International Journal of Food Microbiology 82 (2003) 1 – 11

www.elsevier.com/locate/ijfoodmicro

Susceptibility of Lactobacillus spp. to antimicrobial agents

Morten Danielsen *, Anette Wind
Identification Section, Applied Biotechnology, Chr. Hansen A/S, Bøge Allé 10-12, 2970 Hørsholm, Denmark

Received 9 September 2001; received in revised form 24 May 2002; accepted 14 June 2002

Abstract

Bacteria used as probiotics or in starter cultures may serve as hosts of antibiotic resistance genes, which can be transferred to
pathogenic bacteria. Before launching a starter culture or a probiotic product into the market, it is therefore important to verify
that the single bacterial isolates (strains) do not contain transferable resistance genes. A study has been undertaken to establish
the levels of susceptibility of Lactobacillus spp. to various antimicrobial agents. This is a prerequisite for differentiating putative
transferable resistance from natural resistance. A selection of 62 strains has been screened with the use of the Etest (ABBiodisk,
Stockholm, Sweden) for their susceptibility to 25 antimicrobial agents. The strains belonged to the following species:
Lactobacillus plantarum/pentosus, L. rhamnosus, L. paracasei, L. sakei, L. curvatus and species of the L. acidophilus group: L.
johnsonii, L. crispatus, L. gasseri, and L. acidophilus.
The results from the Etests have shown that the level of susceptibility to the antimicrobial agents is species-dependent. For
the following antimicrobial agents, susceptibility varied several folds between species: vancomycin, teicoplanin, tetracycline,
norfloxacin, ciprofloxacin, fusidic acid, and clindamycin. The differences between the species were more subtle for the rest of
the tested antimicrobial agents. On the basis of the result, it was possible to suggest minimal inhibition concentrations (MICs)
for the individual Lactobacillus species to be used as a microbiological breakpoint when screening strains for transferable
resistance genes.
D 2002 Elsevier Science B.V. All rights reserved.

Keywords: Susceptibility; Lactobacillus spp.; Antimicrobial agents

1. Introduction the risk of transferring the genes to pathogenic bac-

In recent years, increased focus has been given to
food as potential vehicles of antibiotic resistance
genes (Perreten et al., 1997; Franz et al., 1999; Klein,
2000). It is of general belief that starter cultures have
the potential to serve as a reservoir of such genes with
* (updated 2001), a report by the Scientific Committee
Corresponding author. Tel.: +45-45-74-84-54; fax: +45-45-
74-89-94.
E-mail address: morten.danielsen@dk.chr-hansen.com
(M. Danielsen).
teria. The presence of resistance genes in many lactic
acid bacteria (LAB) and the transfer of plasmids and
conjugative transposons to and from LAB have been
reported as reviewed by Teuber et al. (1999).
Probiotics for use in the EU as additives in feeding-
stuffs must comply with the guidelines described in
the EU council directive 87/153/EEC. In 1997
for Animal Nutrition (SCAN) recommended to the
EU commission that the absence of transferable
resistance genes should be an important prerequisite

0168-1605/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved.
PII: S 0 1 6 8 - 1 6 0 5 ( 0 2 ) 0 0 2 5 4 - 4
 2
Table 1
Distribution of MICs of h-lactams (penicillins, cephalosporins, and carbapenems) for Lactobacillus species
Antibiotic Species Number of isolates for which the MIC (Ag/ml) was as follows:
V 0.03 0.06 0.12 0.25 0.5 1 2 4 8 16 32 64 128 z 256 na

