Published Ahead of Print 12 April 2010.

10.1128/AAC.00407-10.
2010, 54(6):2567. DOI: Antimicrob. Agents Chemother.
Egervrn, Mauro Giacomini and Peter Vandamme
Lars Axelsson, Jenni Korhonen, Sigrid Mayrhofer, Maria
Tosi, Morten Danielsen, Ana Beln Flrez, Jaana Mtt,
Geert Huys, Klaas D'Haene, Margo Cnockaert, Lorenzo

Acid Bacteria
Bifidobacteria and Nonenterococcal Lactic
Susceptibility Testing Methods for
Two Commercial Antimicrobial
Intra- and Interlaboratory Performances of
http://aac.asm.org/content/54/6/2567
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ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, June 2010, p. 25672574 Vol. 54, No. 6
0066-4804/10/$12.00 doi:10.1128/AAC.00407-10
Copyright 2010, American Society for Microbiology. All Rights Reserved.
Intra- and Interlaboratory Performances of Two Commercial
Antimicrobial Susceptibility Testing Methods for Bidobacteria
and Nonenterococcal Lactic Acid Bacteria

Geert Huys,
1,2
* Klaas DHaene,
2
Margo Cnockaert,
2
Lorenzo Tosi,
3
Morten Danielsen,
4
Ana Bele n Florez,
5
Jaana Ma tto,
6
Lars Axelsson,
7
Jenni Korhonen,
8
Sigrid Mayrhofer,
9
Maria Egerva rn,
10
Mauro Giacomini,
11
and Peter Vandamme
2
BCCM/LMG Bacteria Collection
1
and Laboratory of Microbiology,
2
Department of Biochemistry and Microbiology, Faculty of
Sciences, Ghent University, B-9000 Ghent, Belgium; Istituto di Microbiologia, Universita Cattolica del Sacro CuorePiacenza,
Piacenza, Italy
3
; Chr. Hansen A/S, Boege Alle 10-12, Hoersholm, Denmark
4
; Instituto de Productos Lacteos de Asturias (IPLA),
CSIC, Carretera de Inesto s/n, Villaviciosa, Asturias, Spain
5
; VTT, Technical Research Centre of Finland, Espoo, Finland
6
;
Noma Mat AS, As, Norway
7
; Department of Biosciences, Nutrition and Food Biotechnology, University of Kuopio, Kuopio,
Finland
8
; Department of Food Science and Technology, University of Natural Resources and Applied Life Sciences-BOKU,
Vienna, Austria
9
; Microbiology Division, Research and Development, National Food Administration,
Uppsala, Sweden
10
; and DIST, University of Genova, Genoa, Italy
11
Received 26 March 2010/Accepted 2 April 2010
In a small-scale harmonization study involving nine laboratories in eight European countries, the intra- and
interlaboratory performances of two commercially available systems, i.e., the VetMIC microplate system and
Etest, for antimicrobial susceptibility testing of nonenterococcal lactic acid bacteria (NELAB) and bidobac-
teria were analyzed. In addition, one laboratory also performed standard broth microdilution as a reference
method. MICs of tetracycline, erythromycin, ampicillin, gentamicin, clindamycin, and streptomycin for the
type strains of 25 species of NELAB and bidobacteria and MICs of vancomycin for a selection of relevant taxa
were determined. The previously described lactic acid bacterium susceptibility test medium (LSM) and related
mixed-medium formulations, all including Iso-Sensitest broth as a basic component, were used as test media.
The overall agreement of median MIC ranges 1 log
2
dilution determined by the VetMIC and Etest methods
with the median MICs determined by the reference method was very good for tetracycline, ampicillin, and
streptomycin (92.3 to 100%) but low for erythromycin (19.5 to 30.7%) and clindamycin (50.0 to 80.8%). There
was a consensus among the participating laboratories that VetMIC was preferred over Etest because of its
lower cost, better growth support, and more uniform criteria for MIC end point reading. With the range for
acceptable intralaboratory reproducibility being dened as the median MIC 1 log
2
dilution, VetMIC results
(with 69.2% of all data sets in the acceptable range) were shown to display greater reproducibility than Etest
results (with 58.8% of all data sets in the acceptable range). Also at the interlaboratory level, the proportion
of MIC values obtained with VetMIC that belonged to the complete agreement category (60.0%) was higher
than the proportion of such values obtained with Etest (47.0%), which indicates a higher degree of interlabo-
ratory reproducibility for the former method. Apart from some agent-specic effects, the majority of VetMIC
and Etest replicate data sets were situated within a 1- to 2-log
2
dilution range, suggesting that the two methods
can be considered to be equivalent for recognizing resistance phenotypes. This multicenter study has further
validated the standard use of LSM and related mixed-medium formulations with commercially available
systems and formed the basis for the ongoing development of the ISO 10932/IDF 223 standard for susceptibility
testing of NELAB and bidobacteria.
Because of their distinctive fermentative, functional, and
potentially health-promoting properties, bidobacteria and
nonenterococcal lactic acid bacteria (NELAB) such as lacto-
bacilli, lactococci, and Streptococcus thermophilus are inten-
sively used in the food industry as starter cultures, adjunct
cultures, and probiotics (29). Although the majority of NELAB
and Bidobacterium species are food-grade organisms, the
large-scale application and deliberate introduction of such cul-
tures into the food chain has opened the debate over whether
or not criteria that document their safety for human and ani-
mal use should be dened (35). Despite the overall low patho-
genic potential of these organisms, several studies have indi-
cated that especially NELAB can act as reservoirs of
potentially transferable antimicrobial resistance genes (1, 10,
15, 31). In the eld of probiotics, the absence of acquired
resistance traits has been recommended as a safety criterion in
the selection of new commercial culture probiotic strains for
human use (12, 27, 28, 33).
