PoIish JournaI oI MicrobioIogy

2007, VoI. 56, No 3, 165168

ORIGINAL PAPER
Introduction
Quantitative antibiotic sensitivity can be estimated
by either agar/broth diIution methods, or by agar diI-
Iusion methods. The minimum inhibitory concentra-
tion (MIC) in diIution methods is not aIIected by the
growth oI standard bacteriaI inocuIums because the
antibiotic is compIeteIy active Irom the beginning oI
the incubation period. On the other hand, in diIIusion
methods (Etest, muItidisc method) the concentration
oI the antibiotic at the edge oI inhibition zone at the
time oI its Iormation reIates to the growing bacteriaI
popuIation. This antibiotic concentration, the actuaI
bacteriaI density and the time when the edge oI inhi-
bition zone is Iounded are caIIed the criticaI concen-
tration (Cc), the criticaI popuIation and the criticaI
time, respectiveIy (Linton, 1961; Cooper, 1963; Barry,
1980; DeIignette-MuIIer and FIandrois, 1994). The
criticaI time generaIIy Iasts Ior a period oI severaI
hours aIter the start oI incubation. In sIow growing
bacteria, the criticaI time is Ionger. The criticaI popu-
Iation is thereIore higher than the standard inocuIum
and, consequentIy, the criticaI concentration can theo-
reticaIIy diIIer Irom MIC. The question we hope to
answer is, does it reaIIy diIIer? II yes, then the Etest
wouIdn`t work properIy. However, the Etest has been
reported to be reIiabIe by many authors, aIthough it
has not been weII studied (KronvaII, 2000). The pur-
pose oI this paper is to uncover the theoreticaI expIa-
nation Ior Etest accuracy.
How the antibiotic criticaI concentration can be
measured? II two or more discrete discs with diIIerent
amounts oI the same antibiotic are used, the criticaI
concentration oI the antibiotic can be caIcuIated
Irom the content oIthe discs and Irom thediameter oI
their respective inhibition zones. The IoIIowing ruIes
appIy to thetheory oI inhibition zone Iormation in the
disc method:
G The criticaI time Ior the same bacteria strain,
antibiotic, and cuItivation conditions is indepen-
dent oI the amount oI antibiotic in the disc (oI
the disc content) (Barry, 1980).
G As soon as the edge oI a zone is Iormed, its
diameter wiII not change (Linton, 1961; Barry,
1980).
G The reIation between the area oI inhibition zone
and naturaI Iogarithm oI antibiotic disc con-
tent is Iinear (Barry, 1980; KronvaII, 1982;
DeIignette-MuIIer and FIandrois, 1994).
Reliability of the Etest in Light of the Correlation between an Antibiotic`s Critical
Concentration (Cc) and MIC Values
MAREK BEDNAR*
Department oI MedicaI MicrobioIogy, CharIes University, 3
rd
FacuIty oI Medicine, Prague, Czech RepubIic
Received 24 February 2007, revised 20 JuIy 2007, accepted 24 JuIy 2007
Ab s t r a c t
The study reIates to the theory oI diIIusion methods Ior antibiotic sensitivity testing. The aim oI the study was to show the reIationship
between the antibiotic criticaI concentration (Cc) and its minimum inhibitory concentration (MIC). The resuIts contribute to the expIana-
tion oI the Etest`s reIiabiIity and support the scientiIic basis Ior MIC determination using agar diIIusion methods. SusceptibiIity among 90
cIinicaI isoIates oI 12 common aerobic bacteriaI species to gentamicin, erythromycin, or oxaciIIin was assessed using the muItidisc method
(Ior Cc), by the agar diIution method (Ior MIC) and by the Etest. The resuIts oI aII three methods were statisticaIIy compared and Iound to
be cIoseIy reIated. The regression equation Ior Cc vaIues and MIC was Iog
2
(MIC) 0.99Iog
2
(Cc)0.13; r 0.99; p0.05; the regression
equation Ior Cc vaIues and Etest-MIC (Et) was Iog
2
(Et) 0.86Iog
2
(Cc)0.34; r 0.96; p0.05; the regression equation Ior Etest-MIC
vaIues and MIC was Iog
2
(MIC) 1.12Iog
2
(Et)0.50; r 0.96; p0.05.
Ke y wo r d s: Etest, muItidisc method, antibiotic criticaI concentration (Cc), antibiotic sensitivity testing
* Corresponding author: M. Bednar, Department oI MedicaI MicrobioIogy, CharIes University, 3rd FacuIty oI Medicine, Ruska 87,
100 00 Prague, Czech RepubIic; phone: (42) 26 7162580; Iax: (42) 26 7162516; e-mail: bednarmcbox.cz
166 Bednar M. 3
G The antibiotic diIIuses into the agar according to
Fick`s second Iaw oI diIIusion (VesterdaI, 1947;
Humphrey and Lightbown, 1952; Koch, 1999).