M. Danielsen, A. Wind / International Journal of Food Microbiology 82 (2003) 1–11

Amoxicillin, L. plantarum/pentosus 5 1 2 1 9
clavulanic acid L. paracasei/rhamnosus 2 3 4 6 15
L. acidophilus group 2 1 1 4
L. sakei/curvatus 0
Ampicillin L. plantarum/pentosusb 1 8 3 1 3 2 18
L. paracasei/rhamnosus 2 3 10 7 22
L. acidophilus group 2 9 4 1 16
L. sakei/curvatus 2 2 1 1 6
Benzylpenicillin L. plantarum/pentosusb 1 4 8 5 Etest max 32c 18
L. paracasei/rhamnosus 2 9 9 1 1 22
L. acidophilus group 1 6 8 1 16
L. sakei/curvatus 2 2 2 6
Oxacillin L. plantarum/pentosusb 1 7 10 18
L. paracasei/rhamnosus 3 9 9 1 22
L. acidophilus group 3 6 6 1 16
L. sakei/curvatus 1 2 3 6
Cephalothin L. plantarum/pentosus 2 3 6 7 18
L. paracasei/rhamnosus 1 2 4 11 3 1 22
L. acidophilus group 3 7 3 2 1 16
L. sakei/curvatus 1 2 1 2 6
Cefoxitin L. plantarum/pentosus 7 7
L. paracasei/rhamnosus 15 15
L. acidophilus group 1 1 1 1 4
L. sakei/curvatus 1 1 2
Imipenemd L. plantarum/pentosus 4 9 1 1 1 2 Etest max 32c 18
L. paracasei/rhamnosus 1 3 4 13 1 22
L. acidophilus group 2 4 7 3 16
L. sakei/curvatus 1 2 2 1 6
a
n = number of strains tested.
b
Haze often observed within inhibition zone.
c
Etest max 32: The concentration on the strips was maximum 32 Ag/ml.
d
Pinpoint colonies often observed.
 M. Danielsen, A. Wind / International Journal of Food Microbiology 82 (2003) 1–11 3

for approval (EC, 2001a). In an opinion from the
SCAN on the 3rd of July 2001, it is explicitly told
how to screen strains for the absence of transferable
resistance genes (EC, 2001b). A number of micro-
biological breakpoints are given on the basis of two
published articles (Barrett and Jones, 1996; Zarazaga
et al., 1999). Any level of susceptibility, which cannot
be accounted for, is subject to further analysis by in
vitro experiments to be proven intrinsic or mutational
(EC, 2001b). The coming EU guidelines for trans-
ferable resistance genes in probiotics for animals will
most likely be extended to probiotics for humans and
starter cultures in the near future (EC, 2000, 2001b).
Similar guidelines might also be implemented in the
US as increased monitoring of antibiotic resistance to
antimicrobial agents is planned (ITFAR, 2000).
Published information comparing MICs of indi-
vidual Lactobacillus species and including a fair
number of isolates, to our knowledge, is limited to
the works of Felten et al. (1999) and Zarazaga et al.
(1999). Both articles stress the need for differentiat-
ing between individual Lactobacillus species. Fur-
thermore, data have been published on the screening
of Lactobacillus delbrueckii subsp. bulgaricus and
lactis (Katla et al., 2001) and L. rhamnosus (Char-
teris et al., 2001). Knowledge about natural resist-
ance for individual Lactobacillus species will lessen
the need for extensive experiments as suggested by
SCAN and increase the safety of new products.
This study has been carried out to make a contri-
bution to the establishment of relevant microbiolog-
ical breakpoints for a number of Lactobacillus
species.
2. Materials and methods
2.1. Bacterial strains
A selection of 62 strains from the Chr. Hansen
Culture Collection was used for the survey. In total,
18 L. plantarum/pentosus, 13 L. paracasei, 9 L. rham-
nosus, 5 L. sakei, 1 L. curvatus, and 16 from the L.
acidophilus group (3 L. acidophilus, 3 L. johnsonii, 6 L.
crispatus, and 4 L. gasseri) were used for the study. L.
paracasei, L. rhamnosus, and L. crispatus have been
identified to species level by 16S rRNA sequencing
(Mori et al., 1997; Pavlova et al., 2002). L. sakei, L.
curvatus, L. acidophilus, L. johnsonii, and L. gasseri
have been identified to species level by rRNA-targeted
oligonucleotide probe hybridisation (Hertel et al.,
1991; Pot et al., 1993). The strains of L. plantarum/
pentosus were identified by 16S rDNA sequencing but
could not be differentiated, as the 16S rDNA sequences
of the type strains are identical. All strains have been
genotyped with pulsed field gel electrophoresis and
have different fingerprints (results not shown).
2.2. Susceptibility testing
The Etest (ABBiodisk) was used according to the
manufacturer’s instructions. As no standards exist for

