Evaluation
of the
E
Test
for Susceptibility
Testing
of
Anaerobic
Bacteria
DIANEM. CITRON,l2* MARGARETAI. OSTOVARI,1ASA KARLSSON,3ANDELLIE J. C. GOLDSTEIN"4
R. M. AldenResearch Laboratory, Santa Monica Hospital Medical Center, SantaMonica, California 904041; Anaerobe Systems, SanJose, California 951312;Departmentof Medicine, Universityof California,
LosAngeles, California 900244; andABBiodisk, Solna, Sweden3
Received 3 June 1991/Accepted 8 July 1991
The susceptibilities of 105 clinical isolates of anaerobic bacteriaweredetermined bya newmethod, theEtest
(ABBiodisk,Solna, Sweden), andwerecomparedwiththeMICs for theseorganisms obtained by the reference
agardilutionmethod by using supplemented brucella and Wilkins-Chalgrenagars.The Etestisaplastic strip
withapredefined antibiotic gradient immobilizedon onesideandaMICinterpretive scale printedontheother side.Stripswithcefoxitin,cefotaxime, imipenem,penicillin, metronidazole, and clindamycinwereused in this
study. A suspension of the test strain equal to the visual turbidity ofa no. 0.5 McFarland standard was
preparedand swabbed onto a 150-mm-diameter plate. Thestrips were applied ina radial fashion, and the
plateswereincubatedunder anaerobic conditions. Aftergrowth had occurred,anellipse of inhibitionwasseen
around each strip. At the point of intersection of the ellipse with the strip, the MIC was read from the
interpretive scale. For most antibiotic-organism combinations, the ellipse was clear and the endpoint was
sharp. The E-test MICs were interpreted after overnight and 48-h incubation for 58 of the strains. After overnight incubation, 87% of the E-test MICswerewithin 1dilution of theagardilutionMICs,and98% were
within 2 dilutions. After 48 h of incubation,agreementwas86and97%respectively. E-test MICs obtained for
theBacteroidesfragiisgroupafterovernight incubationwere morecomparable than those obtained after 48h
ofincubation toagardilutionMICsdeterminedat48hforall drugs exceptclindamycin. On brucella agar,
therewas a2%categoricaldiscrepancyratebetween theE-testMICs andagardilution MICs, which occurred
mostly withcefoxitin. The Etestiseasytoperform and read, is suitableforall anaerobes,canbe usedto test
single patient isolates as needed, and offers the laboratory a reliable method for susceptibility testing of
anaerobic bacteria.
The National Committee for Clinical Laboratory Stan-dards (NCCLS) (16) guidelines for susceptibility testing of anaerobicbacteriarecommend susceptibility testing of indi-vidual patient isolates under certain circumstances. The reference agar dilution method is not readily adaptable for testing individual isolates, and the broth microdilution method has drawbacks because ofdiscrepancies with the reference method (1) and because many anaerobes do not
grow well in broth media (16). The modified broth disk elution method, a 24-h test that is convenient for testing individual isolates (14), has been demonstratedto be
inac-curatefor testing the activity of beta-lactam agents against membersof the Bacteroides fragilisgroup(13, 21) and isno longer recommended by NCCLSas anacceptable procedure
(16). Although patients with anaerobic infectionsareusually treated empirically (7, 8), in certain situations, such as therapy for chronic infections, undrainable abscesses,
bac-teremia,orfailureofinitialtherapy, susceptibilitytesting is
recommended.Asimple, rapid,andreliablemethod thatcan beused to testindividual isolates in these circumstances is
needed.
