Collaborative evaluation of the microbial profile system for quantitative antimicrobial susceptibility testing

0095-1137/83/030436-09$02.00/0

CopyrightC1983, American SocietyforMicrobiology

Collaborative

Evaluation of the Microbial Profile

System

for

Quantitative Antimicrobial Susceptibility

Testing

E. HUGH

GERLACH,`*

RONALD N.JONES,2 AND ARTHUR L. BARRY3

Department of Laboratories, St. FrancisHospital, Wichita,Kansas672141;Department of Pathology,Kaiser

FoundationRegional Laboratory, Clackamas, Oregon 9701S2; and Clinical Microbiology Laboratory, University of California, Davis, Sacramento, California 9S8173

Received 17March1982/Accepted 29 November 1982

This three-center collaborative studywasconducted to evaluatesamplesof the Microbial Profile System (MPS) antimicrobial microdilution panels [previously produced byMinnesotaMining& Manufacturing Co., (3M Co.), St. Paul,Minn. andcurrently produced by Flow Laboratories, Inc., Rockville, Md.). Thiswasa

three-phase study. In phase I, the inter- and intralaboratory agreement was

determinedby using strains with selectedrangesofsusceptibility. The MPS and reference microdilution minimum inhibitory concentrations were within accept-able variation, +1 dilution for 97.7% for the MPS and 98.8% for the reference microdilution panels for the intralaboratory comparisons. The percentage of strains with minimum inhibitory concentrations in the acceptable range for the interlaboratory variation was 96.2% for the MPS and 96.0% for the reference microdilution panels. The phase II studies used strains with known resistance

mechanisms.The percent agreement with these strains was:Enterobacteriaceae,

94.5%; nonenteric gram-negative rods, 95.4%; staphylococci, 92.3%; and strepto-cocci, 96.6%. The overall agreement within acceptable limits was 94.7% with these strains.Whentesting359 clinicalisolates,thefrequencyof strains within the acceptable range of agreement between the twomethods was97.3%. The MPS panelsgaveresultsin eachofthe threestudy phases equivalenttothose obtained with the reference microdilution panels.

The use ofa microdilution procedure forthe

quantitative determination of microbial suscepti-bilitytoantimicrobialagents hasbeenstudiedby

anumber ofinvestigators (5-8, 10). Theusageof this methodology has increased with the avail-ability of semiautomated equipment for dispens-ing the diluted antimicrobial agents in wells of plastictrays.The realincrease in the popularity of this methodology has undoubtedly been due

totheincreasing number of commercialvendors

of prepared microdilution trays. These trays contain diluted solutionsof antimicrobialagents in the frozen state. The comparatively recent entrance into the market of trays containing

different concentrations ofantimicrobial agents dried in the wells would provide improved as-pects of reproducibility, due in part to their increased shelf life (suggested by the manufac-turers to be >12 months). These commercially prepared frozen trays and dried-form products

havebeen evaluated in several studies and found to correlate well with results obtained either

with areference microdilution(RMD) method or with a standardized reference broth dilution method (2, 6, 9, 10-14). The Microbial Profile System (MPS; manufactured byMinnesota

Min-ing & ManufacturMin-ing Co. [3M Co.], St. Paul,

Minn.) is a microdilution tray containing dried reagentsforbiochemicalidentification and serial

concentrations of antimicrobial agents for

sus-ceptibility testing. We present a three-center

evaluation of the panel containing the dried

antimicrobialagents to determine the

compara-tiveaccuracyandreproducibility of these panels

fordetermining the minimuminhibitory

concen-trations (MICs) ofthe agents dried in the tray.

Standardized reference methods were utilized for these comparisons(12).

MATERIALS AND METHODS

Test strains. In the phase I studies, 26 bacterial isolatesweretestedbythe two methods on 3 separate

days in each of the three participating laboratories.

The organisms used and the number tested were:

Acinetobacter calcoaceticus subsp. anitratus, 1;

En-terobacter aerogenes, 1; Enterobacter cloacae, 1;

Escherichia coli, 2; Klebsiella oxytoca, 1; Klebsiella pneumonia, 2;Proteusmirabilis, 2; Providencia rett-geri,1; Providenciastuartii,1; Pseudomonas aerugin-osa, 2; Staphylococcus aureus, 2; Staphylococcus

epidermidis,4; Streptococcus avium, 1; Streptococcus

durans, 1; Streptococcus faecalis, 2; and Streptococ-cus liquefaciens, 2. These strains were selected to

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MICROBIAL PROFILE SYSTEM EVALUATION 437

maximize the number of MICs fallingwithin therange

ofantimicrobial concentrations provided in the MPS panels. Thetestorganismswerealso selected withthe intent ofhavingatleast four on-scale valuesfor each antimicrobialagentandtohave these endpoints deter-mined with four different species. These objectives weremetfor nearly all antimicrobialagents.