Table 2
Distribution of MICs of non-h-lactam cell wall acting antimicrobial agents for Lactobacillus species
Antibiotic Species Number of isolates for which the MIC (Ag/ml) was as follows:
V 0.03 0.06 0.12 0.25 0.5 1 2 4 8 16 32 64 128 z 256 na
Bacitracin L. plantarum/pentosus 1 3 3 11 18
L. paracasei/rhamnosus 2 4 3 1 12 22
L. acidophilus group 2 6 2 3 2 1 16
L. sakei/curvatus 2 1 3 6
Vancomycin L. plantarum/pentosus 6 6
L. paracasei/rhamnosus 15 15
L. acidophilus group 1 12 3 16
L. sakei/curvatus 2 1 3 6
Teicoplanin L. plantarum/pentosus 6 6
L. paracasei/rhamnosus 1 14 15
L. acidophilus group 1 1 1 2 5
L. sakei/curvatus 0
a
n = number of strains tested.
4 M. Danielsen, A. Wind / International Journal of Food Microbiology 82 (2003) 1–11

susceptibility testing of lactobacilli, the following con-
ditions were found to ensure semi-confluent growth
and thereby optimal susceptibility testing. A suspen-
sion with a density of McFarland 1 in saline buffer was
swapped in three directions on 4-mm-thick MRS agar
(Oxoid, Hampshire, England) with a cotton swap. The
plates were incubated in anaerobic jars with Oxoid
AnaeroGen (Oxoid) for 48 h before reading the plates.
MRS agar was used in order to ensure good growth of
the lactobacilli as the standard susceptibility test agars
Mueller – Hinton and Iso-Sensitest agar fail to do so.
MRS agar has been used in other surveys but little is
known about the interaction of MRS and different
antimicrobial agents. MICs for the bacteriostatic drugs
tetracycline, erythromycin, clindamycin, sulphadia-
zine, and trimethoprim/sulphamethoxazole were read
at 80% inhibition, i.e. the first point of significant
inhibition as judged by the naked eye as recommended

Table 3
Distribution of MICs of antimicrobial agents that inhibit protein or mRNA synthesis for Lactobacillus species
Antibiotic Species Number of isolates for which the MIC (Ag/ml) was as follows:
V 0.03 0.06 0.12 0.25 0.5 1 2 4 8 16 32 64 128 z 256 na
Chloramphenicol L. plantarum/pentosus 2 11 5 18
L. paracasei/rhamnosus 4 17 1 22
L. acidophilus group 3 5 8 16
L. sakei/curvatus 3 3 6
Clindamycin L. plantarum/pentosus 2 2 2 1 3 2 4 2 18
L. paracasei 1 6 4 2 13
L. rhamnosus 3 5 1 9
L. crispatus 1 3 1 1 6
L. acidophilus/johnsonii 2 3 1 6
L. gasseri 1 1 2 4
L. sakei/curvatus 2 1 3 6
Erythromycin L. plantarum/pentosus 2 11 5 18
L. paracasei/rhamnosus 1 2 6 10 1 2 22
L. acidophilus group 1 3 6 5 1 16
L. sakei/curvatus 1 1 4 6
Fusidic Acid L. plantarum/pentosus 2 2 4 7 2 1 18
L. paracasei/rhamnosus 15 15
L. acidophilus group 4 4
L. sakei/curvatus 1 1 3 1 6
Rifampicin L. plantarum/pentosus 3 5 6 4 18
L. paracasei/rhamnosus 1 6 12 1 1 1 22
L. acidophilus group 3 3 1 1 2 6 16
L. sakei/curvatus 4 1 1 6
Tetracycline L. plantarum/pentosus 1 2 7 5 1 2 18
L. paracasei/rhamnosus 4 14 3 1 22
L. acidophilus group 1 5 7 1 1 1 16
L. sakei/curvatus 2 3 1 6
Gentamicin L. plantarum/pentosus 3 2 10 3 18
L. paracasei/rhamnosus 1 3 6 7 3 2 22
L. acidophilus group 2 3 1 5 1 3 1 16
L. sakei/curvatus 1 2 2 1 6
Kanamycin L. plantarum/pentosus 11 11
L. paracasei/rhamnosus 1 1 1 12 15
L. acidophilus group 5 5
L. sakei/curvatus 2 1 3 6
Streptomycin L. plantarum/pentosus 1 17 18
L. paracasei/rhamnosus 1 2 3 2 4 10 22
L. acidophilus group 2 2 5 6 1 16
L. sakei/curvatus 6 6
a
n = number of strains tested.
Table 4
Distribution of MICs of antimicrobial agents not presented in Tables 1 – 3 for Lactobacillus species

