International Journal of Food Microbiology 105 (2005) 281 – 295

www.elsevier.com/locate/ijfoodmicro

Review

Antibiotic resistance in food lactic acid bacteria—a review

Shalini Mathur, Rameshwar Singh*
National Collection of Dairy Cultures, Dairy Microbiology Division, National Dairy Research Institute, Karnal, PIN 243 122, India
Received 20 July 2004; received in revised form 18 December 2004; accepted 20 March 2005

Abstract

Antibiotics are a major tool utilized by the health care industry to fight bacterial infections; however, bacteria are highly
adaptable creatures and are capable of developing resistance to antibiotics. Consequently, decades of antibiotic use, or rather
misuse, have resulted in bacterial resistance to many modern antibiotics. This antibiotic resistance can cause significant danger
and suffering for many people with common bacterial infections, those once easily treated with antibiotics. For several decades
studies on selection and dissemination of antibiotic resistance have focused mainly on clinically relevant species. However,
recently many investigators have speculated that commensal bacteria including lactic acid bacteria (LAB) may act as reservoirs
of antibiotic resistance genes similar to those found in human pathogens. The main threat associated with these bacteria is that
they can transfer resistance genes to pathogenic bacteria. Genes conferring resistance to tetracycline, erythromycin and
vancomycin have been detected and characterized in Lactococcus lactis, Enterococci and, recently, in Lactobacillus species
isolated from fermented meat and milk products. A number of initiatives have been recently launched by various organizations
across the globe to address the biosafety concerns of starter cultures and probiotic microorganisms. The studies can lead to
better understanding of the role played by the dairy starter microorganisms in horizontal transfer of antibiotic resistance genes to
intestinal microorganisms and food-associated pathogenic bacteria.
D 2005 Elsevier B.V. All rights reserved.

Keywords: Antibiotic resistance; Lactic acid bacteria; Antibiotic resistance genes

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 282
2. Emergence and spread of antibiotic resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283
3. Antibiotic resistance in lactic acid bacteria isolated from foods . . . . . . . . . . . . . . . . . . . . . . . . . 284
3.1. Antibiotic resistance profiles of LAB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285
3.2. Mobile genetic elements in LAB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288

* Corresponding author. Tel.: +91 184 2259192; fax: +91 184 2250042.
E-mail address: rsndri@gmail.com (R. Singh).

0168-1605/$ - see front matter D 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.ijfoodmicro.2005.03.008
282 S. Mathur, R. Singh / International Journal of Food Microbiology 105 (2005) 281–295

3.3. Plasmids encoding AR genes in LAB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288

3.4. Conjugative transposons encoding AR genes in LAB . . . . . . . . . . . . . . . . . . . . . . . . . . . 289
3.5. Conjugative transfer among LAB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289
4. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291