Due mainly to the limited clinical relevance of NELAB and
bidobacteria, the development and optimization of methods
for antimicrobial susceptibility testing of these organisms have
long been underappreciated. Moreover, the fact that many of
these organisms have specic nutritional and atmospheric re-
* Corresponding author. Mailing address: Laboratory of Microbiol-
ogy, Ghent University, K.L. Ledeganckstraat 35, B-9000 Ghent, Bel-
gium. Phone: 32-9-2645131. Fax: 32-9-2645092. E-mail: Geert.huys
@UGent.be.
Supplemental material for this article may be found at http://aac
.asm.org/.

Published ahead of print on 12 April 2010.

2567
quirements for growth does not allow uniform use of standard-
ized susceptibility test media such as Mueller-Hinton broth
and Iso-Sensitest (IST) broth. There are indications that de
Man, Rogosa, and Sharpe (MRS) medium, which is commonly
used as a growth medium for most of these organisms, may
exhibit antagonistic effects with supplemental antimicrobials in
susceptibility testing (13). To address the apparent limitations
of using single media, a mixed formulation of IST broth (90%,
vol/vol) and MRS broth (10%, vol/vol) referred to as lactic acid
bacterium susceptibility test medium (LSM) was developed
recently (16). This new formulation has proven to support the
growth of a wide taxonomic range of lactobacilli and Bidobac-
terium spp. (when supplemented with 0.03% cysteine) and
minimizes potential antagonism between medium components
and tested antimicrobials. So far, LSM and related mixed-
medium formulations, all containing IST broth as the basic
component, have been successfully used to determine MICs
for members of the genera Bidobacterium, Lactobacillus, Lac-
tococcus, Pediococcus, and Streptococcus by microdilution and
Etest methods (2, 5, 7, 8, 17, 18, 2024, 32).
In the near future, it is expected that the increased use
of LSM as the standard medium for susceptibility testing of
NELAB and bidobacteria will produce a large amount of
MIC data, enabling the denition of epidemiological cutoffs
(ECOFFS). As proposed by the European Committee on
Antimicrobial Susceptibility Testing (EUCAST [http://www
.eucast.org/]), ECOFFS provide an objective basis to differen-
tiate wild-type organisms, which lack acquired and mutational
resistance mechanisms, from non-wild-type members of the
same species that contain one or more mechanisms conferring
antimicrobial resistance. For this purpose, evaluation of the
internal and external quality assurance procedures for refer-
ence methods and commercial systems for susceptibility testing
is absolutely crucial for the correct interpretation of these
ECOFFS. Several harmonization studies of susceptibility test-
ing of clinical (14, 30), veterinary (26, 34), and aquatic (11, 25)
organisms have provided important insights into the reproduc-
ibility of results from standardized methods at the intra- and/or
interlaboratory level. Although such studies would also be
highly valuable to all with an interest in evidence-based bio-
safety assessments of NELAB and bidobacteria for human
and animal use, the performances of susceptibility test meth-
ods using LSM within and across laboratories have to our
knowledge not been evaluated.
Within the framework of an international research project,
the European Union Assessment and Critical Evaluation of
Antibiotic Resistance Transferability in Food Chain (EU-
ACE-ART), a small-scale harmonization study involving nine
laboratories in eight European countries was conducted. The
study set out to examine the performances of two commercial
susceptibility test methods widely used throughout the EU-
ACE-ART project, i.e., the VetMIC system (broth microdilu-
tion) and Etest (agar diffusion), at the intra- and interlabora-
tory levels. For this purpose, MICs of six antimicrobials for the
type strains of 25 NELAB and Bidobacterium species and
MICs of vancomycin for a selection of relevant taxa were
determined using LSM and related mixed-medium formula-
tions as test media.
MATERIALS AND METHODS
Study design and culture conditions. Susceptibility testing by VetMIC and
Etest systems was performed according to standard operating procedures (SOPs)
that were approved by all nine participants in the harmonization study: Labora-
tory of Microbiology, Ghent University, Ghent, Belgium; Istituto di Microbio-
logia, Universita Cattolica del Sacro CuorePiacenza, Piacenza, Italy; Chr.
Hansen A/S, Hoersholm, Denmark; Instituto de Productos La cteos de Asturias,
Villaviciosa, Asturias, Spain; VTT Biotechnology, Espoo, Finland; Noma Mat
AS (previously Matforsk AS), As, Norway; University of Kuopio, Kuopio, Fin-
land; Department of Food Science and Technology, University of Natural Re-
sources and Applied Life Sciences, Vienna, Austria; and Institution of Microbi-
ology, Research and Development, National Food Administration, Uppsala,
Sweden. In addition to taking part in the interlaboratory harmonization study,
one participant also determined the MICs for all strain-antimicrobial combina-
tions (except those with vancomycin) by a noncommercial reference method
based on the use of fresh broth microdilution panels.