M. Danielsen, A. Wind / International Journal of Food Microbiology 82 (2003) 1–11

Antibiotic Species Number of isolates for which the MIC (Ag/ml) was as follows:
V 0.03 0.06 0.12 0.25 0.5 1 2 4 8 16 32 64 128 z 256 na
Sulphadiazine L. plantarum/pentosus 6 6
L. paracasei/rhamnosus 15 15
L. acidophilus group 4 4
L. sakei/curvatus 2 2
Trimethoprim/ L. plantarum/pentosus 1 1 5 Etest max 32b 7
Sulphamethoxazole L. paracasei/rhamnosus 15 15
L. acidophilus group 4 4
L. sakei/curvatus 2 2
Ciprofloxacin L. plantarum/pentosus 8 Etest max 32b 8
L. paracasei/rhamnosus 1 2 7 7 4 1 22
L. acidophilus group 6 6
L. sakei/curvatus 1 5 6
Norfloxacin L. plantarum/pentosus 8 8
L. paracasei/rhamnosus 1 4 5 5 15
L. acidophilus group 4 4
L. sakei/curvatus 0
Nitrofurantoinc L. plantarum/pentosus 1 4 5 1 2 1 4 18
L. paracasei/rhamnosus 1 1 10 3 4 2 1 22
L. acidophilus group 1 2 6 5 1 1 16
L. sakei/curvatus 2 1 1 1 1 6
Metronidazole L. plantarum/pentosus 7 7
L. paracasei/rhamnosus 15 15
L. acidophilus group 4 4
L. sakei/curvatus 2 2
a
n = number of strains tested.
b
Etest max 32: The concentration on the strips was maximum 32 Ag/ml.
c
Pinpoint colonies often observed.

5
6 M. Danielsen, A. Wind / International Journal of Food Microbiology 82 (2003) 1–11

by the manufacturer. For nitrofurantoin and imipenem, 2.3. Antimicrobial agents

pinpoint colonies were sometimes observed within the
inhibition zone. These colonies are not included in the
MICs. Microbiological breakpoints are suggested in
Table 5 based on the susceptibility testing results.
The breakpoints in this paper are defined as the MIC
right above the apparent normal range of the species
for a given antimicrobial agent. Strains with MICs
equal to or higher than the breakpoints are considered
resistant (EC, 2001b).
An initial selection of 25 different antimicrobial
agents was used to screen a subset of the strains.
Subsequently, the number of antimicrobial agents
used was dependent on the species. For those combi-
nations of antimicrobial agents and species where no
or little extra information was gained, the number was
reduced to approximately 15. Two examples of this
reduction are norfloxacin (which gave the same pat-