Note on ruIe 3): the reIation between the radius oI
the inhibition zone and antibiotic disc content is ex-
pressed by the equation
1}
where r
n
is the radius oI inhibition zone in n-th disc
and m
n
is the content oI the disc. A graphic image oI
this dependency is a straight Iine. Its intercept a and
sIope b can be caIcuIated Irom the measured data by
common statisticaI procedures. Measurement oI the
zone radius Irom the center oI discs provides a better
correIation with disc content and size oI inhibition
zone, in terms oI equation 1}, than does measure-
ment oI the zone radius starting Irom the edge oI the
disc (KronvaII, 2000).
Note on ruIe 4): a derived radiaI Iaw Ior two-
dimensionaI cyIindricaI diIIusion appIies to the disc
method (Humphrey and Lightbown, 1952; Koch, 1999).
2}
where Cc

(g/mI) is the criticaI concentration oI anti-
biotic, m
n
(g) is the antibiotic disc contentat the
beginning oI diIIusion, r
n
(cm) is the radius oI the
respective inhibition zone, measured Irom the disc
center D (cm
2
/s) is the diIIusion constant oI the anti-
biotic, t
c
(s) is the criticaI time, d (cm) is the agar
depth, and e is the base oI naturaI Iogarithms. The
denominator 4*Dt
c
d reIIects the drop in antibiotic
concentration in the disc during the criticaI time and
the agar depth. DimensionaI anaIysis oI the IormuIa
yieIds g/mI, which conIirms the mathematicaI vaIid-
ity oI the IormuIa.
It can be proven that b 4Dt
c
. This is in agree-
ment with the research done by others (Cooper, 1963;
Drugeon et al., 1987; DeIignette-MuIIer and FIandrois,
1994; Koch, 1999). In IormuIa 2}, you can substi-
tute sIope b Ior 4Dt
c
and we can substitute a bIn(m
n
)
Ior r
2
n
. By consequent reduction, we arrive at resuIting
IormuIa combining equations 1} and 2}
*3}
where a is the intercept, b is the sIope oI regres-
sion Iine according to equation 1}, and d is the agar
depth. FormuIa 3} caIcuIates the criticaI concentra-
tion oI an antibiotic without knowing its diIIusion
constant or criticaI time (i.e., without caIibration). II
we use at Ieast three discs, the reIiabiIity oI the resuIt
can be ascertained Irom the correIation oI the Ioga-
rithms oI the discs` content andthe sizes oI the re-
spective inhibition zones.
Experimental
Materials and Methods
SusceptibiIity among ninety cIinicaI isoIates oI
common aerobic bacteriaI species to gentamicin,
erythromycin, or oxaciIIin was assessed. We perIormed
90 concurrent sensitivity measurements using the
muItidisc diIIusion method, standard agar diIution
method and Etest. Sensitivity was measured 33 times
Ior oxaciIIin, 27 times Ior gentamicin, and 30 times
Ior erythromycin. These antibiotics were chosen due to
their diverse mode oI action. They aIso provide sharp,
cIear zones oI inhibition in the diIIusion method.
Bacterial strains and culture media. Forty-Iive
strains oI Staphvlococcus aureus, seventeen strains oI
Staphvlococcus epidermidis, six strains oI Pseudomo-
nas aeruginosa, six strains oI Proteus mirabilis, six
strains oI Escherichia coli, Iour strains oI Klebsiella
spp., three strains oI Acinetobacter baumanii, two
strains oI Enterobacter spp. and one strain oI Entero-
coccus faecalis were used. AII the strains were iso-
Iated during routine investigations oI various cIinicaI
specimens ina hospitaI Iaboratory. The strains were
chosen according to the quaIitative sensitivity testing
(NCCLS, 1993a) so as to obtain three sets oI strains
Ior measurements oI the sensitivity to given antibiotic
over a wide range oI MIC vaIues. Erythromycin or
oxaciIIin sensitivity was checked in Gram-positives
whereas gentamicin sensitivity in Gram-negatives. The
actuaI experiment invoIved a quantitative assessment
oI the sensitivity to a particuIar antibiotic in seIected
strains using the muItidisc method, agar diIution
method, and Etest. The tests were run concurrentIy Ior
each inocuIum. We used MueIIer-Hinton agar (Oxoid,
Unipath Ltd., Basingstoke, Hampshire, EngIand)
poured to a depth oI 4 mm in Petri dishes (diameter
90 mm) Ior aII three methods. The inocuIa Ior aII three
methods came Irom coIonies that were suspended in
physioIogicaI saIine soIution to a density oI 0.5 on
the McFarIand scaIe. PIates used Ior the muItidisc
method and the Etest were inocuIated by swabbing.