The test panel comprised the type strains of 18 NELAB and 7 Bidobacterium
species and Enterococcus faecalis ATCC 29212 (LMG 8222) obtained from the
BCCM/LMG Bacteria Collection, Ghent University, Ghent, Belgium (http:
//bccm.belspo.be/about/lmg.php) (Table 1). The strains were distributed to the
respective participating laboratories as lyophilized cultures. Upon arrival in the
labs, cultures were recovered on MRS medium and maintained on cryobeads
during the entire study. Each strain was tested in three different laboratories
using VetMIC and Etest and in a single laboratory using a broth microdilution
reference method. Per strain-antimicrobial combination tested with VetMIC and
Etest, each participating laboratory had to conduct a series of ve independent
consecutive replications starting from new subcultures. For combinations tested
with the broth microdilution reference method, four consecutive replications
were conducted.
Prior to the actual susceptibility assay, strains were precultured overnight (16
to 24 h, except for bidobacteria, which usually required 40 to 48 h) on the agar
version of the test medium under the recommended incubation conditions (Ta-
ble 1). For lactobacilli, the LSM formulation (i.e., 90% IST broth [code no.
TABLE 1. Growth conditions for antimicrobial susceptibility testing
of NELAB and Bidobacterium type strains
a
Strain Medium
Temp
(C)
B. adolescentis LMG 10502
T
LSM-cysteine 37
B. animalis subsp. animalis LMG 10508
T
LSM-cysteine 37
B. bidum LMG 11041
T
LSM-cysteine 37
B. breve LMG 13208
T
LSM-cysteine 37
B. longum subsp. longum LMG 13197
T
LSM-cysteine 37
B. pseudolongum subsp. pseudolongum
LMG 11571
T
LSM-cysteine 37
B. thermophilum LMG 21813
T
LSM-cysteine 37
E. faecalis LMG 8222 IST broth 37
Lb. acidophilus LMG 9433
T
LSM 37
Lb. amylovorus LMG 9496
T
LSM 37
Lb. brevis LMG 6906
T
LSM 28
Lb. casei LMG 6904
T
LSM 28
Lb. delbrueckii subsp. bulgaricus LMG
6901
T
LSM 37
Lb. fermentum LMG 6902
T
LSM 37
Lb. gasseri LMG 9203
T
LSM 37
Lb. helveticus LMG 6413
T
LSM 37
Lb. johnsonii LMG 9436
T
LSM 37
Lb. paracasei subsp. paracasei LMG 13087
T
LSM 28
Lb. pentosus LMG 10755
T
LSM 28
Lb. plantarum LMG 6907
T
LSM 28
Lb. reuteri LMG 9213
T
LSM 37
Lb. rhamnosus LMG 6400
T
LSM 37
Lb. sakei subsp. carnosus LMG 17302
T
LSM 28
Lb. sakei subsp. sakei LMG 9468
T
LSM 28
Lc. lactis subsp. lactis LMG 6890
T
IST broth 32
S. thermophilus LMG 6896
T
IST-M17-lactose 42
a
All strains were grown under anaerobic conditions. LMG strains were ob-
tained from the BCCM/LMG Bacteria Collection, Ghent University, Ghent,
Belgium.
2568 HUYS ET AL. ANTIMICROB. AGENTS CHEMOTHER.
CM0471; Oxoid], 10% MRS broth [code no. CM0359; Oxoid]) was used, whereas
for bidobacteria, LSM broth was supplemented with 0.3 g/liter l-cysteineHCl
(yielding a formulation hereinafter referred to as LSM-cysteine). The pHs of
LSM and LSM-cysteine were adjusted to 6.7 after autoclaving of the prepara-
tions with sterile HCl solution (16). For S. thermophilus, IST broth (90%) was
supplemented with M17 broth (10% [code no. CM0817; Oxoid]) and 1% lter-
sterilized lactose (yielding a formulation hereinafter referred to as IST-M17-
lactose) (32). Pure IST broth was used for Lactococcus lactis subsp. lactis. In
Etest assays, agar versions (Oxoid) of the respective broth media were used,
except for MRS broth, which was supplemented with 15 g/liter bacteriological
agar no. 1 (code no. LP0011; Oxoid).
VetMIC assay. After overnight incubation, agar cultures were checked for
purity. For inoculum preparation, individual colonies were suspended in a sterile
glass or plastic culture tube containing 2 to 5 ml sterile saline (i.e., 0.85% NaCl
solution) until a density corresponding to a McFarland (McF) standard of 1 or
a spectrophotometric equivalent (3 10
8
CFU/ml) was obtained. The inoculated
saline suspension was diluted 1:1,000 (for inoculation of VetMIC plates) or 1:500
(for inoculation of vancomycin microdilution plates) in the appropriate test
medium (Table 1) to obtain a nal concentration of 3 10
5
or 6 10
5
CFU/ml,
respectively, as recommended previously (6).
VetMIC (National Veterinary Institute, Uppsala, Sweden [http://www.sva.se
/en/Target-navigation/ServicesProducts/VetMIC/]) is a commercially available
microtiter-based system comprising dried antimicrobials in serial 2-fold dilutions
that can be stored for 2 years at room temperature. For the ACE-ART project,
a tailor-made VetMIC plate was designed, enabling susceptibility testing of two
strains per plate for the following six agents: tetracycline (0.5 to 128 g/ml),
erythromycin (0.12 to 16 g/ml), streptomycin (2 to 256 g/ml), gentamicin (0.5
to 32 g/ml), clindamycin (0.12 to 8 g/ml), and ampicillin (0.12 to 8 g/ml).