The Etest(AB Biodisk, Solna Sweden)isaplastic strip (5
by 50 mm; antibiotic carrier)witha continuous gradient of
antibiotic immobilizedon oneside and anMICinterpretive
scalecorrespondingto15twofold MIC dilutionsontheother
side.Thesusceptibilitytestisperformed by placingthestrip onthesurfaceofaninoculatedagarplate. Afterincubation,
anellipse ofinhibition is formed aroundthestrip,and at the
*Coffespondingauthor.
point where the ellipse intersects the strip, the MIC of the antibiotic is read from the interpretive scale. It has been foundto beareliable method fordetermining the
suscepti-bilities of fastidious and other aerobic and facultatively anaerobic bacteria (2, 4, 7, 10, 12). In this study we evalu-ated this methodforsusceptibility testing of clinical isolates ofanaerobic bacteria.
(This work was presented in part at the Interscience Conference on Antimicrobial Agents and Chemotherapy, HoustonTexas, 17 to20 September 1989.)
MATERIALS ANDMETHODS
Strains. One hundred five clinical isolates of anaerobic
bacteria were tested. These organisms were selected
be-cause ofgrowth characteristics and previous susceptibility resultstoprovideacomprehensive challengetothe method.
They were Bacteroides fragilis (8 strains), Bacteroides
thetaiotaomicron (7 strains), Bacteroides distasonis (8 strains),Bacteroidesvulgatus (8 strains),Bacteroidesovatus (8 strains),Bacteroidesuniformis (8 strains), other
Bacteroi-desspp. (4 strains), Wolinella sp. (1 strain),Prevotella spp.
(9 strains), Porphyromonas spp. (3 strains),Fusobacterium
mortiferumand F. varium (7 strains),Fusobacterium nucle-atum and F. necrophorum (4 strains), Peptostreptococcus spp. (10 strains), Clostridium perfringens (4 strains), other Clostridium spp. (8 strains), and non-spore-forming
gram-positive rods (8 strains). The organismswere identified by standardmethods(17).Five referencestrainswereincluded:
B.fragilis ATCC25285, B. thetaiotaomicron ATCC 29741, B. vulgatusATCC 29327, C. perfringens ATCC 13124, and 2197
0095-1137/91/102197-07$02.00/0
Copyright ©) 1991, American
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Peptostreptococcus magnus ATCC 29328. All organisms
were maintained at -70°C in 10% skim milk. They were
subcultured at least twice on PRAS brucella blood agar
(Anaerobe Systems,
SanJose,Calif.) prior
totesting. Antimicrobial agents. The following antibiotics, whichwere available on the prototype E-test strips, were tested:
cefoxitin, cefotaxime, imipenem, penicillin, metronidazole,
andclindamycin. The E-teststrips and laboratory standard
powderswere supplied byAB Biodisk. The antibiotic
pow-ders werereconstituted
according
tothe instructions ofthemanufacturerandwereused onthe
day
of thetest.Agardilution MIC determinations. MICsweredetermined
by
theNCCLSreference agardilutionmethod(16)
by using
brucella agar (Difco
Laboratories, Detroit,
Mich.)
supple-mented with vitamin
K,
hemin,
and 5% lakedsheep
blood(BBA)
andWilkins-Chalgren
agar(Difco)
without addedblood
(WCA),
asrecommendedby
NCCLS. BBA and WCAplates containing
serial dilutions ofthe antimicrobial agentsranging
from 256 to0.015,ug/ml
wereprepared
andusedonthe same
day.
The inoculum wasprepared
in ananaerobicchamber
by suspending
severalcolonies froma72-h cultureplate
into brucella brothtoachieve thevisualturbidity
ofa no. 0.5 McFarland standard. A Steersreplicator (Craft
Machine Co. Inc.,
Chester, Pa.)
was used toapply
theorganisms
to theplates
for a final inoculum of105
cfu per spot. Plateswere incubated in anaerobicjars
with GasPak(BBL, Cockeysville,
MD) for48h. TheMICwasdefined asthe lowest concentration of antibiotic that resulted in no
growth,
abarely
visiblehaze,
or onecolony.
Etest. The Etest wasevaluated
by
using
BBAandWCAin 150-mm-diameter
plates
containing
60 to 65 ml of agar.The
plates
wereprepared
in-house. The inoculumfortheEtest wasthesameasthat usedforthe agardilutiontests.This
wasapproximately
108
CFU/ml,
asdescribedpreviouslyfordiskdiffusion
testing
of anaerobes(3, 9, 18).