In the phase II studies, an additional 164 strains

werereceived fromthelaboratoryof C. Thornsberry, Centers for Disease Control, Atlanta, Ga. The

orga-nisms used and the numbertestedwere:A. calcoace-ticussubsp.anitratus,3;E.aerogenes,11; Enterobac-teragglomerans, 1; E.cloacae,5; E. coli,27; Hafnia

alvei, 1; K. oxytoca, 2; K. pneumoniae, 8; Proteus

mirabilis, 10; Proteusmorganii, 1; Proteus vulgari,4;

P. rettgeri,4; P.stuartii,3; P. aeruginosa, 25;

Salmo-nella paratyphiA, 1; Salmonella typhi, 2; Serratia

liquefaciens, 1; Serratia marcescens, 6; Shigella

dy-senteriae, 1; Shigella sonnei, 1; Shigella spp., 1;

enterococci, 14; S. aureus, 20; S. epidermidis, 8; Streptococcus bovis, 3;andStreptococcusequinus,4.

These strains were selected to represent

microorga-nismspossessingmostof the antimicrobial resistance

andsusceptibilitypatterns knowntooccurin clinical isolates ofbacteria.

Antimicrobial susceptibility testing. The RMD test

panelswerepreparedaspreviously described(2, 6, 9, 10, 12) in each of the three laboratories with the

Dynatech MIC 2000system (DynatechLaboratories,

Inc., Alexandria, Va.)orthe Quick SpenseII(Sandy

SpringInstrumentCo., Ijamsville, Md.) for filling the trays. Adisposable multiple-pronged inoculating

de-vicewasusedtoinoculatethesetrays.The MICswere independently determined by two experienced

tech-nologists. Ifadisagreement in endpoint determination occurred,anindependent determinationwasmade by athirdtechnologist.

Allofthestock strains used in phases I andIIwere transferred atleast twotimeson agar plate mediato assurepurity of colonialtypes.To verifythe purity of the inocula, a sample of each was taken from the

control well of the inoculatedtrayby usinga1-,I loop

andstreakedon asuitableagarmedium.Therequired

number of colonies perplateforacceptable inoculum

densitywas

10'

CFU/ml. Thesewereexamined after incubationfor theappearanceof dissimilarcolonies.If anyappeared,thetestwasrepeated from the original culture.

TheMPSpanelsweresuppliedasplastictrayswith

thedifferentconcentrationsof thetestdrugsdried in

the wells. The trays were rehydrated with cation-supplementedMueller-Hinton broth medium ina

dis-pensing device provided by 3M (12). This device

dispensed100,ulof brothmediumsimultaneouslyinto

eight wells. The tray was moved along under the

dispensing manifold until all of the test wells were filled. Standardized inocula (12) were prepared by inoculatingthreetofivecoloniesof thetestorganisms into 0.5 ml of a brain heart infusion broth (BBL Microbiology Systems, Cockeysville, Md.) medium.

After 4 h of incubation at 35°C, 50 Rl of the broth culture was transferred to a 25-ml tube of water

containing0.02%polysorbate80. Afterthorough

mix-ing,theresulting suspension containing approximately

106CFU/mlwaspouredinto the inoculumtray.Witha

sterilemultiple-pronged plastic inoculator,about5,ul

of inoculumwas transferredtoeachwell. Theplates

TABLE 1. MPS antimicrobial test panel concentrations afterrehydrationwith 100plof broth

Panelconcentration range Antimicrobial agent

(1±g/ml)'

Gram Gram

positive negative Urne

Amikacin b 2-128

Ampicillin 0.25-16 1-64 128

Carbenicillin - 8-512 1,024

Cephalothin 1-64 1-64 128

Chloramphenicol 1-64 1-64 Clindamycin 0.25-16

Colistin 0.5-32

Erythromycin 0.5-32

Gentamicin 0.25-16 1-64

Kanamycin 0.5-32 2-128

Nalidixic acid 16

Nitrofurantoin - 64

Oxacillin 0.25-16 Penicillin 0.03-2

Tetracycline 0.25-16 0.25-16 64

Tobramycin 1-64

Trimethoprim- 0.5-32 0.5-32 sulfamethoxazole

Vancomycin 1-64

aThe values listedaretheinitial and finalserialtest

concentrations of the antimicrobial agents in each panel. The single concentrations listed in the urine column are the single high concentrations tested on

each traydesignforevaluating urinarytractisolates. b ,Nottested.

were then covered with a plastic lid and incubated overnight(18 to 22 h). Thepanels were then read in the MPS plate reader, which automatically recorded the MIC for each drug.

Twoseparate MPS antimicrobialpanelswere used in this study: one for testing gram-positive bacteria andonefor testinggram-negativebacteria. The antimi-crobial agents and ranges oftestconcentrations used in each panelare listed in Table 1. The study deter-mined the incidence of MIC agreement between the two methodologies. Each RMD MIC was compared with thematching MPS panel MIC for that drug. The resultswereexpressedasMPSMICIRMD MIC ratios. If theMICs by both methodswereidentical,theratio was 1. If the RMDMICwasoneconcentrationlarger, the ratiowas0.5; iftwoconcentrationslarger,theratio was 0.25, etc. If theRMDpanelMICwassmaller,the

ratios would be2.0, 4.0, etc.Ratios of0.5, 1.0,and 2 wereconsideredtobewithinanacceptable range.