Table 5
Microbiological breakpoints for Lactobacillus species used in food or as probiotics
Antibiotic Speciesa Proposed breakpoint, MIC (Ag/ml)
This article SCAN Other articles
Amoxicillin, clavulanic acid All 2 8b
Ampicillin All 4 2 or Intrinsic 0.5c
Benzylpenicillin All 4 1d, 8b
Ciprofloxacin All > 32 4 or Intrinsic >32c, 4 or >32e
Chlorampenicol All 16 16 16c,d
Clindamycin par 0.5
sak, cur 1
rha 2
aci (except L. gasseri) 2
pla, pen 32
L. gasseri 64
Erythromycin aci, sak, cur 1 4 2c, 0.25e
par, rha 2
pla, pen 4
Gentamicin par, pla, pen, rha, sak, cur 128 1 >32b, >256c
aci 256 8b
Kanamycin All >256 32
Linezolid All n.d.f 4
Oxacillin aci 8
par, pla, pen, rha, sak, cur 16
Quinupristin/dalfopristin All n.d.f 4 1c
Rifampicin aci, cur, par, rha, sak 2 32 1d
pla, pen 4
Streptomycin All >256 16 >256c
Tetracycline par, rha, aci 4 16 4c
sak, cur 8
pla, pen 64
Trimethoprim All >32 32 >32c,d
Vancomycin aci 4g 4 4c,d
a
aci = acidophilus group, cur = curvatus, par = paracasei, pen = pentosus, pla = plantarum, rha = rhamnosus, sak = sakei.
b
Felten et al. (1999): L. acidophilus group, L. paracasei, and L. rhamnosus.
c
Katla et al. (2001): L. delbrueckii subsp. lactis and subsp. bulgaricus.
d
Charteris et al. (2001): L. rhamnosus.
e
Zarazaga et al. (1999): L. plantarum, L. paracasei, and L. brevis.
f
n.d.: not determined.
g
Maximum observed MIC was 3 and the breakpoint of 4 can be used if the mid-values of the Etest are considered.
 M. Danielsen, A. Wind / International Journal of Food Microbiology 82 (2003) 1–11
tern of resistance as ciprofloxacin) and sulphadiazine
(for which all strains were resistant). The concentra-
tion on the strips was 0.016 – 256 Ag/ml with the
exception of nitrofurantoin: 0.032 – 512 Ag/ml and
benzylpenicillin, ciprofloxacin, imipenem, and trime-
thoprim/sulphamethoxazole (1/19): 0.02 – 32 Ag/ml.
The complete list of antimicrobial agents is as fol-
lows: amoxicillin and clavulanic acid, ampicillin,
bacitracin, benzylpenicillin, cephalothin, cefoxitin,
chloramphenicol, ciprofloxacin, clindamycin, erythro-
mycin, fusidic acid, gentamicin, imipenem, kanamy-
cin, metronidazole, nitrofurantoin, norfloxacin,
oxacillin, rifampicin, streptomycin, sulphadiazine, tet-
racycline, teicoplanin, trimethoprim/sulphamethoxa-
zole, and vancomycin.
3. Results
In Tables 1 – 4, the results are grouped together on
the basis of the mechanisms of action (Yao and
Moellering, 1999). The h-lactams are presented in
Table 1. In Table 2 are the non-h-lactam cell wall
synthesis acting antimicrobial agents. The antimicro-
bial agents that inhibit protein synthesis or mRNA
synthesis are presented in Table 3. The remaining
antimicrobial agents are presented in Table 4. The
results for L. sakei and L. curvatus are presented
together as the strains of these two species did not
differentiate for any antimicrobial agent (data not
shown). The two species L. rhamnosus and L. para-
casei were formerly classified as subspecies within the
same species but are now regarded as separate species.
For clindamycin, L. rhamnosus had MICs between
0.25 and 1 Ag/ml, while most strains of L. paracasei
were slightly more susceptible with MICs between
0.03 and 0.25 Ag/ml (Table 3). For all other antimi-
crobial agents, the strains of the two species were
similar and are presented together in the tables. The
strains of the species within the L. acidophilus group
also showed a tendency to fall within different MIC
ranges for clindamycin. Three of the four L. crispatus
strains had the lowest observed MICs (0.03 – 0.12 Ag/
ml), and the four strains of L. gasseri had the highest
resistance to clindamycin with MICs between 8 and
32 Ag/ml (Table 3). For all other tested antimicrobial
agents, it was not possible to distinguish between the
species of the L. acidophilus group and the results of
7
these antimicrobial agents are therefore presented
together (Tables 1 –4).
A haze could sometimes be observed within the
Etest inhibition zone of ampicillin, benzylpenicillin,
and oxacillin for L. plantarum/pentosus. Since either
no viable cells could be isolated or the isolated cells
showed the same resistance profiles (unpublished