The inoculumwas
applied
withasterilecottonswab,
using
afresh swabfor eachplate.
The antibiotic carriers were thenapplied
in aradialpattern, withsix
strips applied
toeachplate.
Inocula-tion of the
plates
wasperformed
outside the anaerobicchamber. Plates were incubated in an incubator within an
anaerobicchamber
(Anaerobe Systems)
orinanaerobicjars
at
36°C.
The E-testplates
incubated in the anaerobiccham-berwereevaluated afterincubation
overnight
(approximate-ly
20h)
andfor48h,and those injars
were examined onlyafter48 hofincubation. After
incubation,
anelliptical
zoneof
growth
inhibitionwas seenaroundthestrip.
TheMICwasread from thescale at the intersection ofthe zonewith the
strip.
The E-test MICs that were between the standard twofolddilution stepswere rounded to the nexthigherstep forcomparison
withagardilutionMICs. Theprinciple oftheEtestis illustrated in Fig. 1.
A
categorical
discrepancy was defined as a>2-dilution
difference that resulted in a change of interpretation from
susceptible
to resistant or vice versa. The breakpoints (inmicrograms
per milliliter)used forinterpretation assuscep-tible were as follows: cefoxitin, c32; cefotaxime, s32;
imipenem,
s8;penicillin,
c2 metronidazole, s16; andclindamycin,
c4(16). (The activity of penicillin on the E-teststrips
is expressed in micrograms per milliliter, and thebreakpoint
has been adjustedaccordingly).RESULTS
E-test MICs were readafter overnight incubation for 68 of the
strains, including
51 strains of the B. fragilis group andclostridia
plus
17otherslower-growing
strains. The readingN N
e
/
H
-//. 4H'.
FIG. 1. The E-testprinciple. TheE test uses astripcoatedwith
a logarithmic gradient of an antimicrobial agent applied to an inoculatedplate. Afterincubation,anellipseofinhibitionisformed.
Attheintersectionof theellipse with thestrip,theMIC isread from
theinterpretivescale.
at 48 h included all strains that grew and that had on-scale
endpoints. The E-test MIC was compared with the MIC
obtained byagardilution with thesameagar. Acomparison of the overnightand 48-h E-test MICs versus the 48-h agar
dilutionMICs for allorganismstested ispresentedin Tables
1 and 2. The E-testMICswereeasy to read and had clear,
sharp endpointsfor most of theorganism-antibiotic
combi-nations. For
P-lactamase-producing
strains, in some in-stances, therewas haziness andpinpoint growth that oblit-erated theellipse.This occurredmostlywithsomemembers of the B.fragilis
group with cefoxitin on WCA. Figure 2depicts E-testMIC determinations for B. thetaiotaomicron.
Most of the strains of clostridia grew and displayed clear
endpointsafterovernight incubation,withlittlechangein the
MIC after additional incubation. After 48 hofincubation, 59
of68 (87%) of E-test MICs determined on BBA and WCA
were within 1dilutionofthe agardilution MICs. Therewas
a tendency for imipenem, metronidazole, and penicillin
MICs determined by agar dilution to be about 1 dilution
higher.
SincetheMICswereverylow, however, this didnotchange the interpretive result. Clindamycin E-test MICs
increasedby 1dilution afterday2of incubation for 4 of the 12 strains. For six strains, cefoxitin agar dilution MICs
rangedfrom 32to128
jig/ml.
Forfive of these strains, E-testMICswereconsistently1to2dilutionshigher than the agar
dilutionMICs, althoughthis did not result in any interpretive
discrepancies.This occurred on both agars.