Susceptibility to vancomycin was tested in a separate broth microdilution assay
for bidobacteria and for members of the Lactobacillus acidophilus group, i.e.,
Lb. acidophilus, Lb. amylovorus, Lb. johnsonii, and Lb. gasseri.
One hundred microliters of the 3 10
5
CFU/ml inoculum was added to each
well (yielding 3 10
4
CFU/well) in columns 1 to 6 (strain 1) or 7 to 12 (strain
2) of individual VetMIC plates within 30 min after the preparation of the
standardized inoculum. According to the VetMIC manufacturer, no additional
homogenization step was required because the antimicrobial compound in each
well dissolves easily in the test medium and diffuses to achieve equilibrium
throughout the well. Microplates were incubated under the species-specic con-
ditions listed in Table 1. When anaerobic jars were used, plates were piled with
a lid between every two plates to generate a homogeneous environment through-
out the jar. Depending on whether the tests were performed at 28 or 37C, Lb.
plantarum ATCC 14917
T
(LMG 6907
T
) or E. faecalis ATCC 29212, respectively,
was included as a control strain during each susceptibility assay. As positive and
negative controls, a standardized inoculum and uninoculated test medium, re-
spectively, were added to wells without an antimicrobial compound.
For vancomycin microdilution assays, a stock solution of 1,280 g/ml vanco-
mycin (product no. V2002; Sigma) in water was prepared, and samples of this
solution were diluted in LSM broth (for members of the Lb. acidophilus group)
or LSM-cysteine (for bidobacteria) to obtain intermediate concentrations over
the range from 0.25 to 256 g/ml. Subsequently, 50-l aliquots of the nal
solutions were dispensed into microdilution plates, which were then sealed in
plastic bags and immediately frozen at or below 20C until needed. Upon
usage, plates were thawed under anaerobic conditions. Each well containing 50
l of a serial vancomycin dilution was inoculated with 50 l of the bacterial
inoculum (6 10
5
CFU/ml of LSM broth [for members of the Lb. acidophilus
group] or LSM-cysteine [for bidobacteria]), resulting in a nal bacterial con-
centration of 3 10
5
CFU/ml (3 10
4
CFU/well) and in a nal antibiotic
concentration of 0.12 to 128 g/ml. The last row in each plate was used as a
sterility control and was inoculated with pure LSM broth or LSM-cysteine. The
same control strains used for VetMIC plates were used.
After 48 h of incubation, growth in VetMIC and vancomycin microdilution
assay systems was evaluated visually by comparing the pellet at the bottom of a
well with the positive and negative controls. Any series of wells in which discon-
tinuity in growth was observed were discarded. Irrespective of the bactericidal or
bacteriostatic mechanism of the tested agent, the MIC was dened as the lowest
antimicrobial concentration for which at least 80% visual reduction in growth
was reported.
Etest. Strain inocula with densities corresponding to a McF standard of 1 or a
spectrophotometric equivalent (3 10
8
CFU/ml) were prepared as described
above. A sterile cotton swab was dipped into the standardized inoculum and used
to inoculate an agar plate of the appropriate test medium (Table 1). Inoculated
plates were allowed to dry for approximately 15 min before application of the
Etest (AB bioMe rieux, Solna, Sweden) and incubated under the recommended
conditions (Table 1). Etest strips with preformed antimicrobial gradients in the
test range from 0.016 to 256 g/ml were applied for tetracycline (article no. 5100
2258; AB Biodisk), erythromycin (article no. 5100 1058; AB Biodisk), strepto-
mycin (article no. 5100 2188; AB Biodisk), gentamicin (article no. 5100 1258; AB
Biodisk), clindamycin (article no. 5100 0958; AB Biodisk), ampicillin (article no.
5100 0158; AB Biodisk), and (for members of the Lb. acidophilus group and
Bidobacterium spp.) vancomycin (article no. 5100 2558; AB Biodisk).
After 48 h of incubation, the MIC was dened as the value corresponding to
the rst point on the Etest strip where growth did not occur along the inhibition
ellipse. For bacteriostatic agents (e.g., tetracycline, erythromycin, and clindamy-
cin), the MIC was read at the point where growth was inhibited by 80% (i.e., the
rst point of signicant inhibition as judged by the naked eye).
Broth microdilution reference assay. Cultures at a McF standard of 1 were
prepared as described for the VetMIC assay. The resulting saline culture sus-
pension was diluted 1:500 in the appropriate test medium (Table 1) to obtain a
nal concentration of 6 10
5
CFU/ml.
The MICs of the following antimicrobial agents were determined: tetracycline
(range, 0.5 to 128 g/ml [product no. T3383; Sigma]), erythromycin (range, 0.12
to 16 g/ml [product no. E5389; Sigma]), streptomycin (range, 2 to 256 g/ml
[product no. S6501; Sigma]), gentamicin (range, 0.5 to 32 g/ml [product no.
G1264; Sigma]), clindamycin (range, 0.12 to 8 g/ml [product no. C5269;
Sigma]), and ampicillin (range, 0.12 to 8 g/ml [product no. A0166; Sigma]). For
each agent, two freshly prepared sterile stock solutions were diluted in the
appropriate test medium to obtain 2-fold dilution series, each encompassing a
range of four concentrations. Each well containing 50 l of a serial antibiotic
dilution was inoculated with 50 l of the bacterial inoculum (6 10
5
CFU/ml of
the appropriate broth), resulting in a nal bacterial concentration of 3 10
5
CFU/ml (3 10
4
CFU/well). The last row in each plate was used as a sterility
control and was inoculated with pure broth. The control strains were the same
used for the other assays. After 48 h of incubation, MICs were determined as
described for the VetMIC assay.