RESULTS

The studyconsisted of threephases. Inphase I,eachlaboratorytested 26strains ofbacteriaby usingthe

appropriate panel

(i.e.,

gram-positive

or

gram-negative)

on 3 separate days. Data

submitted byall three

participating

laboratories are summarized in Table

2,

which lists the percentage

frequency

of the MICratios for each

drug. Theratios werecalculated

by using

MIC valuesbetweenthetwoconcentrationextremes

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438 GERLACH, JONES, AND BARRY

TABLE 2. Comparison of MPS and RMD MIC ratiosby antimicrobial agenta

No.of Ratio'

Antimicrobialagent tests

20.25

0.5 1 2 -4

Amikacin 35 2.9 11.4 65.7 17.1 2.9

Ampicillin 149 0.7 18.8 71.8 8.0 0.7

Carbenicillin 68 7.4 39.7 44.1 8.8

Cephalothin 113 2.6 24.3 60.0 12.2 0.9

Chloramphenicol 200 1.5 17.5 64.0 11.5 5.5

Clindamycin 51 2.0 15.6 68.6 11.8 2.0

Colistin 31 9.7 6.5 71.0 12.9

Erythromycin 41 2.5 43.9 46.3 7.3

Gentamicin 89 6.7 48.3 36.0 4.5 4.5

Kanamycin 72 20.8 58.3 16.7 4.2

Oxacillin 43 9.3 83.7 7.0

Penicillin 75 2.7 8.0 74.7 5.3 9.3

Tetracycline 79 7.6 22.8 45.6 22.8 1.2

Tobramycin 51 5.9 86.3 5.9 1.9

Trimethoprim- 37 48.6 43.2 8.2

sulfamethoxazole

Vancomycin 43 23.3 72.1 4.6

aMatchedpairsoftests wereperformedin three separatelaboratoriesonthree separatedays. Onlyon-scale endpointsareincluded.

bRatioswerecalculatedby dividingtheMPS MICby theRMD MIC. Modal ratiosareboldfaced. andwerereferredtoastheon-scale results. The

addition of off-scale results would not have altered the findings. The number of on-scale endpointsperdrug varied from 200 for

chloram-phenicolto only 31 for colistin. The best

com-parison wasobtained with oxacillin and

vanco-mycin, in which 100% of the resultswerewithin

theacceptable limits oftestvariation(i.e., MIC ratiosof0.5, 1, and 2). The results with

trimeth-oprim-sulfamethoxazole and vancomycin were

also all withinacceptable limits, although these

were skewed toward lower MICs by the MPS

method. The results with the aminoglycosides

werealsoquite variable, with gentamicin having

11.2% of the MIC ratios outside of the

accept-able limits of variation compared to only 1.9% for tobramycin. The results with gentamicin

were significantly (P < 0.01) skewed toward lower MICresults obtained with the MPS

meth-odology, as indicated by a modal ratio of 0.5.

Amikacin, kanamycin, and tobramycin did not

show this feature. The results witherythromycin

andcarbenicillin werealso significantly skewed

toward lower MPS MICs. The poorest inter-methodcorrelationswereobtained whentesting

penicillin (88.0%), gentamicin (88.8%), colistin (90.3%), and tetracycline (91.2%). The MICs of nalidixic acid and nitrofurantoin were obtained

by testinga single concentration of those drugs,

and these resultsgave100%correlation between

the two methods. That is, when inhibition

oc-curred in the RMD, it also occurred in the MPS

testpanels. Eveniftheratiosfor these lattertwo

drugs were discounted from the overall

accept-ablerates,thepercent agreementwould only be lowered 1.2% to94.7% agreement.

The intralaboratory variability of each

proce-durewas analyzed by comparing the three MIC

TABLE 3. Summaryof intralaboratory variation

MIC method % MIC atlog2dilution variation of: % With

andLaboratory 0 1 2 .3 acceptable

MPS

St. Francis 87.1 11.4 1.13 0.3 98.5

Kaiser 82.7 15.4 1.5 0.4 98.1

U.C. Davis 81.1 15.5 1.38 2.0 96.5

Total 83.6 14.1 1.3 0.9 97.7

RMD

St. Francis 91.3 7.3 1.3 0.1 98.6

Kaiser 85.8 13.7 0.5 0.0 99.5

U.C. Davis 78.3 20.0 1.5 0.2 98.3

Total 85.1 13.7 1.1 0.1 98.8

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MICROBIAL PROFILE SYSTEM EVALUATION 439