Ofthenon-B.fragilis groupBacteroides spp., Prevotella spp., and Porphyromonas spp., some strains were very
susceptibletothe agents tested, and the MICs determined by
both methods were off-scale. Some of the strains grew
poorlyornot atallonWCA, and MICs determined on WCA
weregenerally 1to 2dilutions lower than MICs determined
on BBA. Ofthe strains forwhich the MICs were on-scale, the agreementwas generally good with no categorical
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TABLE 1. Comparison of E-test MICs determined afterovernight incubation with agar dilution MICs for all anaerobic strains
% of E-testMICs withinthefollowingconcentration(log2) No. of
Antibiotic Medium strainsa agardilutionMIC: %within categofical
>-2 -2 -1 Same +1 +2 >+2 discrepancies
Cefoxitin BBA 54 0 2 26 48 19 4 2 93 2
WCA 56 2 4 9 36 27 18 5 72 10
Ceftizoxime BBA 49 8 6 18 51 14 2 0 83 1
WCA 53 9 8 25 36 21 2 0 82 1
Imipenem BBA 49 2 10 45 41 2 0 0 88 0
WCA 52 0 8 21 63 8 0 0 92 0
Penicillin G BBA 56 0 7 27 59 7 0 0 93 0
WCA 52 0 8 13 54 17 6 2 84 0
Metronidazole BBA 54 0 7 26 50 13 4 0 89 0
WCA 56 0 5 34 45 16 0 0 95 0
Clindamycin BBA 45 4 20 27 47 2 0 0 74 2
WCA 49 10 16 37 33 4 0 0 74 2
All BBA 307 2 9 28 49 10 10 0 87 5
WCA 318 3 8 23 45 16 4 1 83 13
a Number of strains for which MICswerefinite.
crepancies. After 48 h of incubation, MICs for 58 of 66 (85%) change from haze to real growth was easily seen by the E of the testsdetermined on BBA and MICs for 40 of 49 (82%) test,while agar dilution MIC endpoints were more difficult to ofthe testsdeterminedon WCA were within 1 dilution of the interpret. After 48 h of incubation, 36 of 46 (78%) of the E MICs determined by agar dilution. testson BBA and 36 of 48 (75%) of Se E tests on WCA were
The fusobacteria exhibited a tailing phenomenon for the within 1 dilution of the MICs determined by agar dilution.
agar dilution MICs with the beta-lactam antibiotics that Onemajordiscrepancy occurred with a
,-lactamase-produc-madereading ofendpoints of growth somewhat ambiguous. ing strain of F. nucleatum for which the penicillin agar
Inmost cases in the E-test MIC determinations, the strains dilution MIC was 8 to 16 ,ug/ml and the E-test MIC was 1
exhibitedan areaof heavy growth with good demarcation of ,ug/ml. The test was repeated several times, with the same
the ellipse and then a haze extending into the ellipse. result.
Examination of a Gram stain of this haze revealed gram- For the non-spore-forming gram-positive bacilli, agar
di-negativebizarre forms and debris suggestive of L forms. The lution and E-test MICs showed good agreement. Two strains
TABLE 2. Comparison ofE-testMICsdeterminedafter 48hofincubation withagardilutionMICsforallanaerobic strains
% of E-test MICs within thefollowingconcentration(log2) No.of
Antibiotc Medium No. of of agardilution MIC: % within
No.egofia
Antibiotic Medium ~strains' ±1dilution
dicreategoical
>-2 -2 -1 Same +1 +2 >+2 discrepancies
Cefoxitin BBA 91 1 3 25 40 20 9 2 85 7
WCA 90 2 7 17 29 19 10 17 65 17
Cefotaxime BBA 91 4 11 16 45 20 2 1 81 1
WCA 89 3 5 18 39 20 14 1 77 4
Imipenem BBA 81 2 19 46 31 2 0 0 79 0
WCA 78 1 10 32 49 9 0 0 90 0
Penicillin G BBA 89 3 6 35 44 12 0 0 91 2
WCA 85 2 7 18 51 18 4 1 87 1
Metronidazole BBA 92 1 16 30 43 5 2 1 78 0
WCA 85 0 16 40 34 6 4 0 80 0
Clindamycin BBA 79 0 1 10 58 28 1 1 96 2
WCA 77 0 5 26 52 12 5 0 90 2
All BBA 523 2 9 27 44 15 1 1 86 12
WCA 504 1 8 25 42 14 6 3 81 24
aNumber of strainsfor whichMICswerefinite.