results), MICs for these drugs were read at the
interception on the strips between good growth and
the haze. When testing for nitrofurantoin susceptibil-
ity, pinpoint colonies were often observed within the
inhibition zone of the Etest. These colonies are
resistant mutants as Etests of these colonies showed
a stable resistance.
If most strains within a species have MICs above
the maximum of the Etest strip, then this is interpreted
as high natural resistance. This was the case for 13 of
the 25 antimicrobial agents with variation in suscept-
ibility between the species. The antimicrobial agents
are bacitracin, cefoxitin, ciprofloxacin, fusidic acid,
kanamycin, metronidazole, nitrofurantoin, norfloxa-
cin, streptomycin, sulphadiazine, teicoplanin, trime-
thoprim/sulphamethoxazole, and vancomycin.
For some combinations of antimicrobial agents and
Lactobacillus species where high natural resistance
was not observed, it is possible to suggest MICs as a
maximum guideline which tested strains should fall
below. Further testing to clarify the nature of the
resistance should be carried out if higher MICs are
observed. These guideline MICs are summarised in
Table 5. It should be noted that these guidelines are
suggestions that might change as more strains are
tested. Cephalothin and imipenem are not included in
Table 5 as either an unusual broad range of MICs
(cephalothin) or pinpoint colonies (imipenem) have
been observed.
4. Discussion
A study of the surveys of antimicrobial agent
resistance in lactobacilli showed that a variety of
methods had been used (Etest: Croco et al., 1994;
Herra et al., 1995; Felten et al., 1999; Charteris et al.,
2001; Katla et al., 2001; agar dilution: Dutta and
Devriese, 1981; Zarazaga et al., 1999; Goldstein et
al., 2000; disk diffusion: Sozzi and Smiley, 1980;
Charteris et al., 1998; and microbroth: Klein et al.,
8 M. Danielsen, A. Wind / International Journal of Food Microbiology 82 (2003) 1–11
2000). In order to have as much information from the
susceptibility testing as possible, we have chosen the
Etest because it is a quantitative and simple to use
macro method. Pinpoint colonies and the haze within
the inhibition zone that we in some cases observed are
important observations that might not have been
detected by nonquantitative or micro methods. The
pinpoint colonies often observed with imipenem and
nitrofurantoin could be explained by a high sponta-
neous mutation rate to resistance (Tables 1 and 4). In
our experience, the Etest was found to give reliable
and reproducible results. Reading of the strip was very
easy on MRS agar. One exception was the tetracycline
MICs of L. plantarum/pentosus. These species
showed a growth pattern where the reading of the
Etest at 80% inhibition needed a trained eye (Section
2.2).
Lactobacilli seem to be sensitive to the penicillins
and more resistant to oxacillin, cefoxitin, cephalo-
thin, and imipenem (Table 1). These results are in
concordance with Goldstein et al. (2000). Both
Goldstein et al. (2000) and Croco et al. (1994) found
high resistance to cephalosporins (cefoxitin and cef-
triaxone) and lower resistance to piperacillin and
ampicillin. Zarazaga et al. (1999) found a high
portion of their investigated L. plantarum to be
resistant to penicillin. This is in contrast to our
observations where the L. plantarum/pentosus strains
had low MICs but were able to give a haze of
growth in the presence of penicillin, but with no
viable cells (Table 1). The explanations for these
divergent observations might be found in Bayer et al.
(1978) reporting very high minimal bactericidal
concentrations (MBC) to MICs ratios for h-lactams
and lactobacilli.
The observed levels of glycopeptide resistance
(vancomycin and teicoplanin, Table 2) are in accord-
ance with former observations (Dutta and Devriese,
1981; Handwerger et al., 1994; Ferain et al., 1996;
Billot-Klein et al., 1997; Hamilton-Miller and Shah,
1998). Resistance to vancomycin in the L. acid-
ophilus group has been described (Vescovo et al.,
1982; Charteris et al., 1998). These observations are
not supported by this survey and might be due to the
