned by others (3),sufficient clinicalexperiencedoes not yetexisttopermit definition of absolute in vitro breakpointsforCTXandPseudomonasspp. For these reasons it isrecommended that results of disk diffusiontestswith the 30-,ugCTXdisknot be usedinmaking therapeutic decisions regard-ing Pseudomonas spp. and CTX.

LITERATURE CITED

1. Barry, A.L., and C. Thornsberry. 1980. Susceptibility testing: diffusion test procedures, p. 463-474.In E. H.

Lennette,A.Balows, W. J.Hausler, Jr.,andJ. P.Truant

(eds.), Manual of clinical microbiology, 3rd ed.American

Societyfor Microbiology, Washington, D.C.

2. R. R. Facklam. 1980.Streptococci andaerococci, p.

88-110. In E. H. Lennette, A. Balows,W.J. Hausler, Jr..

and J. P. Truant(eds.), Manual of clinical microbiology, 3rd ed. AmericanSociety forMicrobiology. Washington, D.C.

3. Fuchs, P. C., A. L. Barry, C. Thornsberry,R. N.Jones, T. L. Gavan, E. H. Gerlach, and H. M. Sommers. 1980. Cefotaxime: in vitro activity and tentative interpretive standards for disk susceptibility testing. Antimicrob. AgentsChemother. 18:88-93.

4. Heymes, R., A. Lutz,and E. Schrinner. 1978. Experimen-talevaluation of HR 756,anewcephalosporin derivative, p. 823-824. In W. Siegenthaler and R. Luthy (ed.), Currentchemotherapy. American Society for Microbiolo-gy, Washington, D.C.

5. Hoechst-Roussel Pharmaceuticals, Inc. 1981. Claforan (ce-fotaxime sodium) Sterile. Product information package insert, ed. 10/81. Hoechst-Roussel Pharmaceuticals,Inc.. Somerville, N.J.

6. Hoechst-Roussel Pharmaceuticals, Inc. 1982.Claforan (ce-fotaxime sodium) Sterile. Product Information package insert, ed. 2/82. Hoechst-Roussel Pharmaceuticals, Inc.. Somerville, N.J.

7. Kloos, W. E., and P. B. Smith. 1980. Staphylococci, p. 83-87. In E. H. Lennette, A. Balows, W. J.Hausler.Jr., andJ. P. Truant (ed.), Manual of clinical microbiology, 3rd ed. AmericanSociety for Microbiology, Washington, D.C.

8. National Committee for Clinical Laboratory Standards Subcommittee on Antimicrobial DiscSusceptibility Testing. 1981. Performance standards for antimicrobic disc sus-ceptibility tests, 2nd ed., first supplement (NCCLS stan-dardM2-A2). National Committee for Clinical Laboratory Standards Publication 1:141-156.

9. Peters, G., and G. Pulverer. 1980. Comparative in vitro activity of cefotaxime (HR 756). Chemotherapy 27:177-183.

10. Washington, J. A., and V. L. Sutter. 1980. Dilution sus-ceptibilitytest:agar and macro-broth dilutionprocedures. p.453-458. In E. H.Lennette, A. Balows, W. J.Hausler. Jr., and J.P. Truant(ed.), Manual of clinical microbiolo-gy, 3rd ed. American Society forMicrobiology, Washing-ton,D.C.

VOL. 16, 1982

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