Etest. Etest strips (Gentamicin Iow range; Erythro-
mycin; OxaciIIin) were used according to the manu-
Iacturer`s instructions (AB Biodisk, SoIna Sweden).
Agar pIates were incubated at 35C. ResuIts were
read aIter 24 hours according to the manuIacturer`s
reading guide.
Antibiotics used. Antibiotic soIutions were pre-
pared by diIution oI injectabIe preparations: Gentami-
cin LEK PharmaceuticaIs and ChemicaI Co., SIovenia
(gentamicin), Erythrocin Abbott Laboratories, USA
(erythromycin), and ProstaphiIin BristoI-Myers Squibb
S.p.A., ItaIy (oxaciIIin).
Critical concentration (Cc) determinations
(multidisc diffusion method). Four 6 mm diameter
bIank paper discs (Oxoid, Unipath Ltd., Basingstoke,
r
2
n
a b In(m
n
)
* Equation 3} used in this work was brieIIy described in the
MedicaI Science Monitor (2000) 6: 168170.
Cc e

m
n
4*D
t
, d
r
2
n
4Dt
c
Cc e

a
b
1
*bd
167 ReaIibiIity oI Etest, correIation between MIC and antibiotic Cc 3
Hampshire, EngIand) were pIaced onto the inocuIated
agar. An appropriate amount oI antibiotic, dissoIved
in 20 I distiIIed water, was dropped onto discs using
a pipette. The amount oI antibiotic on the discs was
estimated on the basis oI previous quaIitative sensi-
tivity testing oI aII strains (NCCLS, 1993a) so as to
achieve a minimum oI two measurabIe yet distinct in-
hibition zones when using Iour discs (TabIe I). Agar
pIates were incubated at 35C Ior 24 hours. The
diameters oI inhibition zones were measured using an
eIectronic caIIiper. Non-rounded zone diameters and
data on antibiotic disc content were used to caIcuIate
the sIope b and intercept a according to equation 1}.
These constants were subsequentIy empIoyed Iorthe
caIcuIation oI the criticaI concentration oI the antibi-
otics using IormuIa 3}. Where at Ieast three inhibi-
tion zones Iormed, the percentage oI variation in the
zones expIained by the disc content Iogarithms (coeIIi-
cient oI determination R
2
written as a percentage) was
aIso caIcuIated to show the reIiabiIity oI the resuIt.
MIC determination. The assessment oI MIC us-
ing the agar diIution method was perIormed in accor-
dance with NCCLS guideIines (NCCLS, 1993b). The
antibiotics were the same as those used in the
muItidisc diIIusion method. FinaI concentrations oI
antibiotics in the agar ranged Irom 0.012 to 512 mg/I.
The diIutions were based on a geometricaI order (Iac-
tor 2) and were reIated to concentrations oI 1mg/I and
1.5 mg/I (0.012, 0.016, 0.023, 0.031, 0.047, 0.063,
0.094, 0.125, 0.19, 0.25, 0.38, 0.5, 0.75, 1.0, 1.5, 2, 3,
4, 6, 8, 12, 16, 24, 32, 48, 64, 96, 128, 192, 256, 384,
512 mg/I). The inocuIum was appIied to the agar sur-
Iace by means oI a pin repIicator. Agar pIates were
incubated at 35C. ResuIts were read aIter 24 hours.
Statistical evaluation of results. The resuIts were
cIassiIied by the appIied method onIy, not by bacte-
riaI strain or the antibiotic used. The correIations oI
Etest resuIts withMIC, muItidisc diIIusion method re-
suIts with MIC and muItidisc diIIusion method resuIts
with Etest resuIts, were expressed using the Spearman
rank correIation coeIIicient. AIter transIorming the re-
suIts into base 2 Iogarithms, we expressed them by
means oI the Pearson correIation coeIIicient. Statisti-
caI vaIues were caIcuIated by Statistica Ior Windows
(StatSoIt Inc.). DiIIerences no greater than a twoIoId
diIution Iactor between the MIC and the Etest or be-
tween the MIC and the muItidisc method, were used
to caIcuIate agreement (PIaIIer et al., 2000).
Results
Agreement oI Cc with MIC was observed in 89
out oI 90 concurrent criticaI concentration measure-
ments. Regression straight Iine is shown in Figure 1.