use of the disc diffusion test. Disc diffusion has been
reported as nonreliable for vancomycin resistance
testing in Staphylococcus aureus (Tenover et al.,
1998). However, Vescovo et al. (1982) reported the
curing of vancomycin resistance from a L. acid-
ophilus strain by removal of plasmids, which indi-
cate transferable resistance. The observed levels of
bacitracin susceptibility vary to a large extent (Table
2). This was also observed by Katla et al. (2001) in
L. delbrueckii isolates from starter cultures. Dutta
and Devriese (1981) correlated the high level of
resistance to bacitracin to strains isolated from cattle
and poultry where bacitracin is used as a growth
promoting agent and did not find high levels of
resistance in strains isolated from pigs. No pinpoint
colonies were observed when testing for bacitracin,
indicating that mutation to bacitracin resistance
occurs at a very low rate or is non-detectable with
the Etest.
Within the group of antimicrobial agents that
inhibit the protein synthesis, a few of the tested strains
have MICs clearly above the range of MICs found for
the majority of strains within their respective species
(Table 3). The strains belong to L. rhamnosus (chlor-
amphenicol: one strain), L. crispatus (erythromycin/
clindamycin: one strain; tetracycline: one strain), L.
johnsonii (tetracycline: one strain), and L. plantarum
(tetracycline: two strains). Resistance determinants to
these antimicrobial agents have previously been found
in lactobacilli. Examples of the identified determinants
include chloramphenicol (L. reuteri: Lin et al., 1996;
L. plantarum: Ahn et al., 1992), erythromycin (L.
fermentum: Fons et al., 1997; L. reuteri: Axelsson et
al., 1988; Tannock et al., 1994; Lin and Chung, 1999;
Lactobacillus sp. strain 100-33: Rinckel and Savage,
1990) and tetracycline (L. plantarum: EC, 2001c;
Lactobacillus spp.: Roberts and Hillier, 1990). The
results stress the need for MICs for separate species,
because the L. johnsonii strain with an MIC of 16 Ag/
ml does not have a high MIC compared to the L.
plantarum/pentosus strains, whereas the other strains
of the L. acidophilus group to which it belongs are
found to have MICs of 0.25 – 2 Ag/ml (Table 3). High
level of resistance to aminoglycosides is found for all
investigated lactobacilli (Table 3). There is a tendency
to lower MICs for gentamicin compared to kanamycin
and streptomycin. One L. crispatus strain had an MIC
of more than 256 Ag/ml for gentamicin and this might
need further testing for transferability as the other
strains had MICs of 4 – 128 Ag/ml. Aminoglycoside
resistance has been observed by many other investi-
gators (i.e. Charteris et al. 1998; Katla et al., 2001).
 M. Danielsen, A. Wind / International Journal of Food Microbiology 82 (2003) 1–11
Charteris et al. (2000) described the loss of resistance
to aminoglycosides for most investigated lactobacilli
in the presence of conjugated bile salts. A synergistic
effect between penicillin/ampicillin and gentamicin/
streptomycin on lactobacilli has been described by
Bayer et al. (1980). In both cases, it is believed that h-
lactams and conjugated bile salts increase permeabil-
ity of the cell envelope thereby increasing the suscept-
ibility to aminoglycosides. Felten et al. (1999) found
lower MICs than the ones from this study when using
gentamicin Etest strips on Mueller – Hinton agar
plates. Furthermore, a high spontaneous mutation rate
to resistance to kanamycin and streptomycin in lacto-
bacilli has been reported (Curragh and Collins, 1992).
The proposed breakpoint from the EU for amino-
glycosides are very low compared to all the above-
mentioned studies (EC, 2001b). Fusidic acid resist-
ance in L. paracasei/rhamnosus and the L. acidophilus
group is in accordance with the results of Felten et al.
(1999). Sims (1968) described differentiating lactoba-
cilli in sensitive hetero- and resistant homofermenta-
tive species based on fusidic acid resistance. Our
results show that L. paracasei/rhamnosus are resistant
to fusidic acid (Table 3). Since these species are also
heterofermentative, our results do not support the
observations by Sims (1968).