TABLE 4. Summary of interlaboratory variation

MIC method % MIC atlog2dilution variationof: %With

and laboratory ₀1₁ 2_{2 -3}3

_{~~~~~~~~~~~~~~~~~variation}

acceptable

MPS

St. Francis 73.5 20.7 4.7 1.16 94.1

Kaiser 85.6 12.3 1.8 0.3 97.9

U.C. Davis 83.4 13.3 1.8 1.5 96.7

Total 80.8 15.4 2.8 1.0 96.2

RMD

St. Francis 74.3 21.7 2.6 1.48 96.0

Kaiser 81.6 15.6 2.3 0.59 97.1

U.C. Davis 77.0 18.0 4.0 1.1 95.0

Total 77.6 18.4 2.9 1.1 96.0

results obtained in phase I of the study. The

MIC resultsof trialA to B, A toC, and B to C,

expressed inlog2 dilutionsteps, werecompared

for each drug. Table 3 shows that there was

acceptable consistency

in each laboratory for

both methods. Theintralaboratory reproducibil-ity for the MPS varied from 96.5%at the U.C. Davis Centerto98.5%attheSt. Francis

labora-tory. The RMD method was 1.1% more

repro-ducible; although asmall difference, itwas

sta-tisticallysignificant. The RMD showedagreater

incidence of absolute agreement (85.1% versus

83.6%)

betweenintralaboratory trials.The

inter-laboratory

comparisons

are summarized in

Ta-ble4.These statisticswereanalyzed

by

compar-ing the MIC results of the individual laboratory

to the modal MIC for that antimicrobial agent

and

organism. Variations

wereexpressed in

log2

dilutionstepsfrom that modal MIC,

i.e., 0,

1,2, and

.3.

The results

by

theMPS and RMDwere verysimilar

(96.2%

versus

96.0%, respectively).

The St.Francis laboratory hadthebest

intralab-oratory correlation and the poorest correlation in theinterlaboratory

analysis.

Thiswas general-ly due to some of their results

being

skewed toward values lower than those from the other

two

laboratories.

TheKaiser

laboratory

had the

best results overall in both the intra- and inter-laboratoryevaluations.

In the phase II

studies,

the 164 strains from theCenters for Disease Control

laboratory

were

tested

singly

ineach

laboratory

by

the MPS and RMD methods. The distribution of the MIC

ratios representing

on-scale resultsare listed in

Table 5. The best results were obtained when

testing

clindamycin,

vancomycin, tobramycin,

and

amikacin,

asallof the ratioswerewithin the

acceptable

range

(i.e., 0.5,

1, or

2).

Theresults

with

erythromycin

and

tobramycin

each showed

percentages

indicating

that

only

one strain was

outside

of the

acceptable

range for each

drug.

The modal ratio of 1 occurred for each

drug

TABLE 5. Comparisonof MPS and RMD MIC ratiosbyantimicrobialagent for thephaseII strains

Of tU+.;+ kXVP Tf'/n MTr'rtine

nfa-Antimicrobialagent No. of_tests

-0.25 0.5 2

Amikacin Ampicillin Carbenicillin Cephalothin Chloramphenicol Clindamycin Colistin Erythromycin Gentamicin Kanamycin Oxacillin Penicillin Tetracycline Tobramycin Trimethoprim-sulfamethoxazole Vancomycin 123 209 140 215 327 36 100 27 147 193 68 52 154 86 89 24 2.4 5.7 2.3 2.1 3.0 2.0 2.6 2.9 4.5 10.1 11.4 17.7 25.0 17.7 18.3 25.0 29.0 22.2 26.5 16.6 16.2 3.8 15.6 10.5 28.1 70.7 62.7 57.9 67.5 67.6 69.4 60.0 55.6 55.8 62.7 64.7 75.0 53.3 73.2 48.3 8.3 70.9 14.6 14.3 11.4 10.2 9.2 5.6 7.0 18.5 11.6 16.0 13.2 15.4 20.8 15.1 12.4 20.8 3.3 2.9 2.3 2.8 1.0 3.7 4.1 2.1 3.0 5.8 5.8 1.2 1.1

aThepercentageoftestsineach ratiocategorywas: <0.25, 2.7%;0.5, 18.7%; 1,63.1%; 2, 12.9o;24,2.6%.

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TABLE 6. Comparison of MPS and RMD MIC ratiosby organism groupastested inphase II studies in the three laboratories

Organism group and No.of % Frequency of the MPS-MIC/RMD-MIC ratios % Within

antimicrobialgroup tests -0.25 0.5 1 2 -4 ±1log

Enterobacteriaceae 94.5

P-Lactams 368 4.1 21.2 61.1 12.0 1.6

Aminoglycosides 222 1.8 12.6 67.1 15.3 3.2

Others 327 2.8 20.8 61.9 11.9 2.8

NonentericGNB' 95.4

P-Lactams 85 0 22.3 67.1 9.4 1.2

Aminoglycosides 215 0.9 17.2 74.0 7.0 0.9

Others 183 5.5 24.6 56.3 9.8 3.8

Staphylococci 92.3

,-Lactams 83 2.4 16.9 49.4 21.7 9.6

Aminoglycosides 74 1.4 17.5 36.5 39.2 5.4

Others 143 3.5 17.5 65.0 11.9 2.1

Streptococci 96.6

P-Lactams 95 2.1 8.4 80.0 9.5 0

Aminoglycosides 38 2.6 42.1 47.4 2.6 5.3

Others 157 1.3 15.3 66.9 14.6 1.9

aGNB,Gram-negativebacilli. Strainsweretested singlyineachlaboratory.