Data processing and statistical analysis. For each participating laboratory,
MIC data and remarks on end point reading were led in a standard table format
and sent to a central laboratory, where they were collected in a central database.
For evaluation of intralaboratory reproducibility, MIC data generated with
VetMIC and Etest were rst converted to log
2
values. In the case of Etest
readings, MIC data that ranked in between 2-fold dilutions were rounded up to
the next 2-fold dilution before conversion to the log
2
scale. For each series of ve
replicate MIC measurements for a specic strain-antimicrobial combination, the
median of the log
2
series and the log
2
value of the median were determined.
Finally, the mathematical differences between each of the ve log
2
values in the
replicate series and the log
2
value of the median were summed. Analysis of these
summed log
2
distances (SLD) across all strain-antimicrobial combinations al-
lowed classication of the variations in MIC data from the individual participat-
ing laboratories into different SLD categories. Intralaboratory reproducibility
was considered acceptable for data sets classied into categories with an SLD of
0 or 1, corresponding to the range encompassing the median MIC 1 dilution
step.
To assess the interlaboratory variation among results from each group of three
laboratories that tested the same strain-antimicrobial combinations, the non-
parametric Kruskal-Wallis one-way analysis of variance was used because no
normalized distribution of MIC data could be assumed (19). Based on the
dened threshold level, the degree of variance per strain-antimicrobial combi-
nation was expressed as one of three categories: (i) total agreement (i.e., results
from all three laboratories were under the threshold), (ii) partial agreement (i.e.,
results from two of the three laboratories were under the threshold), and (iii)
total disagreement (i.e., results from all three laboratories were above the thresh-
old). In addition, the chi-square test was used to check if the two methods
performed in similar ways. The condence interval corresponded to P values of
0.05.
RESULTS
Protocol and study design agreement. In the initial phase of
the study, each of the nine participating laboratories evaluated
the test conditions described in the draft SOPs using Lb. plan-
tarum ATCC 14917
T
or E. faecalis ATCC 29212 for suscepti-
bility assays at 28 or 37C, respectively. During this test phase,
several laboratories reported that accurate reading of Etest
end points was not always possible due to different shapes of
inhibition ellipses. Especially for Etest reading of clindamycin
VOL. 54, 2010 SUSCEPTIBILITY TESTING OF LAB AND BIFIDOBACTERIA 2569
MICs, both bulb-like and narrowing ellipses were observed. In
order to ensure that all ellipse types were read at 80% inhibi-
tion, it was agreed that bulb-like ellipses would be read at the
intersection where the head of the bulb ended in order to
exclude microcolonies. If the ellipse was narrowing, clindamy-
cin MICs were read at the actual intersection and distinct
individual colonies within the lower part of the narrowing
ellipse were included if they were situated very close to the
strip.
Once the SOPs were nalized, Lb. plantarum ATCC 14917
T
and E. faecalis ATCC 29212 were included as control strains in
all assays in the actual harmonization trial. A crossover scheme
was developed to ensure that each laboratory tested seven to
nine type strains and that each strain was tested in three dif-
ferent laboratories using VetMIC and Etest methods. Labora-
tory 5 used only VetMIC, and this arrangement was partially
compensated for by laboratory 2s conducting a higher number
of Etest assays than the other laboratories. Per strain-antimi-
crobial combination, a set of ve independently obtained MIC
values were collected, resulting in totals of 2,875 and 2,755
MIC values generated by VetMIC and Etest procedures, re-
spectively. In addition, laboratory 8 also determined MICs
using the broth microdilution reference method, resulting in
624 MIC values.
Comparison of VetMIC and Etest with the broth microdi-
lution reference method. In order to compare MIC data ob-
tained by commercial VetMIC and Etest assays with those
obtained by the broth microdilution reference method, the
median value in the series of replicate MIC measurements for
each strain-antimicrobial combination analyzed in a given lab-
oratory was determined. Interlaboratory ranges of the median
values for VetMIC and Etest MICs were then compared to the
median values for broth microdilution MICs determined in
laboratory 8 (see Table S1 in the supplemental material), and
percentages of agreement were calculated (Table 2). Depend-
ing on the antimicrobial tested, marked differences in the per-
centages of agreement between the median MICs from the two
commercial assays and those from the reference method were
found. For tetracycline, streptomycin, and ampicillin, 92.3 to
96.2% and 96.2 to 100% of the median MICs determined with
the reference method were within the interlaboratory range of
the median values for VetMIC and Etest MICs 1 log
2
dilu-
tion, indicating very good and excellent agreement, respec-
tively. In contrast, low-level (19.5 to 30.7%) agreement
between the results from commercial assays and those from the
reference method was observed for erythromycin. In between
these two groups of antimicrobials, moderate agreement for
gentamicin (69.2 to 73.1%) and clindamycin (50.0 to 80.8%)
was found. Except for clindamycin MICs, however, no pro-
nounced differences between VetMIC and Etest in the level of
agreement with the reference method were observed.
Intralaboratory performance of susceptibility testing. Each
series of ve independent replicate determinations per strain-
antimicrobial combination submitted by an individual labora-
tory was classied into an SLD category expressing the intra-
laboratory variation between replicate MIC results (Table 3).