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I
FIG. 2. Susceptibility of B. thetaiotaomicrontometronidazole (MZ),cefotaxime (CT),
clindamycin
(CM),penicillin
G(PG), imipenem(IP),and cefoxitin(CX) determinedbythe E test.
ofPropionibacterium acnesweretoosusceptibleto mostof theantimicrobialagentsforcomparison.For the strains that
grewafterovernight incubation, therewas little increasein the MIC after additional incubation. Five of these strains
were at least moderately resistant to metronidazole, and therewas agoodcorrelation in the MICs obtainedbythe two
methods, with 13 of 15 (87%)of MIC determinations being
within 1dilution of each other. Overall,at48 h ofincubation,
36 of 40(90%)ofE-testMICs determined onBBA and 35 of 40 (88%) E-test MICs determined on WCA were within 1 dilution of the agardilutionMICs. One categorical discrep-ancy occurred with one strain of Eubacterium limosum for
which the cefoxitinagardilution MICwas 32 ,ug/ml and for
which the E-testMICwas128 ,ug/ml after overnight
incuba-tion butthatincreasedto256 ,ug/ml after 48 h of incubation.
There was good agreement between the methods for peptostreptococci. After48 hof incubation, 28 of 35 (80%) of E-test MICs on BBA and 26 of31 (84%) E-test MICs on
WCAwerewithin1dilution ofthe MICsdeterminedbyagar
dilution. Metronidazole MICs determined by the Etestwere
2dilutionslower than theagardilutionMICsfor three strains
and were 3 dilutions lowerfor afourth strain. This did not
result in acategorical discrepancy.
Mostofthe B.fragilisgroup grew after overnight
incuba-tion and yielded readable endpoints. The comparison of
E-test MICs obtained after overnight incubation with agar
dilution MICs is presented in Table 3. The +1-dilution
agreement between the methods for imipenem, penicillin, andmetronidazole was verygood afterboth overnight and
48-h incubation, with a tendency for E-test MICs to be lower. When the E-test clindamycin MICs obtained after
overnight incubation were compared with the agar dilution
MICs, only61to69%werewithin 1
dilution; however,
after48 h ofincubation,agreementwas98%onBBA and 90%on
WCA. The E-test MICs were several dilutions lower than the agardilution MICs. Toclarifythisobservation, 15 strains that exhibited this discrepancy were selected and MIC
determinationswererepeated by bothmethods, except that
the agardilution MICswerealsointerpretedafterovernight incubation and again after 48 h of incubation to allow a
comparisonbetween the methods andtodetermine whether
the agardilution MICs also increased with continued
incu-bation. The agar dilution MICs determined afterovernight
incubation were 1 to 4 dilutions lower than the MICs measured after 48 h. When theovernightE-testMICswere
comparedwith theovernightagardilutionMICs,agreement
wasimproved between themethods, with 13 of 15(87%) of
the previously discrepant MICs being within 1 dilution.