without the significant skewing that occurred in the phase I studies. When we reviewed the

results by organism group versus antimicrobial

group (Table 6), the highest overall agreement (96.6%) occurred when testing the streptococci. The poorest overall agreement occurred when testing the staphylococci, principally due to

variations occurring when testing the

beta-lac-tamdrugs. In the Kaiser and St. Francis

labora-tories, when testing the staphylococci against gentamicin, the modal ratio was 2.0, indicating

lower MICs by the RMD method for these strains. The results, including on-scale results with nalidixic acid, nitrofurantoin, or both,

slightly raised the overall percent agreement (data presentedinparentheses).

Those strains from the Centers for Disease Control collection which caused significant

ab-TABLE 7. Bacterial strainsproducing significanterrorsbetween methods in thephase II studies

Organism Antimicrobial agent

MPS

RMD

Laboratory

MPS RMD

E.agglomeransN1057 Ampicillin 8 32 Kaiser

K.pneumoniae N1177 Ampicillin 8 32 U.C. Davis

E.agglomeransN1057 Cephalothin 8 32 Kaiser

P. mirabilisN1093 Cephalothin 4 16 Kaiser

K.pneumoniae N1174 Tobramycin 16 4 Kaiser

K.pneumoniae N1174 Gentamicin 16 2 Kaiser

E. coli N1100 Chloramphenicol 64 16 U.C. Davis

S. liquefaciensN1020 Chloramphenicol 8 32 U.C. Davis

P.aeruginosa N1143 Colistin 16 1 ' Kaiser

P.aeruginosaN1035 Colistin 2 16 U.C. Davis

P.aeruginosa N1023 Colistin 8 2 U.C. Davis

P. aeruginosaN1142 Colistin 2 8 U.C. Davis

P.aeruginosa N1133 Colistin 4 16 Kaiser

P.aeruginosa N1107 Amikacin 32 8 U.C. Davis

P.aeruginosaN1023 Gentamicin 16 4 U.C. Davis

S.faeciumP1145 Gentamicin 4 16 U.C. Davis

S.epidermidis P1007 Oxacillin 4 16 Kaiser

S.epidermidis P1007 Oxacillin 4 1 U.C. Davis

S. aureus P1168 Oxacillin 16 1 U.C. Davis

S. aureus P1168 Oxacillin 4 1 Kaiser

S.aureusP1052 Cephalothin 8 32 Kaiser

S.aureusP1081 Gentamicin 8 1 U.C. Davis

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MICROBIAL PROFILE SYSTEM EVALUATION 441 TABLE 8. CorrelationofoxacillinMICs of methicillin-resistant staphylococci

MIC(>Lg/ml)

Organism Kaiser U.C. Davis St. Francis

MPS RMD MPS RMD MPS RMD

S. epidermidis P1150 4 4 8 16 >16 >16

S. epidermidis P1151 8 >16 8 16 4 8

S. aureus P1167 2 4 2 4 16 16

S.aureusP1168 4 1 1 16 0.5 1

S. epidermidisP1101 1 2 2 '0.25 1 2

S. epidermidis P1007 4 16 4 1 2 2

S.epidermidis P1045 1 1 1 '0.25 1 0.5

S.aureusP1052 16 >16 >16 '0.25 16 8

S.aureusP1060 1 2 >16 '0.25 2 1

S.aureusP1081 4 8 >16 '0.25 8 8

errant MIC ratios are listed in Table 7. These

organism-antimicrobial agent combinations

were responsible for seven very major (false

susceptible), eight major (false resistance), and six minor interpretive errors. The organism-antimicrobialcombinations causing theseerrors were: S. epidermidis, oxacillin; S. aureus,

oxa-cillin, cephalothin, and gentamicin; P.

aerugino-sa, colistin, amikacin, and gentamicin; and S.

faecium, gentamicin. Those members of the Enterobacteriaceae which caused significantly false susceptibility variations by the MPS data

were limited to six strains. The strains and antimicrobial combinationswere: E.

agglomer-ans,ampicillin andcephalothin; K. pneumoniae

(2strains), ampicillin; P. aeruginosa, colistin; S.

faecium, gentamicin; and S. aureus,

cephalo-thin. When testingP. aeruginosa with colistin, there seemedtobeanequal distribution of major

andvery majorerrors.