1. Introduction maintained and spread through bacterial populations

About 50 years ago, antibiotics were introduced for
the treatment of microbial diseases. Since then, the
greatest threat to the use of antimicrobial agents for
therapy of bacterial infections has been the develop-
ment of antimicrobial resistance in pathogenic bacte-
ria. Antibiotic resistance has been shown to have
occurred rarely in bacteria collected before the anti-
biotic era (Hughes and Datta, 1983). Shortly after the
introduction of each new antimicrobial compound,
emergence of antimicrobial resistance is observed
(Levy, 1997). The magnitude of the problem is signif-
icantly increased by the possibility of bacteria to trans-
fer resistance determinants horizontally and by the
mounting increase in the use (over-use and misuse)
of antibiotics, which has created an enormous selective
pressure towards resistant bacteria (Levy, 1992). Scott
(2002) concluded that gene transfer occurs widely in
vivo between gastrointestinal tract bacteria, and be-
tween gastrointestinal tract bacteria and pathogenic
bacteria, as identical resistance genes are present in
diverse bacterial species from different hosts. In fact
we face the frightening probability that most patho-
genic bacteria that threaten human health will soon be
resistant to all known antibiotics.
However, for several decades studies on the selec-
tion and dissemination of antibiotic resistance have
focused mainly on clinically relevant bacterial species.
The evolution of antibiotic resistant food borne patho-
gens has been amply documented in recent years
(Witte, 1998; Ridley and Threlfall, 1998; Teuber,
1999; Teuber and Perreten, 2000; Threlfall et al.,
2000; White et al., 2002). Recently many investigators
have speculated that commensal bacteria may act as
reservoirs of antibiotic resistance genes similar to those
found in human pathogens (Perreten et al., 1997a; Levy
and Salyers, 2002) and are thus very important in our
understanding of how antibiotic resistance genes are
(Levy and Miller, 1989). The main threat associated
with these bacteria is that they can transfer resistance
genes to pathogenic bacteria. Such reservoir organisms
could possibly be found in various foods and food
products containing high densities of non-pathogenic
bacteria as a result of their natural production process.
The food chain can be considered as the main route
of transmission of antibiotic resistant bacteria between
the animal and human population (Witte, 1997). More
specifically, fermented dairy products and fermented
meats that are not heat-treated before consumption
provide a vehicle for antibiotic resistant bacteria with
a direct link between the animal indigenous microflora
and the human gastrointestinal tract. In 1998, the
Reservoirs of Antibiotic Resistance (ROAR) network
project [http://www.apua.org]) was set up in order to
promote the studies on the selection and dissemination
of non-pathogenic antibiotic resistant bacteria in
humans, during food production and agricultural pro-
cesses, and in the environment. Although most food-
associated lactic acid bacteria (LAB) have acquired the
dGenerally Regarded As SafeT (GRAS) status, the
potential health risk, due to the transfer of antibiotic
resistance genes from LAB reservoir strains to bacteria
in the resident microflora of the human gastrointestinal
tract and hence to pathogenic bacteria, has not been
fully addressed.
Fermented milk products use lactic starter cultures,
and these bacteria enter into our intestines in large
numbers where they interact with the intestinal micro-
flora. Commercial introduction of probiotics contain-
ing antibiotic resistance strains may also have negative
consequences, for example, when resistance is trans-
ferred to intestinal pathogens. There is little informa-
tion regarding the presence of antibiotic resistance
genes on starter strains and their potential to transfer
the resistance genes to pathogens. A number of initia-
tives have been recently launched across the globe to
 S. Mathur, R. Singh / International Journal of Food Microbiology 105 (2005) 281–295
address the biosafety concerns of starter cultures and
probiotic microorganisms.
In the current report, extensive study is presented
to review the distribution and origin of antibiotic
resistance in LAB, the potential mechanisms of trans-
fer and their role in the dissemination of antibiotic
resistances in animal and human population.
2. Emergence and spread of antibiotic resistance
The greatest threat to the use of antibiotics is the
emergence and spread of resistance in pathogenic
bacteria that consequently cannot be treated by previ-
ously successful regimens. Extensive recent reviews of
the application of antibiotics in human and veterinary
medicine (Levy, 1997; WHO, 1997), agriculture (Na-
tional Research Council and institute of Medicine US,
1998; MAFF, 1998; Falkiner, 1998) and aquaculture
(Reilly and Kaferstein, 1997) have documented the
evolution and enrichment of antibiotic resistant bac-
teria: the phenomenon is regularly observed upon the
introduction of a new antibiotic (Levy, 1997). Devel-
opment of antibiotic resistance in bacteria is mainly
based on two factors, the presence of resistance genes
and the selective pressure by the use of antibiotics
(Levy, 1992).
Prior to discussing these two factors, a distinction
between intrinsic and acquired resistance has to be
made. Resistance to a given antibiotic can be intrinsic
to a bacterial species or genus (inherent or natural
resistance) that results in an organism’s ability to thrive
in the presence of an antimicrobial agent due to an
inherent characteristic of the organism. Intrinsic resis-
tance is not horizontally transferable, and poses no risk
in non-pathogenic bacteria. In contrast, acquired resis-
tance is present in some strains within a species usually
susceptible to the antibiotic under consideration, and
might be horizontally spread among bacteria. Ac-
quired resistance to antimicrobial agents can arise
either from mutations in the bacterial genome or
through the acquisition of additional genes coding
for a resistance mechanism. These genetic changes
alter the defensive functions of the bacteria by chang-
ing the target of the drug by changing the membrane
permeability, by enzymatic inactivation of antibiotic
(e.g. by h-lactamases, aminoglycoside acetyl-, nucleo-
tidyl- and phosphoryl-transferases), by active transport
283
of antibiotics (e.g. by membrane inserted ATP-depen-
dent efflux systems), by target modification (e.g. meth-
ylation of 23S rRNA, mutation of aminoacid sequence
of topoisomerase) (Davies, 1997), or by routing meta-
bolic pathways around the disrupted point (Poole,
2002). Resistances are likely to have developed long
before the clinical use of antibiotics. Such resistance
genes may originate from the antimicrobial producers
that carry resistance genes for protecting themselves
from their antimicrobial products (Davies, 1997). Po-
tentially, another origin of resistance genes may be