Therewas littledifference notedforcefoxitin andpenicillin agardilution MICs measuredafter overnight and 48-h incu-bation. For2 of 30 strains, therewas afourfold increase in the cefotaxime MIC between overnight and 48-h
incuba-tions. For cefoxitin, agreement between the methods was
verygood when BBAwas used. After overnight incubation, 29of 31 (94%)of E-test MICs werewithin1 dilution of the agardilution MICs. After 48 h, 37 of 45(82%) E-testMICs
were within 1 dilution, with the E-test MICs showing a
tendencytoincrease withprolonged incubation.Twostrains
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TABLE 3. Comparison of E-test MICs determined after overnight incubation with agar dilutionMICs for the B. fragilisgroup
%of E-test MICs within the following concentration(log2) No.of
Antibiotic Medium strains'No. of of agar dilution MIC: ±%1dilutionwithin
dicreategoical
catego.ical>-2 -2 -1 Same +1 +2 >+2 discrepancies
Cefoxitin BBA 33 0 0 18 61 15 3 3 94 2
WCA 38 3 3 11 26 32 18 8 69 9
Cefotaxime BBA 33 9 3 18 45 15 3 0 78 1
WCA 38 5 8 26 39 18 3 0 83 1
Imipenem BBA 33 3 9 45 42 0 0 0 87 0
WCA 39 0 5 23 64 8 0 0 95 0
Penicillin G BBA 33 0 6 12 72 9 0 0 93 0
WCA 37 0 8 11 51 22 8 3 84 0
Metronidazole BBA 35 0 3 33 54 11 0 0 98 0
WCA 38 0 5 34 47 13 0 0 94 0
Clindamycin BBA 28 11 29 32 29 0 0 0 61 2
WCA 30 17 13 33 33 3 0 0 69 2
All BBA 185 4 8 26 51 8 1 1 85 5
WCA 210 4 7 23 43 16 5 2 82 12
a Number of strains for which MICs were finite.
yielded discrepant results. For both strains, agar dilution
MICs were 32 ,ug/ml, and E-test MICs were 128 and 256
,ug/ml afterovernight incubation. After 48 h of incubation,
four additionalstrainsfor which the agar dilution MICs were
16 to 32
p,g/ml,
the E-test MICs were 64 to 256 ,xg/ml.Cefoxitin MICs determinedon WCA had a lower correlation
between the methods. After overnight incubation, 27 of 38
(69%) of E-test MICs were within 1 dilution of the agar
dilutionMICs.After 48 h of incubation, 26 of 45 (58%) were
within 1 dilution. Nine strains yielded higher E-test MICs
afterovernight incubation, and after 48 h of incubation, 16
strains yielded higherE-testMICs than agar dilution MICs,
resulting in categorical discrepancies. Cefotaxime yielded
acceptableagreementbetween the methods. After overnight
incubation, 78%of E-test MICs on BBA and 83% of E-test
MICs on WCA were within 1 dilution ofthe agar dilution
MICs,
withonecategorical discrepancyoneachmedium.At48h,34of45(76%)E-testMICs on BBA and 30 of 45(67%)
E-test MICs on WCA were within 1 dilution. At 48 h, two
categorical discrepancies occurred on BBA and three
oc-curredon WCA.
The American Type Culture Collection control strains
were tested at least five times with each antibiotic and on eachagar. The reproducibility ofthe E test was 528 of539 tests
(98%)
within a 2-dilution interval and 100% within a3-dilution interval. In
comparison
with agar dilution testswith penicillin, metronidazole, imipenem, and
clindamycin,
overnight E-test MICs exhibited 94% agreement within 1
dilution,
withnointerpretive discrepancies.
B.thetaiotaomi-cron yielded discrepant results when it was tested with
cefoxitin and cefotaxime on WCA. Cefoxitin E-test MICs
were 1 to 3dilutionshigherthan the agar dilutionMICs.The mode E-test MIC of cefoxitinwas64
,ug/ml
afterovernight
incubation and 256
,ug/ml
after 48 h of incubation. Theacceptablerangeis8 to32
,ug/ml.
E-testMICsof cefotaximewere 256and >256 P,g/ml, withan
acceptable
range of 16to64
pug/Inl.
For C.perfringens,
agardilutionMICswere2to4 dilutionshigherthan the E-testMICs; however,
thereweresingle colonies present within the
ellipse
thatwere isolatedandretested.The repeat E tests on these coloniesexhibited
MICsthat were within 1dilution of the agar dilution MICs. P.magnusdidnotgrow on WCA after overnight incubation, andMICs for P. magnus at 48 h were 1 to 2 dilutions lower
thanthosemeasuredon BBA by the agar dilution and E-test
MIC methods.