The main(major andvery major) errors with

the staphylococci (Table 8) resulted from tests

for susceptibility to oxacillin. Except for one

strain, S. epidermidis P1007, the RMD method produced lower MIC values with the strains of S. aureus and S. epidermidis. Staphylococcal resistance to methicillin wasevaluated by

com-paring the oxacillin susceptibilityof 10 methicil-lin-resistant strains ofstaphylococci. Ifa

resist-antMICbreakpoint of >2 ,ug/mlis accepted for oxacillin, then neither microdilutionsystem

con-sistently detected methicillin-resistant strains

amongthe staphylococci.

In phase III of the study, a collaborative

evaluation of the antimicrobial susceptibility testing ofrecent clinical isolates with the MPS and with the RMD was done at three medical

centers. A total of 359 bacterial isolates were

tested by each method. The strains were all recent isolates (i.e., less than 48 h), and they

weretohave been selectedsothat thecollection

wouldnotincludemorethan 25% E. coli andno morethan15% S. aureus. Ascanbe seenfrom

footnote ainTable9, thispercentage wasmet, thereby providing a good distribution of test

species. The test strains were identified to the species level by either the API system or a

comparable method (4). Organisms susceptible and resistanttoeach testedantimicrobialagent

werefound in the study population.

The resultsof the comparison studies with the clinical isolatesareshowninTables9and10. Of the1,538 on-scale data pairs, 97.3% of the ratios

were in the accepted ratios (0.5, 1, and 2) for

comparative evaluation. Ifthe total4,536 pairs, which would represent both on- and off-scale

values, were considered, only 2.6% of those

valueswere off-scale.

TABLE 9. Datasummaryof the 359organismstested with MIC pairsonthe dilutionscalea No.of No. oftests(%)with MPSMIC/RMD MIC ratios of:

strains 0.25 0.5 1 2 -4

Enterobacteriaceae 209 12(1.4) 179(20.9) 521 (60.9) 139(16.2) 5(0.6) Nonenteric rods 39 5(2.4) 51 (24.8) 125(60.7) 24(11.7) 1(0.4) Streptococci 64 1 (0.5) 31 (15.1) 108(52.7) 51 (24.9) 14(6.8)

Staphylococci 47 2(0.7) 52(19.2) 188(69.4) 28(10.3) 1 (0.4)

aIncludes the strains from the following genera (with the numbers obtained at the Kaiser Foundation

Laboratory, U.C. Davis Medical Center, and St. Francis

Hospital, respectively,

given within parentheses): Escherichia(16,38, 8),Klebsiella(20, 10,19),Enterobacter(10, 11, 11),Proteus(17, 10, 10),Serratia(5,6, 2), Citrobacter(1, 3, 7),Providencia(0, 0,3),Aeromonas(2, 0, 0),Pseudomonas(10,13, 9), Acinetobacter (5,0,0), Staphylococcus (32,22, 10),andStreptococcus (16,11,20).

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442 GERLACH, JONES, AND BARRY

TABLE 10. Data tabulation from all 359organisms tested at the three collaborating laboratoriesagainst 17 antibiotics'

No.oftest % with MPS MIC/RMD MIC ratio of6:

Antimicrobialagent pairs

-0.25

0.5 1 2

.4

Amikacin 44 2.2 11.4 75.0 11.4 0

Ampicillin 214 1.9 15.9 61.7 20.5 0

Carbenicillin 94 1.1 36.2 48.9 13.8 0

Cephalothin 189 0.5 16.9 64.6 18.0 0

Chloramphenicol 307 0.3 20.5 65.2 12.7 1.3

Clindamycin 31 0 22.6 74.2 3.2 0

Colistin 123 3.2 30.9 48.0 17.9 0

Erythromycin 22 0 31.8 63.6 4.6 0

Gentamicin 93 3.2 34.4 46.3 11.8 4.3

Kanamycin 133 0 8.3 69.2 16.5 6.0

Oxacillin 37 0 16.2 78.4 0 5.4

Penicillin 43 0 11.6 67.5 20.9 0

Tetracycline 106 1.9 21.7 46.2 28.3 1.9

Tobramycin 35 2.9 11.4 77.1 5.7 2.9

Trimethoprim- 40 5.0 17.5 55.0 22.5 0

sulfamethoxazole

Vancomycin 27 0 18.5 81.5 0 0

aAtotalof 1,538 MIC datapairswereused.Both MICswereon-scalevalues,i.e.,MIC

.1

dilution from the extremesof thelog2 dilution sequence. The addition of the off-scale results (4,536 total pairs) did notsignificantly alter theanalysis.

bThepercentage oforganismsin each ratiocategorywas <0.25, 1.3%; 0.5,20.4%; 1, 61.2%; 2, 15.7%;.4, 1.4%.