genes of which the products play a role in the bacterial
metabolism. Such genes may undergo stepwise muta-
tions, which change the substrate spectrum from sub-
strates of biosynthetic or biodegradative pathways to
antibiotics (Davies, 1994).
Antibiotic resistance determinants may be vertically
or horizontally spread in natural microbial communi-
ties. A vertical dissemination is mediated by the clonal
spread of a particular resistant strain. For horizontal
gene transfer in bacteria three mechanisms have been
identified (Davison, 1999): natural transformation, in-
volving the uptake and incorporation of free DNA from
the extracellular medium; conjugation, a cell contact
dependent DNA transfer mechanism found to occur in
most bacterial genera; and transduction, a transfer me-
diated by bacteriophages. The evolution of antibiotic
resistance in microbial communities is enhanced by the
horizontal transfer of resistance genes over species and
genus border by conjugative plasmids, transposons, the
possession of integrons and insertion elements, as well
as lytic and temperate bacteriophages (Davies, 1994).
The relative contribution of these different mechanisms
is unknown, but conjugation is thought to be the main
mode of antibiotic resistance gene transfer (Salyers,
1995). One reason for thinking this is that many anti-
biotic resistance genes have been found on mobile
elements like plasmids and conjugative transposons.
A second reason is that conjugation allows DNA to
move across genus and species lines, whereas transfor-
mation and transduction are usually restricted to within
the same species.
The selective pressure imposed by the use of anti-
microbial agents plays a key role in the emergence of
resistant bacteria. Whenever a mixed bacterial popu-
lation is exposed to antimicrobial agents, it is likely
that there will be bacteria that are resistant to the
respective drugs at the concentration applied. Under
284 S. Mathur, R. Singh / International Journal of Food Microbiology 105 (2005) 281–295
selective pressure, the numbers of these will increase
and some may pass their resistance genes to other
members of the population (Aarestrup, 1999).
A single antibiotic may not only select for resis-
tance to that particular drug. It can also include resis-
tance to other structurally related compounds of the
same class; e.g. resistance to tetracycline by tet (M)
includes also resistance to oxytetracycline, chlortetra-
cycline, doxycycline and minocycline (Chopra and
Roberts, 2001; Chopra, 1994). When antibiotics of
different classes share the same target site, and this
target site is modified by the product of a resistance
gene, cross-resistance between structurally unrelated
antibiotics is observed; e.g. combined resistance to
macrolides, lincosamides and streptogramin B by the
erm genes (Roberts et al., 1999). In addition, a number
of plasmids have been identified which carry multiple
resistance genes, resulting in co-transfer (Levy, 1992).
3. Antibiotic resistance in lactic acid bacteria
isolated from foods
Lactic acid bacteria (LAB) are a group of Gram-
positive bacteria, which excrete lactic acid as a main
fermentation product into the medium. This biochemical
definition associates lactic acid bacteria of different phy-
logenetic branches of bacterial evolution: the blow GCQ
taxa, e.g. Enterococcus, Lactobacillus, Lactococcus,
Leuconostoc, Pediococcus and Streptococcus and the
bhigh GCQ genus Bifidobacterium (Schleifer and Lud-
wig, 1995). Some LAB strains from animal and
human intestinal microflora have been adopted as
dprobioticT food supplements including Enterococcus
faecium, Lactobacillus plantarum, Lactobacillus acid-
ophilus, Lactobacillus casei subsp. rhamnosus, and
several Bifidobacterium and Propionibacterium spe-
cies (Tannock, 1998). Lactic acid bacteria widely used
as probiotics or in starter cultures have the potential to
serve as a host of antibiotic resistance genes with the
risk of transferring the genes in many lactic acid
bacteria and other pathogenic bacteria.
In recent years, increased focus has been given to
food as vehicles of antibiotic resistance genes (Perreten
et al., 1997a; Franz et al., 1999; Klein et al., 2000).
However, there have been very few systematic studies
to investigate acquired antibiotic resistance in LAB
from food. Most data exist on opportunistic pathogenic
enterococci, while the number of reports on lactococci
and lactobacilli is limited. Vancomycin resistant en-
terococci (VRE) have emerged in the last decade as a
frequent cause of nosocomial infections. Of consider-
able concern is the possibility that VRE, selected and
enriched by the use of avoparcin (with cross resistance
to vancomycin) as a growth promoter in animal hus-
bandry, are spread via the food chain (Wegener et al.,
1997; Klein et al., 1998; Pavia et al., 2000; Van Den
Braak et al., 1998; Giraffa and Sisto, 1997). Entero-
coccal food isolates (mainly Enterococcus faecalis and
E. faecium) were analysed for resistances to a broader
range of different antibiotics using phenotypic suscep-
tibility testing, both in raw meat (Klein et al., 1998;
Quednau et al., 1998; Knudtson and Hartman, 1993;
Robrido et al., 2000; Davies and Roberts, 1999) and
fermented milk and meat products (Teuber and Perre-
ten, 2000; Franz et al., 2001; Batish and Ranganathan,
1986; Giraffa, 2002). Their data suggest a high preva-
lence of (multiple) antibiotic resistant enterococci in
foods, which nevertheless were mostly susceptible to
the clinically relevant antibiotics ampicillin and vanco-
mycin. Butaye et al. (2000) studied the in vitro suscep-
tibility and resistance of E. faecium strains isolated
from raw poultry meat, cheese, raw pork, and prepara-
tions of cheese and raw pork to growth promoting
antibacterials used in animals and antibiotics used
therapeutically in humans. Resistance against bacitra-
cin, virginiamycin, narasin and tylosin, a macrolide
antibiotic, was found to be high among strains from
poultry meat. Only one poultry strain showed high
resistance to ampicillin. In another study enterococci
isolated from Portuguese dairy products (milk and
cheese) were screened for gentamicin resistance
(Lopes et al., 2003). Although enterococci are gener-
ally regarded as being intrinsically resistant to low
levels of gentamicin, a high-level gentamicin resistance
was detected in many dairy isolates. Donabedian et al.
(2003) evaluated the molecular mechanisms of genta-
micin resistance in Enterococcus isolates from animals,
foods and humans. It was suggested that there are
similarities in gentamicin resistance in enterococci iso-
lated from humans, retail food, and farm animals from
geographically diverse areas and there is evidence of
spread of gentamicin resistant enterococci from ani-
mals to humans through the food supply. Recently,
Huys and coworkers (2004) have reported the preva-
lence of tetracycline resistance in Enterococcus isolates
 S. Mathur, R. Singh / International Journal of Food Microbiology 105 (2005) 281–295 285