The category with an SLD of 0, in which no variation among
the ve replicate values was recorded, contained a clearly
higher proportion of the VetMIC data sets for strain-antimi-
crobial combinations (47.8%) than of the Etest data sets
(30.9%). This difference was apparent for all labs (except lab-
oratory 5) but was not necessarily observed for all strains in the
test panel. For instance, for control strain E. faecalis ATCC
TABLE 2. Percentages of agreement between results from
commercial VetMIC and Etest assays and results from
the broth microdilution reference method
Antibiotic
% Agreement
a
between results from
broth microdilution and:
VetMIC Etest
Erythromycin 19.5 30.7
Tetracycline 96.2 96.2
Streptomycin 92.3 96.2
Ampicillin 92.3 100
Gentamicin 73.1 69.2
Clindamycin 50.0 80.8
Total 70.5 78.8
a
Values are percentages of agreement between the median MICs determined
by broth microdilution (the reference method) in laboratory 8 and the interlabo-
ratory range of the median MICs (1 log
2
dilution) determined by the VetMIC
and Etest methods.
TABLE 3. Intralaboratory reproducibility of VetMIC and Etest results expressed as SLD values
a
Laboratory
No. of combinations tested
(no. of values) for:
No. (%) of data sets with SLD of:
0 1 2 3 3
VetMIC Etest VetMIC Etest VetMIC Etest VetMIC Etest VetMIC Etest VetMIC Etest
1 69 (345) 68 (340) 23 18 9 14 27 23 4 4 6 9
2 60 (300) 79 (395) 29 22 12 29 14 25 4 2 1 1
3 65 (325) 65 (325) 35 20 20 16 6 22 4 4 3
4 94 (470) 94 (470) 56 20 22 30 13 33 3 8 3
5 42 (210) ND 11 ND 9 ND 13 ND 5 ND 4 ND
6 68 (340) 68 (340) 12 11 13 15 18 14 15 12 10 16
7 75 (375) 75 (375) 38 34 21 24 15 17 1
8 52 (260) 52 (260) 39 27 9 15 4 10
9 50 (250) 50 (250) 32 18 8 11 10 13 7 1
Total 575 (2,875) 551 (2,755) 275 (47.8) 170 (30.9) 123 (21.4) 154 (27.9) 120 (20.9) 157 (28.5) 36 (6.3) 37 (6.7) 21 (3.6) 33 (6.0)
a
Laboratories were assigned numbers in random order. All MIC results were read after 48 h. ND, not determined.
2570 HUYS ET AL. ANTIMICROB. AGENTS CHEMOTHER.
29212, the difference in reproducibility of results between
methods was evident, with 69.0% of the series of results ob-
tained with VetMIC situated in the category with an SLD of 0,
compared to only 36.6% of the series of results obtained with
Etest (data not shown). In contrast, no difference for control
strain Lb. plantarum ATCC 14917
T
was recorded, as approxi-
mately 37% of the submitted series of results from both meth-
ods were in the category with an SLD of 0.
The lower number of Etest results for individual strain-
antimicrobial combinations in the category with an SLD of 0
was compensated for by the numbers of Etest data sets as-
signed to categories with SLD of 1 and 2 (Table 3). These
categories contained proportionally more results for strain-
antimicrobial combinations assayed with Etest (27.9 and
28.5%, respectively) than for those assayed with VetMIC (21.4
and 20.9%, respectively). In these categories, intralaboratory
variation was reected by the fact that one of the ve repeated
MIC measurements deviated by 1 log
2
dilution from the me-
dian (SLD 1) or that one MIC value deviated by 2 log
2
dilutions from the median or that two MIC values deviated by
1 log
2
dilution from the median (SLD2). The proportions of
strain-antimicrobial combinations with the highest range of in-
tralaboratory variation (those with data sets with an SLD of
3) were relatively comparable for the VetMIC (9.9%) and
Etest (12.7%) methods. Laboratory 6 submitted the highest
proportional numbers of data sets with SLD of 3 (25 of 57 for
VetMIC and 28 of 70 for Etest). Regardless of the antimicro-
bial tested, the combinations tested in laboratory 6 that yielded
an SLD of 3 involved mainly Lactobacillus strains, as no
bidobacteria were included in the test panel for this labora-
tory.
Interlaboratory performance of susceptibility testing. The
performances of both susceptibility test methods across all
participating laboratories were classied into three variance
categories representing total agreement, partial agreement, or
total disagreement among results from each set of three labo-
ratories that tested the same strain-antimicrobial combinations
(Table 4). Globally, a higher proportion of MIC values ob-
tained with VetMIC (60.0%) than of those obtained with Etest
(47.0%) belonged to the complete agreement category, which
indicates higher interlaboratory reproducibility of results from
the VetMIC method. Overall, 50% of VetMIC values for all
antimicrobials except tetracycline (42.3%) belonged to this
category. Conversely, 50% of the Etest MIC values for all
antimicrobials except tetracycline (61.5%), ampicillin (57.7%),
and vancomycin (66.7%) were in complete agreement among
three laboratories. The complete disagreement category con-
tained a higher proportion of Etest MIC values (28.0%) than
of VetMIC MIC values (18.5%). For all antimicrobials except
tetracycline (15.4%) and vancomycin (16.7%), 20% of the
Etest MIC values were in total disagreement among three
laboratories. In contrast, 20% of the VetMIC values ob-
tained for all agents except tetracycline (30.8%), streptomycin
(23.1%), and gentamicin (26.9%) belonged to this category.