The 41 strains with MIC ratios that were

outside oftheacceptable limits(ratiosofc0.25 and

.4)

represented only 2.7% of the total

on-scale ratios. These accounted foronly 0.5%very

major interpretive errors (false-sensitive MICs by the MPS method). The antimicrobial

agent-organism combinations resulting in these very

majorerrorswere:oxacillin, S. aureus; ampicil-lin, S. marcescens; ampicillin, M. morganii; ampicillin,P. rettgeri; gentamicin, P.

aerugino-sa; aminoglycosides, A. calcoaceticus subsp.

anitratus (2 strains); and chloramphenicol, K.

pneumoniae. Changes in the interpretive criteria didnotoccurwhen theMIC ratios were within

theacceptable limits.

Theresults with nalidixicacid and

nitrofuran-toinalsowerenotincluded intheoverall percent agreementbecause onlyoff-scaleMIC ratios of 1.0werepossiblesince these were tests of single

concentrationsof either drug. Even when added to the total results, they increased the

accept-able totals by <1.0%. Therefore, 97.3% of all testswere in acceptable agreement.

The occurrence of a well with growth of the testorganismsthat ispreceded andfollowedby wells in which growth has been inhibited is

referredto as the skipped-wellphenomenon (5,

8, 9). This phenomenon occurred frequently in the MPS panels; e.g., 106 and 142 incidents in

phaseIIandphase III,respectively.The

major-ity of incidents occurred when testing the

ami-noglycosides

in the KaiserandU.C. Davis

labo-ratories. In both laboratories, the problem

occurred only in the wells designatedas contain-ing the highestconcentrations of amikacin,

gen-tamicin, or

kanamycin.

The U.C. Davis

labora-tory

assayed

the

aminoglycoside

contentof the

wellsinthegram-negative MPStrays andfound that many of the

high-concentration

wells did

notcontainanydetectable drug.TheSt.Francis laboratory had noticed a similar occurrence (in earlier product development) in the wells

con-tainingthelowestconcentrations of penicillinG.

The St. Francis laboratory subsequently

as-sayed all the wells in three lots of trays and found that the antimicrobial agent content was

consistently between 90and102% ofthe desig-nated content. This variation is within the

ac-ceptable

range of variation for such products.

The trays in a fourth production lot of

gram-negative

panels

were assayed forthewell con-tent of aminoglycoside by bioassay,

high-per-formance liquid chromatography, and fluorescent immunoassaymethods(1). The

ami-noglycoside content was consistently within

10%of the stated value.

DISCUSSION

The accuracy and

reproducibility

ofMIC re-sultsobtainedwith the MPStrays were tested in three

participating

laboratories. This

compara-tiveevaluationwasmadeby

using

singlelots of RMDtrays

produced

inthelaboratories ofeach

participating investigator

(12). The MIC ratios

were within the acceptable range (indicating

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MICROBIAL PROFILE SYSTEM EVALUATION 443

results in the range of±1 log2 dilution

concen-tration) for 95.9, 96.3, and 97.3% fortwo groups

ofstock cultures (phase I and II) and 359recent

clinical isolates (phase III), respectively. The

MPS-MIC/RMD-MIC ratios for the stock and

challengestrains compared favorably withthose

obtained with the Sensititre and Sceptor sys-tems,both of which contain antimicrobialagents

in adried form (6, 9, 10). The results from all

three studies show betteragreement than those

fromacommerciallyprepared frozen tray (2, 6,

9, 10). The intralaboratory and interlaboratory reproducibilityoftheMPStrayswas acceptable

for all three laboratories. The results were most

similartothoseobtained with theRMDmethod.

The intralaboratory variationoutside of the

ac-ceptablerange wasonly4.1and3.7% for thetwo groups of stock strains and 2.7% for therecent

clinical

isolates.

TheMPS and RMD methods bothaccurately

categorized the resistant and susceptible strains within the variousbacterialgenera and species. Therewerenotably loweraminoglycoside MPS

MIC results when testing enterococci, but this

was not considered a significant clinically rele-vantproblem, asthose organisms arenot effec-tively treated by

aminoglycosides

alone. Alow

incidence

(<0.5%)

ofverymajor MPS

interpre-tive errors was found in all study phases. This

was especially evident when testing

beta-lacta-mase-producing

Enterobacteriaceae and the

aminoglycoside-resistant

enteric and

nonfer-mentative gram-negative bacilli. The MPS

re-sults with methicillin-resistant staphylococci

wereinconsistent compared with the other anti-microbial agents. These results indicate that oxacillin isnot an

optimal

reagentfor

detecting

methicillin resistance of

staphylococci;

howev-er, morestudieson this

subject

are

required.

Theoccurrenceof the

skipped-well

phenome-non incommercial

dry-form products

has been

reported

by

others

(5, 9;

R.

Jones,

manuscript

in

preparation).

An earlier report

by

Jones and

colleagues

offered reasonable

explanations

of

this

infrequent problem

(9).

Inthis

study,

how-ever, the

high

frequency

of

skipped

wells was

explained

by

the

findings

of antimicrobial

agent-free wells. This

problem

was mechanical and limitedtothe

high-concentration

wells of

amika-cin, gentamiamika-cin,

and

kanamycin.