from European cheeses and studied the phenotypic and
genotypic assessment of tetracycline resistance. The
tetracycline resistance could be linked to the presence
of tet (M) genes in Enterococcal isolates, all of which
also harbored a member of the Tn916–Tn1545 con-
jugative transposon family. Cataloluk and Gogebaken
(2004) reported the prevalence of tet (M) and erm (B)
genes in majority (61.961%) of lactobacilli of human
and dairy origin isolated from Turkey. The resistant
strains belonged to Lb. acidophilus, Lactobacillus cris-
patus, Lactobacillus gasseri and Lb. plantarum. It
shows that drug resistance may be acquired in the
intestinal tract during passage and spread to dairy
products by the hands of workers during production.
An overview of antibiotic resistances reported in the
other food-associated LAB is given in Table 1, which
can be summarized by stating that only a limited num-
ber of papers have reported the prevalence of antibiotic
resistance in mainly Lactobacillus spp. isolated from
raw meat and fermented food products. A few studies
have reported an overall susceptibility to antimicrobial
agents (with the exception of intrinsic resistances) in
strains used as meat starter cultures (Raccach et al.,
1985; Holley and Blaszyk, 1997) or dairy starter cul-
tures (Katla et al., 2001; Reinbold and Reddy, 1974).
3.1. Antibiotic resistance profiles of LAB
The knowledge of intrinsically coded resistance of
LAB to common antibiotics is necessary to recognize
acquired resistance traits. Enterococci are intrinsically
resistant to cephalosporins and low levels of amino-