DISCUSSION
The E testoffers a simple method for susceptibility testing
of all typesofanaerobic bacteria. Itis applicable for testing
single isolates in those instances in which this information is
of vital importance for selecting the mostappropriate
ther-apy. Results forrapidly growingorganisms, such as the B.
fragilis group and clostridia, are
available
after overnightincubation. Eighty-six percent of E-test MICs determined
afterovernight incubationwerewithin 1 twofold dilution of
the agardilution MIC when BBA was used.
There was atendency for the E-test MICs tobe slightly
lower than those measured
by
the agar dilution method,especially with imipenem, metronidazole, and to a lesser
degree, penicillin. This could be explained in part by a
difference in the inoculum that is actually delivered to the
plate. Spots of inoculumdelivered bythe Steers
replicator
are approximately 5 mm in diameter and have an area of
about 2
mm2;
thus, thefinalinoculum isapproximately
5 x104
CFU/mm2.
One-tenthofamilliliter ofthis sameinocu-lum spread over a 15-cm-diameter
plate
results in a finalinoculum of
approximately
6 x 10 CFU/mm2, which isalmostafull
log1o
unit lower. Inoculumeffectsare variabledependingonthe
antibiotic,
theorganism,
and theagar baseused for
testing.
In astudy
thatcompared
the effects ofinoculumandmediumonMICsforB.
fragilis
groupstrains,
Wexleret al. (20)
reported
thatimipenem
geometric meanMICs for theB.
fragilis
groupwerehalf ofatwofold dilution lower from an inoculum of 105 to 104organisms
perspot
when BBA was used. This is similar to the difference
observedinour
study.
Although
aninoculum of108CFU/mlhas beenused fordisk diffusion testswith anaerobes
(3,
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18),
theuseofaninoculum of109 CFU/mlmight yield
E-test MICs morecomparable
to the agar dilutionMICs for some ofthese agents and deservesfurtherinvestigation.
Adifferencein the cefoxitin MICs for theB.
fragilis
groupobtained
by
the two methods was evident on WCA.Agar
dilution MICs for9of38ofthesestrainswere16to32
Fg/ml
and E-test MICs were 128 to >256
,ug/ml
afterovernight
incubation. It is
unlikely
that theorganisms degraded
thecefoxitin in the
plates,
since a very smallproportion
ofstrainspossess
cefoxitin-degrading
enzymesandcefoxitinisrelatively
stabletotheP-lactamases
present in Bacteroides spp.(19).
Thisphenomenon
also occurredwithfivestrainsofnon-p-lactamase-producing
Clostridium spp. andone strainof Eubacterium for which cefoxitin MICs were 32 to 64
,ug/ml,
as measuredby
agar dilution and E-test MICs that wereconsistently
1to2twofolddilutionshigher.
Strainsforwhich MICs were lower than 8
,ug/ml
did not exhibit thistendency.
Because this did not occuron BBA to the same extent, wepostulate
that WCA contains aningredient
thatdestabilizestheantibiotic
gradient
forcefoxitinatthehigher
concentration.
MIC determinations for fusobacteria with beta-lactam
agents
yielded interesting
results.Agar
dilution teststypi-cally produce tailing endpoints
which make accurate MICdeterminations difficult. Johnson et al.
(11)
studied thisphenomenon
andfoundthe hazestoconsist of Lforms,
andthey
concluded that since there is noconvincing
evidence that Lformshave anyclinicalsignificance,
thehazy growth
should be
ignored
whenreading
agar dilution MICplates.
Ourgroup
(5)
studiedtheeffectof differentagarbasesonthehazing
andfound that different agars influencethe extentoftailing endpoints. Regardless
ofthebaseused,
thereading
ofagar dilution MIC
endpoints
issubject
to morevariability
and
inconsistency
forthis group oforganisms
than itis forotheranaerobes. The E-test MICswere
comparatively
easytoread. Therewas a muchclearer demarcationbetween the haze and the real
growth,
andthus,
an accurateendpoint
could be determined. While the agreement between the
methods was within 1 twofold dilution in most
instances,
therewas
again
atrendfor the Etest toyield slightly
lowerMICs. One
major discrepancy
occurredwith a,B-lactamase-producing
strainofF. nucleatum andpenicillin.