Theassays of

subsequent MPS

production

lotsshowed

appro-priate

concentrations of antimicrobial agents in

each

well,

indicating

that this

problem

hasbeen

eradicated.

MPS is the fourth

dry-form

product

to be evaluated. The first

product,

the

AST,

was evaluated

by

MacLowry

and Marsh

(11)

and Tilton and

Isenberg (13).

In thereport

by

Tilton

and

Isenberg,

they

cited

approximately

90%

correlation of AST

susceptibility

results with

those of the disk diffusion, agar dilution, and broth microdilution methods.The continued

ex-cellent accuracy of other products containing

dried antimicrobial agents indicates that this methodology should be readily accepted by clin-ical laboratories. The minor disadvantages en-countered in rehydrating the trays are vastly outweighed by conveniences such as long shelf

life (>12 months) and room temperature stor-age. These trays also allow laboratories to use several broth media andtoroutinelyuse cation-supplemented Mueller-Hinton broth, which is generally unavailable in the frozen trays (12).

This lattercapability is provided in the unique mediumfound in the Sceptorsystem (9).

We conclude that the MPSfor broth

microdi-lution antimicrobial agent susceptibility testing gives MICs equivalent to those of the National Committee for Clinical Laboratory Standards reference brothmicrodilution method (12). The complete MPS system contains dried reagents

for simultaneous biochemical identification and antimicrobialsusceptibility testing. This capabil-ity should provide a significant reduction in

labor andan increased capability for data han-dling, thus providing more accurate laboratory

reports and an epidemiological system of

bio-types and antibiograms previously available

onlyby extended effortonthepartof laboratory personnel. Theaccuracy of the MPS biochemi-calsystemhasyet to be reported.

LITERATURE CITED

1. Anhalt, J. P., and S. D. Brown. 1978. High performance liquid chromatographic assay of aminoglycoside antibiot-ics in serum.Clin. Chem. 24:1940-1947.

2. Barry,A.L., R. N.Jones, and T. L. Gavan. 1978. Evalua-tion of theMicro-Mediasystemfor quantitative antimicro-bial drug susceptibility testing: a collaborative study.

Antimicrob. AgentsChemother. 13:61-69.

3. Ericsson,H.M., and J. C. Sherris. 1971. Antibiotic

sensi-tivity testing: report of an international collaborative study. Acta Pathol. Microbiol. Scand. Sect. B.

217(Suppl.):90-176.

4. Fuchs, P. C. 1976. The replicator method foridentification

andbiotypingof common bacterial isolates. Lab. Med. 6:6-11.

5. Gavan, T.L., and D. A. Butler. 1974. An automated microdilution methodfor antimicrobial susceptibility

test-ing,p.88-93. In A. Balows (ed.),Current techniques for antibioticsusceptibility testing. Charles C Thomas,

Pub-lisher,Springfield,Ill.

6. Gavan,T.L.,R. N.Jones,and A. L.Barry. 1980. Evalua-tion of theSensititresystemforquantitative antimicrobial drug susceptibility testing:acollaborative study. Antimi-crob.AgentsChemother.17:464-469.

7. Gavan, T.L., and M. A. Town. 1970. A microdilution method forantibioticsusceptibility testing:anevaluation. Am.J.Clin. Pathol. 53:880-885.

8. Gerlach, E. H. 1974. Microdilution I: a comparative

study, p. 63-76. In A.Balows(ed.), Currenttechniques

for antibiotic susceptibility testing. Charles C Thomas, Publisher, Springfield,Ill.

9. Jones,R.N.,T. L.Gavan,and A.L.Barry.1980. Evalua-tionof the Sensititre microdilution antibioticsusceptibility

system against recentclinical isolates: three-laboratory collaborativestudy.J. Clin.Microbiol. 11:426-429.

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10. Jones, R. N., C. Thornsberry, A. L. Barry, and T. L. Gavan. 1981. Evaluation of the Sceptor microdilution antibiotic susceptibility testing system: a collaborative investigation. J. Clin.Microbiol. 13:184-194.

11. MacLowry, J. D., and H. H. Marsh. 1968. Semiautomatic microtechnique for serial dilution antibiotic sensitivity testing in the clinical laboratory. J. Lab. Clin. Med. 72:685-687.

12. National Committee for Clinical Laboratory Standards. 1980. Proposed standard: M7-P. Standard method for

J.CLIN. MICROBIOL.

dilution antimicrobial susceptibility tests for bacteria which growaerobically. National Committee for Clinical

Laboratory Standards, Villanova,Pa.

13. TUlton,R.C.,andH. D.Isenberg. 1977. Evaluation of the performance parameters of a prediluted, quantitative anti-bioticsusceptibilitytestdevice. Antimicrob.Agents Che-mother. 11:271-276.

14. Tilton, R.C., and H. D. Isenberg. 1977. In-use evaluation of a prediluted quantitative antibiotic susceptibility test device. Antimicrob.AgentsChemother. 12:470-473.

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