Table 1
Overview of antibiotic resistances reported in the food-associated LAB
Foods Species Resistance References
Raw meat products
Poultry Lb. reuteri G4 cat Lin et al., 1996
Raw ground pork Lb. reuteri 100-63 erm(T) Tannock et al., 1994
Lb. plantarum caTC2R Cm Ahn et al., 1992
Raw ground pork and beef Lb. sakei, Lb. curvatus, Tetracycline (69%); Vidal and Collins-
Lb. plantarum, Lb. brevis, chloramphenicol (3%); Thompson, 1987
Leuco. mesenteroides methicillin (85%)

Fermented products
Raw milk soft cheese
Greek cheese
Yoghurt starter cultures
Nigerian fermented foods
and beverages
Fermented dry sausages
Turkish yoghurts
European probiotic products
Lc. lactis strain K214 Str–tet (S)–cat Perreten et al., 1997b
Lb. acidophilus ACA-DC 243 Penicillin Charteris et al., 1998
S. thermophilus and Neomycin, polymyxin B Sozzi and Smiley, 1980
Lb. delbruekii ssp. bulgaricus
Lb. pentosus, Lb. acidophilus, Tetracycline (42.5%) Olukoya et al., 1993
Lb. casei, Lb. brevis, Erythromycin (17.5%)
Lb. plantarum, Lb. jensenii Ampicillin (47.5%)
Cloxacillin (80%);
penicillin (77.5%);
Lactobacillus species Tetracycline Gevers et al., 2003
Gentamicin (79%)
Penicillin G (64%)
Kanamycin (79%)
S. thermophilus Vancomycin (65%) Aslim and Beyatli, 2004
Lb. acidophilus, Lb. rhamnosus, Tetracycline (26%) Temmerman et al., 2002
Lb. casei, Lb. johnsonnii, Penicillin G (23%)
Lb. plantarum, Lb. reuteri, Erythromycin (16%)
Lb. delbreukii spp. bulgaricus Chloramphenicol (11%)