We observed two disagreements between the MIC and
Fig. 1. CorreIation oI resuIts oI sensitivity measurements by muItidisc method (Cc criticaI concentration) and agar diIution
method (MIC minimum inhibitory concentration). Broken Iines indicate the 95 conIidence band; some points overIap
TabIe I
Antibiotic disc content in individuaI discs A, B, C, and D
in muItidisc method rounded to three signiIicant digits
In order that the number oI discs in the muItidisc method can be reduced
to Iour, strains were initiaIIy subdivided into groups that were either
sensitive or resistant using a routine quaIitative disc method. Because oI
practicaI reasons, the ratio between the neighboring discs` contents is
usuaIIy (but not obIigatory) reguIar.
Erythromycin 1000 200 40.0 8.00 200 40.0 8.00 1.60
Gentamicin 800 256 64.0 16.0 256 64.0 4.00 1.00
OxaciIIin 5000 714 102 14.6 102 14.6 2.08 0.298
Antibiotic
Disc content (g)
Resistants Sensitives
D C B A D C B A
168 Bednar M. 3
the Etest. AII disagreements occurred among resistant
strains. Both the muItidisc method and the Etest
correIated weII with the diIution agar method: both
the Spearman and Pearson coeIIicients reached at Ieast
0.9. The reIation between the resuIts oI the muIti-
disc method (Cc) and MIC was Iog
2
(MIC) 0.99
Iog
2
(Cc)0.13; r 0.99; p0.05. The reIation between
the Etest resuIts (Et) and MIC was Iog
2
(MIC)
1.12Iog
2
(Et)0.50; r 0.96; p0.05. The reIation be-
tween Cc and Et was Iog
2
(Et) 0.86Iog
2
(Cc) 0.34;
r 0.96; p0.05. The reIiabiIity oI the muItidisc
method expressed as the average percentage oI varia-
tion in the zones expIained by the disc content Ioga-
rithms was 98.85 (92.36100.00). The b vaIue in
IormuIa 1} was not reIated to the sensitivity oI the
strains. Thus, the criticaI time was independent Irom
strain sensitivity. Neither species-dependent nor anti-
biotic type-dependent irreguIarities in Cc-MIC reIa-
tionship were Iound.
Discussion
According to the cIassicaI theories (Cooper, 1963;
Barry, 1980; Hedges, 1999), the bacteriaI growth rate
impacts the inhibitory zone diameter. In the Kirby-
Bauer quaIitative disc diIIusion method, this pheno-
menon is soIved by the interpretative standards
(NCCLS, 1993a). The cruciaI question is whether
such 'inaccuracy substantiaIIy inIIuences the Etest
resuIt. The resuIts oI our work show that it does not,
because the antibiotic (criticaI) concentration under-
neath the edge oI the Iorming zone practicaIIy equaIs
MIC. So in Etest the bacteriaI growth rate may
impact the inhibitory zone shape but not its point oI
intersection with the scaIe on the strip.
In the muItidisc method, the concentration oI anti-
biotic on each additionaI discs is, optimaIIy, Iour to
seven times Iower than on the preceding disc (depend-
ing on the type oI antibiotic). Because the experimen-
taI design initiaIIy divided the strains into either sen-
sitive or resistant, we were abIe to reduce the number
oI discs used to Iour and stiII obtain at Ieast two mea-
surabIy distinct zones oI inhibition Ior an accurate caI-
cuIation oI the criticaI concentration. Without this ini-
tiaI categorization, Iive or six discs wouId be required
to test over the IuII scaIe oI an Etest strip. Such increas-
ing disc number brings the muItidisc method cIoser to
the Etest, which can be imagined as a chainoI antibio-
tic discs with exponentiaIIy growing antibiotic con-
tent. The primary data in both methods are the criticaI
concentrations oI antibiotics. The Etest and muItidisc
method do have simiIarities the zones (incIuding the
zero zones) are aIways Iormed aIter the criticaI time
passes. In the case oI the Etest, the criticaI concentra-
tion iI r 0 estimates MIC using a printed scaIe.
The correIation between the criticaI concentration
and MIC is not a new Iinding. NevertheIess, the extent
oI the correIation is surprising. It impIies that in diI-
Iusion quantitative methods, bacteriaI growth up untiI
the criticaI time does not inIIuence the resuIt. This
observation contributes to an understanding oI the
accuracy oI the Etest on a wide variety oI organisms,
and indicates that the resuIts obtained with quantita-
tive diIIusion methods (E-test, muItidisc method) can
be expressed as MICs without any conversion.
Acknowledgement
The author wouId Iike to thank Marie Duskova and Zorka
Haasova Ior their technicaI Iaboratory assistance and Jiri Horacek
Ior the soItware production. The program Ior the criticaI concen-
tration caIcuIation is downIoadabIe on http://www.II3.cuni.cz/
ustavy/mikrobioIogie/downIoad/atbcc.zip; Iast accessed 7/07/07.
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