The reason for thisdiscrepancy
isunclear,
but itmight
be related todifferencesin
growth
ratebetweenbothmethods,
therateof,B-lactamase
production,
or the diffusion ofpenicillin
from the E-teststrip.
The otherP-lactamase-nonproducing
F.nucleatum strain and two strains ofF.
necrophorum
wereexquisitely susceptible
topenicillin.
MICs for these organ-isms were <0.015jig/ml
by
both methods. Because thecurrent NCCLS document
(16)
lists thebreakpoint
forpenicillin
as4U/ml(or
2,xg/ml),
wesuggestthatlaboratoriesperform
P-lactamase
testing
on Fusobacterium isolates forwhich
penicillin
MICsare>0.25jig/ml.
Additionaltestswith moreP-lactamase-producing
strains would be ofinterest.The
clindamycin
MICs obtained by both methods afterovernight
incubation were 2 to 3 doubling dilutions lower thanthoseobserved after48 hof incubationfor many of theB.
fragilis
group strains. Thus, when the overnight E-test MICswerecompared
with agardilutionMICsobtained after 48 h ofincubation,
severaldiscrepanciesoccurred. Barry etal.
(3)
evaluated a 24-h disk diffusion method forsuscepti-bility
testing
of the B.fragilis
group and modified thereference
agardilutionmethodby readingthe results at 24 hto make itmore
comparable
tothe diskdiffusiontechniqueforall antibiotics in the
study. Overnight
agardilutionMICdeterminations
inourstudy
weremore comparable toover-nightE-test MICs thanagardilution MICs determined after
the standard 48 h of incubation were.
Broth microdilution is the only method available for
testingindividual anaerobes. Panels forsusceptibility
testing
of anaerobes are commercially available, but many do not
have the selection of antimicrobial agents thatare relevant for the individualpatient. Although the broth microdilution method is considered an acceptable method foranaerobes,
Aldridge and Sanders (1) reported that for 72% of the B.
fragilis group isolates tested by broth microdilution, cefti-zoximeMICs were -fourfold lower than the MICs obtained by agar dilution. Significant variation also occurred with cefoxitinand clindamycin. Inaddition,quality control MICs in the acceptable range for the reference strains have not
been established for microdilutiontechniques (16).
Brook (6) reported that ceftizoxime MICs obtained by agardilution correlated withefficacy in the eradication of B. fragilis and B. thetaiotaomicron from subcutaneous ab-scessesin a mouse model, while the MICs obtainedby broth microdilution did not. Additionally, Lorian (15) has sug-gested that organisms growing on agar moreclosely simulate their state in the body. Therefore, agar methods may be more clinically relevant than broth methods. Additional studies to establish clinical correlation are needed.
The E test provides a simple and rapid method for quantitative susceptibility testing that is suitable for all anaerobes. Antibiotics can be selected for the individual patient. Fastidious organisms can be inoculated onto supple-mented BBA, which supports the growth of almost all anaerobes. After application of the antibiotic carriers, incu-bation time is dependent on the rate of growth of the organism, and in many instances, MICs are available after 20 h. E-test MICs are generally in very good agreement with those obtainedby the agar dilution method. BBAproduced the fewest discrepancies with all the strains, with 97.8% categorical agreement, while supporting optimum growth. WCA was not suitable for testing cefoxitin with the B. fragilis group. The E test is a reliable overnight method for susceptibility testing of rapidly growing anaerobic bacteria.
Slower-growing strains yield good results after 48 h of
incubation.
ACKNOWLEDGMENTS
Thisstudywassupported inpartbygrantsfromMerckSharp & Dohme and theMauriceGoldstein PublicHealth ResearchFund.
Wethank Ellen Jo Baronforreview of the manuscript. REFERENCES
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