Others
Maize silage Lb. plantarum 5057 tet (M) Danielsen, 2002
Gene names and abbreviations: cat: chloramphenicol acetylase gene; erm: erythromycin resistance gene; Cm: Chloramphenicol; tet: tetracycline
resistance gene; str: streptomycin adenylase gene. Lb: Lactobacillus; Lc: Lactococcus; Leuco: Leuconosstoc; S: Streptococcus.
286 S. Mathur, R. Singh / International Journal of Food Microbiology 105 (2005) 281–295
glycoside and clindamycin (Teuber et al., 1999; Knudt-
son and Hartman, 1993). Lactobacilli, pediococci and
Leuconostoc spp. have been reported to have a high
natural resistance to vancomycin, a property that is
useful to separate them from other Gram-positive bac-
teria (Hamilton-Miller and Shah, 1998; Simpson et al.,
1988). Some lactobacilli have a high natural resistance
to bacitracin, cefoxitin, ciprofloxacin, fusidic acid,
kanamycin, gentamicin, metronidazole, nitrofurantoin,
norfloxacin, streptomycin, sulphadiazine, teicopla-
nin, trimethoprim/sulphamethoxazole, and vancomy-
cin (Danielsen and Wind, 2003). For a number of
lactobacilli a very high frequency of spontaneous mu-
tation to nitrofurazone (10-5), kanamycin and strepto-
mycin was found (Curragh and Collins, 1992). From
these data it is clear that intergenus and interspecies
differences exist, and consequently identification at
species level is required in order to interpret phe-
notypic susceptibility data. Another study was un-
dertaken to establish the levels of susceptibility of
Lactobacillus spp. to various antimicrobial agents
(Danielsen and Wind, 2003) and it was shown to be
species-dependant. For the following antimicrobial
agents, susceptibility varied several folds between spe-
cies: vancomycin, teicoplanin, tetracycline, norfloxa-
cin, ciprofloxacin, fusidic acid, and clindamycin. The
differences between the species were more subtle for
the rest of the tested antimicrobials.
In a study undertaken by Temmerman et al. (2002),
a total of 55 European probiotic products were eval-
uated with regard to the identity and the antibiotic
resistance of the bacterial isolates recovered from
these products. Using the disc diffusion method, an-
tibiotic resistance among 187 isolates was detected
against kanamycin (79% of the isolates), vancomycin
(65%), tetracycline (26%), penicillin G (23%), eryth-
romycin (16%) and chloramphenicol (11%). Overall
68.4% of the isolates showed resistance against mul-
tiple antibiotics including intrinsic resistances.
The resistance spectrum of Bifidobacterium was
described by Charteris et al. (1998). The investigated
(probiotics) bifidobacteria are susceptible to ampicil-
lin, penicillin G, cephalosporin, bacitracin, chloram-
phenicol, erythromycin, clindamycin, nitrofurantoin
and tetracycline. Resistances–some being most likely
intrinsic–exist towards vancomycin, gentamicin,
kanamycin, streptomycin, fusidic acid, trimethoprim,
norfloxacin, nalidixic acid, metronidazole, polymyxin
B and colistin. The mechanisms of resistances are
unknown.
The resistance profiles of enterococci (E. faecium)
isolated from food or patients are widely varying,
containing many acquired traits. A possible baseline
is provided by E. faecium strain 68, which is used as a
probiotic for man as well as for animal, and as a silage
inoculant. This strain was isolated around 1920 in the
pre-antibiotic area. It is susceptible to erythromycin
(15 Ag disc), framycitin (100 Ag), streptomycin/peni-
cillin (streptopen 35 Ag), gentamicin (10 Ag), penicil-
lin, tetracycline (30 Ag) and chloramphenicol (30 Ag).
It is intrinsically resistant to kanamycin (30 Ag), strep-
tomycin (10 Ag), and oxacillin (5 Ag).
Investigated strains of Lactococcus lactis were sen-
sitive to amikacin, ampicillin, 1st generation cephalo-
sporin, chloramphenicol, erythromycin, gentamicin,
imipenem, oxacillin, penicillin, pipericillin, sulphona-
mide, tetracycline, trimethoprim/sulfomethoxazole,
and vancomycin (de Fabrizio et al., 1994). A slightly
lowered susceptibility was observed towards carbeni-
cillin, ciprofloxacin, dicloxacillin and norfloxacin. In-
trinsic resistances were recorded towards colistin,
fosfomycin, pipemidic acid and rifamycin. Recently,
multiple drug efflux proteins were discovered in Lc.
lactis subsp. lactis MG1363 (van Veen and Konings,
1998), one being an ABC transporter (lmr A), and the
other proton motive force dependent drug transporter
(lmr P). Both are responsible for a resistance to high
concentrations of ethidium bromide. The natural sub-
strates are not known. Twenty-six strains of Lc. lactis
subsp. cremoris and subsp. lactis were all resistant to
trimethoprim and almost all to sulfathiazole. Resis-
tances to gentamicin, kanamycin, lincomycin, nafcil-
lin, neomycin, nisin, rifampin and streptomycin varied
(Orberg and Sandine, 1985).
Thirty-one strains of Lactobacillus delbrueckii