Notably, cases of complete disagreement tended to occur more
frequently among strain-antimicrobial combinations including
a strain of bidobacteria (38 of 98 combinations [38.8%]) than
among those including a strain of lactobacilli (39 of 200 com-
binations [19.5%]) (data not shown).
The interlaboratory reproducibility of results from the two
methods was also statistically analyzed using the chi-square test
(Table 4). This analysis further substantiated the results from
the variance analysis, indicating that VetMIC and Etest essen-
tially performed differently among labs for all tested antimi-
crobials except gentamicin. Across all antimicrobials, however,
the difference between the two methods was not found to be
statistically signicant (P 0.034).
DISCUSSION
Most NELAB and bidobacteria used in food and feed
production have a long history of safe use (35). In this context,
producers usually rely on the GRAS (generally recognized as
safe) denition used in the United States and the QPS (qual-
ied presumption of safety) approach of the European Food
Safety Authority (http://www.efsa.europa.eu/en.html) for the
risk assessment of commercial strains. However, the facts that
acquired antimicrobial resistance mechanisms are known to
occur in NELAB and bidobacteria (1, 10, 15, 31), that the
antimicrobial resistance pattern is strain specic rather than
species specic, that relatively few resistance data are available
for these bacterial groups, and that standardization of suscep-
tibility test methods is currently lacking have raised some con-
cern about the practical implementation of GRAS and QPS
TABLE 4. Interlaboratory reproducibility of VetMIC and Etest results expressed as degrees of variance per antimicrobial agent
Degree of variance Procedure
% of MICs
b
of:
Tetracycline
(26)
Erythromycin
(26)
Streptomycin
(26)
Gentamicin
(26)
Clindamycin
(26)
Ampicillin
(26)
Vancomycin
(12)
All agents
(168)
Complete agreement VetMIC 42.3 69.2 53.8 53.8 69.2 73.1 50.0 59.5
Etest 61.5 38.5 34.6 42.3 38.5 57.7 66.7 47.0
Partial agreement VetMIC 26.9 19.2 23.1 19.2 15.4 23.1 33.3 22.0
Etest 23.0 23.1 26.9 26.9 34.6 19.2 16.7 25.0
Complete disagreement VetMIC 30.8 11.5 23.1 26.9 15.4 3.8 16.7 18.5
Etest 15.4 38.5 38.5 30.8 26.9 23.1 16.7 28.0
P
a
0.104 0.198 0.083 0.029 0.182 0.099 0.099 0.034
a
P values of 0.05 were within the condence interval.
b
All MICs were read after 48 h. Numbers in parentheses after names of agents are numbers of strains tested.
VOL. 54, 2010 SUSCEPTIBILITY TESTING OF LAB AND BIFIDOBACTERIA 2571
approaches. The present study is the rst to determine the
intra- and interlaboratory performances of two protocols for
antimicrobial susceptibility testing of NELAB and bidobac-
teria by using appropriate growth-supporting test media with
IST broth as a basic component.
The general remarks received from the participating labo-
ratories indicated that the preparation and use of LSM and
other mixed-medium formulations were very straightforward
and that VetMIC was preferred over Etest for a number of
practical reasons. First, it was argued that the VetMIC system,
although it offers less exibility in choice and concentration
range of test agents, clearly is less expensive than Etest. Also,
better overall growth of the selected NELAB and Bidobacte-
rium strains was observed in the broth version (used for
VetMIC) than on the agar version (used for Etest) of a given
test medium. For instance, several laboratories reported suf-
cient growth of Lb. amylovorus LMG 9496
T
and Lb. brevis
LMG 6906
T
in LSM broth but very weak growth on LSM agar.
For either of the two methods, 48 h of incubation was required
because several NELAB strains and especially bidobacteria
failed to produce visible growth on LSM after 24 h during
initial growth trials (data not shown). Likewise, Egerva rn and
colleagues (6) observed previously that end points in LSM-
based microdilution testing of Lactobacillus species were more
easily read after 48 h of incubation. Finally, accurate reading of
Etest clindamycin MICs, in contrast to VetMIC clindamycin
MICs, was not always possible due to different shapes of inhi-
bition ellipses and required the addition of specic guidelines
in the SOPs.
Unlike the participants in the present study, other research-
ers (3, 24) have observed ingrowth of resistant microcolonies in
elliptical inhibition zones during Etest analyses of NELAB.
Possibly, this may be due to the high rate of spontaneous
mutation after prolonged (24- to 48-h) incubation with sub-
inhibitory antibiotic concentrations. In a recent study of Lb.
acidophilus group members in which VetMIC was compared to
the agar-based disk diffusion and Etest methods, a minor sat-
isfying level of agreement was specically found for results of
susceptibility testing with bacteriostatic agents such as clinda-
mycin (24). In the present study, VetMIC and Etest were
compared to broth microdilution as a noncommercial refer-
ence method, and MICs of tetracycline, streptomycin, genta-
micin, and ampicillin determined by the two assays were in
good agreement with those determined by the reference
method. In contrast to what could be expected given the re-
ported difculties with reading of Etest results, a higher per-
centage of agreement with the reference MICs within a range
of 1 log
2
dilution was observed for Etest results than for
VetMIC results. The low levels of agreement between the
MICs of erythromycin determined by the commercial assays
and the reference method were surprising and require further
attention. It is the lack of congruence observed for clindamycin
and erythromycin MICs that largely accounted for the differ-
ences in overall agreement with the reference method between
VetMIC (70.5%) and Etest (78.8%). On the other hand, it
should be kept in mind that the broth microdilution reference
method was conducted at only one of the participating labo-
ratories and that, ideally, equivalent numbers of MIC measure-
ments by different methods are needed to compare different
test systems.