The observed resistance to metronidazole, sulpho-
namides, and trimethoprim (Table 4) is in accordance
with other surveys (e.g. Charteris et al., 1998; Katla
et al., 2001). Lactobacillus spp. have decreased
susceptibility to trimethoprim because of a trimetho-
prim-insensitive dihydrofolate reductase (Huovinen,
1987). Resistance of lactobacilli to metronidazole
might be because of the absence of hydrogenase
activity (Church et al., 1996). L. paracasei/rhamno-
sus have, in agreement with this study (Table 4),
been reported as more susceptible to fluoroquino-
lones than other Lactobacillus species (Felten et al.,
1999; Zarazaga et al., 1999). Different levels of
susceptibility to nitrofurantoin were observed
between strains of the same species (Table 4). The
mutants we isolated might explain these differences.
A high mutation rate to nitrofurazone, a related drug,
has been observed in lactobacilli before (Curragh and
Collins, 1992).
The SCAN has so far concluded that three pro-
biotic products pose a risk to human and animal health
because they include bacterial strains having trans-
9
ferable resistance genes (EC, 2001a). The data of this
survey indicate that six strains might have transferable
resistance genes on the basis of their resistance to
chloramphenicol, erythromycin/clindamycin, and tet-
racycline (Table 3). One strain of L. rhamnosus has an
elevated MIC for oxacillin (Table 1). The genetic
basis of this kind of resistance can be due to either
mutations in the penicillin-binding proteins or due to
the presence of a h-lactamase (Massova and Mobash-
ery, 1998). These 7 strains out of the total 62 strains
should be subject to genetic analysis and transfer
experiments in order to determine whether they have
transferable resistance genes.
We recommend using the method described in this
study together with the microbiological breakpoints
listed in Table 5 when screening lactobacilli for
potentially transferable resistance to chlorampheni-
col, clindamycin, erythromycin, gentamicin, rifam-
picin, tetracycline, and one or more of the h-lactams:
ampicillin, benzylpenicillin, and oxacillin. The L.
acidophilus group should furthermore be screened
for vancomycin resistance. Resistance to rifampicin
is often due to mutations (Ezekiel and Hutchins,
1968) and is therefore not likely to be transferable.
Though MICs can vary dependent on the method and
media applied, we propose a re-evaluation of the
microbiological breakpoints set by SCAN as these
breakpoints especially for aminoglycosides are not in
accordance to this and other studies (Table 5).
Furthermore, these results and the results of Zarazaga
et al. (1999) and Felten et al. (1999) clearly show the
need for differentiating between Lactobacillus spe-
cies, which is not done in the opinion published by
SCAN (EC, 2001b). Transferable resistance genes
might not be well expressed in an investigated strain
or might be present in all strains of a given species.
A phenotypic test can therefore not guarantee the
absence of transferable resistance genes, but it will
increase the likeliness of the absence of transferable
resistance genes in the strain. With the use of genetic
methods, it might be possible to screen for the
known genes conferring resistance to sulphonamides,
trimethoprim, glycopeptides, and the aminoglyco-
sides streptomycin and kanamycin. As the number
of known resistance genes still increases, detection of
resistance genes with the use of PCR/Southern blot-
ting/hybridisation will demand a huge amount of
work (Tenover and Rasheed, 1999). The advent of
10 M. Danielsen, A. Wind / International Journal of Food Microbiology 82 (2003) 1–11
chip technology might make genetic testing more
feasible as has been shown on a small experimental
scale by Westin et al. (2001).
Acknowledgements
We thank Kelli Doherty and Elise Pedersen for
excellent technical assistance, Kim I. Sørensen for
careful reading of the manuscript, and Elke Brock-
mann for the providing the data on the species
identification. This work was supported by the Danish
Academy of Technical Sciences grant EU 843.
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