subsp. bulgaricus as components of yoghurt cultures
showed intrinsic resistance towards mycostatin,
nalidixic acid, neomycin, polymyxin B, trimetho-
prim, colimycin, sufamethoxazol and sulphonamides.
Susceptibilities to cloxacillin, dihydrostreptomycin,
doxcycline, furadantin, novobiocin, oleandomycin,
oxacillin and streptomycin were prominent while
kanamycin and streptomycin susceptibilities varied
(Sozzi and Smiley, 1980). Many strains of Lb. plan-
tarum, Lb. casei, Lactobacillus salivarius, Lactoba-
cillus leishmannii, Lb. acidophilus carry intrinsic
 S. Mathur, R. Singh / International Journal of Food Microbiology 105 (2005) 281–295
resistance towards vancomycin which is due to the
presence of d-alanine: d-alanin ligase-related enzymes
(Elisha and Courvalin, 1995). Fifteen strains of Strep-
tococcus thermophilus from yoghurt cultures showed
varying resistance levels to colimycin, gentamicin,
kanamycin, mycostatin, nalidixic acid, neomycin,
polymyxin b, trimethoprim/sulfamethoxazol, strepto-
mycin and sulfonamides (Sozzi and Smiley, 1980). In
a study by Aslim and Beyatli (2004) 34 S. thermo-
philus strains isolated from Turkish yoghurts were
examined for their antibiotic resistance patterns and
plasmid carriage. Most strains of S. thermophilus were
found to be resistant to gentamicin (79%) and penicil-
lin G (64%) and susceptible to chloramphenicol (94%)
and tetracycline (88%); however, no correlation was
observed between the resistance to antibiotics and the
occurrence of plasmids in some strains. Recently Citak
and coworkers (2004) studied the antibiotic resistance
and incidence of Enterococcus species in Turkish
white cheese. Resistance to streptomycin, erythromy-
cin, oxacillin and vancomycin was frequently found in
these enterococci, and resistance to vancomycin was
found in 96.8% of E. faecalis isolates and 76% E.
faecium. This examination indicated poor sanitary
conditions during production and processing and a
significant health risk for consumers. Furthermore,
Maskell and Pead (1992) reported an increasing inci-
dence of isolation of lactobacilli from patients in Eng-
land and Wales and the detection of oflaxacin
resistance in these isolates. In another study seven
Lb. acidophilus strains were examined for their anti-
biotic sensitivity against various chemotherapeutic
agents and all were found to be sensitive to novobio-
cin, chloramphenicol, ampicillin, erythromycin, oxy-
tetracycline and chlorotetracycline, whereas all were
resistant to norfloxacin and nalidixic acid (Gupta and
Mittal, 1995). The authors recommended that cultures
should be tested for their sensitivity toward commonly
used chemotherapeutic agents to be able to survive in
the gut even during antibiotic treatment.
LAB are also used as additives in animal nutrition.
The safety issues concerning the use of these organ-
isms have been addressed by various regulatory bod-
ies in different countries. The presence of acquired
antibiotic resistance factors is considered highly un-
desirable in Europe but of lesser issue in the USA. In
the United States of America, the Food and Drug
Administration (FDA) classifies certain microorgan-
287
isms as Generally Recognised as Safe (GRAS). An
organism or a product with a GRAS status is exempt
from the statutory premarket approval requirements.
An organism can be in the GRAS list either on the
basis of a history of safe use. However, the European
view point is more stringent as the Scientific Com-
mittee on Animal Nutrition (SCAN) proposed that any
listing should be qualified, allowing the general safety
of the organism/group of organisms to be concluded
provided that certain specifics are met. In the case of
many of the live organisms currently used in the
manufacture of, or added to, dairy products, this
may simply be a requirement to demonstrate the
absence of acquired resistance factors. It is recom-
mended to develop a system that would allow a
qualified presumption of safety (QPS). In a QPS
system the safety assessment of food LAB could be
limited to the presence of transmissible antibiotic
resistance markers as other tests are not relevant for
lactic bacteria. (European Commission, 2004) Opin-
ion of the Scientific Committee on Animal Nutrition
(SCAN) on the criteria for assessing the safety of
microorganisms resistant to antibiotics of human clin-
ical and veterinary importance was adopted on 3 July
2001 and later revised on 18 April 2002 (European
Commission, 2002). According to SCAN all bacterial
products intended for use as feed additives must be
examined to establish the susceptibility of the com-
ponent strain(s) to a relevant range of antibiotics. Such
tests must be made in a consistent manner using
internationally recognized and standardised methods.
Determination of the minimum inhibitory concentra-