The apparent differences in the support of test organism
growth and the uniformity of end point reading between
VetMIC and Etest were also reected in the mathematical
scoring of their performances within and across laboratories.
Based on the criterion that only data sets categorized as having
SLD of 0 or 1 (i.e., those within the range of the median
MIC 1 dilution step) were considered to have acceptable
intralaboratory reproducibility, VetMIC (with 69.2% of data
sets meeting this standard) appeared to display better repro-
ducibility of results than Etest (with 58.8% of data sets meeting
this standard). Clearly, the category with an SLD of 0 had the
largest impact on this difference, i.e., 16.9% of all replicate
series that exhibited no intralaboratory variation when tested
with VetMIC showed a variation of 1 log
2
steps at the in-
tralaboratory level using the Etest system. Accordingly, there
was a shift of the number of Etest series allocated into cate-
gories with SLD of 1 and 2, which was 14.1% higher than the
corresponding number of VetMIC series. A more global anal-
ysis indicates that 87% of all VetMIC (87.3%) and Etest
(90.1%) replicate data sets were situated within a 1 to 2 log
2
deviation range from the median, thus demonstrating that re-
sults from the two methods correlate well at the intralabora-
tory level. Previous comparisons between Etest and microdi-
lution methods revealed differences of up to 2 log
2
dilutions
but indicated the two methods to be equivalent in recognizing
resistance phenotypes (4, 9).
Analysis of interlaboratory performance was based on a
study with each strain-antimicrobial combination being tested
by three laboratories. Although results from such small-scale
studies have to be interpreted with caution, variance analysis
per agent indicated that for all antimicrobials except gentami-
cin, VetMIC appeared to be more suitable for interlaboratory
harmonization than Etest. However, when both VetMIC and
Etest were compared to a reference method (i.e., broth mi-
crodilution), the preference for VetMIC was no longer pro-
nounced. Despite the availability of well-dened SOPs, the fact
that experienced as well as nonexperienced users of Etest con-
tributed to the harmonization trial is likely to have inuenced
the outcome of the study. In most cases, reading of Etest
results is straightforward, but in cases of ingrowth and/or dif-
fuse inhibition zone edges, precise reading may require the
assistance of an experienced user. Also, simultaneous analyses
of intra- and interlaboratory performances of different suscep-
tibility test methods for bacterial groups with complex growth
requirements such as NELAB probably requires a larger set of
MIC measurements. Preferably, future harmonization studies
should include a higher number of participants to more reli-
ably investigate the inuence of previous working experience
on the test performances.
In this study, we opted to include mainly type strains pri-
marily because of their universal availability in public culture
collections worldwide and their well-dened taxonomic status.
However, because most of these type strains appeared to be
susceptible to the tested antimicrobials, it is not clear to what
extent the performance differences observed between VetMIC
and Etest for susceptibility testing of NELAB and bidobac-
teria may affect the recognition of resistance phenotypes. In
this respect, the inclusion of reference strains with phenotyp-
ically and/or genotypically documented resistance in future
harmonization studies would certainly help to assess the im-
2572 HUYS ET AL. ANTIMICROB. AGENTS CHEMOTHER.
pact of these intra- and interlaboratory differences. Undoubt-
edly, accurate and reproducible determination of antimicrobial
MICs remains crucial in the scientic communication between
food/feed industries and regulatory authorities (33). On the
other hand, it is unlikely that intermethod deviations of 1 to 2
log
2
dilutions as commonly measured in the present study will
lead to failure to differentiate between susceptible wild-type
populations and non-wild-type resistant populations, for which
the respective MIC ranges are usually 4 log
2
dilution steps
apart. Clearly, this discrimination will also depend on the avail-
ability, agreement, and interpretation of ECOFFs based on
data for a sufciently large number of geographically and bio-
logically diverse members of a species.
In conclusion, this multicenter study has further validated
the use of LSM and related mixed-medium formulations for
susceptibility testing of bidobacteria and NELAB with
commercially available assays. Noteworthily, the general
protocol that was followed throughout this harmonization
study has been used as a basis to develop the ISO 10932/IDF
223 standard Milk and Milk ProductsDetermination of
the Minimal Inhibitory Concentration (MIC) of Antibiotics
Applicable to Bidobacteria and Non-Enterococcal Lactic
Acid Bacteria (LAB) (http://www.iso.org/iso/iso_catalogue
/catalogue_tc/catalogue_detail.htm?csnumber46434). Pro-
vided that this new standard becomes widely adopted for
susceptibility testing of NELAB and bidobacteria, the def-
inition of ECOFFs is the next step toward a more standard-
ized approach in assessing the biosafety of these organisms
in food and feed production. The recent transfer of all
approved MIC data from the ACE-ART project to
EUCAST for incorporation in an on-line MIC distribution
database (G. Kahlmeter, personal communication) is ex-
pected to stimulate that process in the near future.
ACKNOWLEDGMENTS
This work was funded by the Sixth Framework Programme of the
European Commission and was carried out as part of the project ACE-
ART (CT-2003-506214). G. Huys is a postdoctoral fellow of the Fund for
Scientic ResearchFlanders (F.W.O.Vlaanderen), Flanders, Bel-
gium.
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