tion (MIC) of the antibiotic expressed as mg/l or Ag/
ml has been suggested along with breakpoints cate-
gorizing bacterial species as resistant. It has been
mentioned that determination of MIC is not necessary
for species designated as inherently resistant to the
antibiotic. A major obstacle in proposing breakpoints
for lactic acid bacteria is the general lack of relevant
data. For some genera of lactic acid bacteria, such as
Lactobacillus, there are no generally accepted stan-
dard procedures for MIC determination and informa-
tion on MIC ranges is rather limited. Accordingly the
breakpoints suggested by SCAN may be seen as a
pragmatic response to introduce consistency in the
separation of strains with acquired transferable resis-
tance form susceptible strains. In view of the SCAN,
in each case identification of an MIC value at or above
288 S. Mathur, R. Singh / International Journal of Food Microbiology 105 (2005) 281–295
the given breakpoints should require further investi-
gation. The European Commission SCAN documents
(EC Health and Consumer Protection) are available on
the Internet site of EC.
3.2. Mobile genetic elements in LAB
A prerequisite to acquire antibiotic resistance
genes from other bacteria is the potential of LAB to
communicate actively and passively with these bacte-
ria with the aid of conjugative plasmids and transpo-
sons. Plasmids are common in LAB, and differences
are found in size, function and distribution (Davidson
et al., 1996; Wang and Lee, 1997). The functions
found on plasmids include hydrolysis of proteins,
metabolism of carbohydrates, amino acids and citrate,
production of bacteriocins and exopolysaccharides,
and resistance to antibiotics, heavy metals and phages.
At least 25 species of lactobacilli contain native plas-
mids (Wang and Lee, 1997), and often appear to
contain multiple (from 1 to 16) different plasmids in
a single strain. Plasmids are common in enterococci,
lactococci, leuconostocs, pediococci, and present in
some strains of lactobacilli and bifidobacteria (Della-
glio et al., 1995; Deveriese and Pot, 1995; Sgorbati et
al., 1995; Simpson and Taguchi, 1995; Teuber, 1995).
Although several S. thermophilus plasmids have been
sequenced (Mercenier, 1990; Janzen et al., 1992;
Somkuti et al., 1998; Geis et al., 2003), only recently,
a few genes other than those necessary for plasmid
replication have been identified (Geis et al., 1999;
O’Sullivan et al., 1999; Somkuti et al., 1998). Certain
conjugative antibiotic resistance plasmids, such as
pAMh1 and PIP501, are capable of both mobilization
and intergeneric conjugation (Tannock, 1987; Lan-
gella et al., 1993). Conjugative transposons (broad
and narrow host rage) have been described in entero-
cocci, lactococci and streptococci (Clewell, 1993;
Salyers et al., 1995).
3.3. Plasmids encoding AR genes in LAB
R-plasmids encoding tetracycline, erythromycin,
chloramphenicol, or macrolide–lincomycin–strepto-
gramin resistance have been reported in Lb. reuteri
(Vescovo et al., 1982; Axelsson et al., 1988; Lin et al.,
1996; Tannock et al., 1994), Lactobacillus fermentum
(Ishiwa and Iwata, 1980; Fons et al., 1997), Lb. acid-
ophilus (Vescovo et al., 1982), and Lb. plantarum
(Ahn et al., 1992; Danielsen, 2002), isolated from
raw meat, silage and faeces. Most of these R-plasmids
had a size smaller than 10 kb (5.7–18 kb). Lb. fermen-
tum isolated from pig faeces carried a 5.7 kb plasmid
with an erm gene conferring high level erythromycin
resistance which was 98.2% identical to the gene of the
enterococcal conjugative transposon Tn1545 (Fons et
al., 1997). The reported prevalence of antibiotic resis-
tance genes such as erythromycin, vancomycin, tetra-
cycline, chloramphenicol, and gentamicin resistance
genes, on transferable genetic elements in enterococci
is more extensive, both on plasmids (Christie et al.,
1987; Rice et al., 1998; West and Warner, 1985; Cle-
well et al., 1974; Murray et al., 1988) and transposons
(Perreten et al., 1997b; Clewell et al., 1995; Rice and
Marshall, 1994).
A multiple antibiotic resistance plasmid pK214
was reported in a Lc. lactis strain K214 isolated
from raw milk soft cheese (Perreten et al., 1997a),
encoding streptomycin, tetracycline and chloramphen-
icol, and drug efflux gene mef 214. The tetracycline-
resistant gene (providing ribosome protection) is
99.8% homologous to tet (S) from Listeria monocy-
togenes. Characterization of mef 214 demonstrated
that it mediated multiple antibiotic resistance hence
recently it has been renamed mdt (A) for multiple drug
transporter. In Lc. lactis the gene mediated increased
resistance to erythromycin and tetracycline (Perreten
et al., 2001). Two other multidrug transporters have
been described in Lc. lactis. The first (LmrA) is a
member of the ABC superfamily (Bolhuis et al.,
1994); the second (LmrP) is a proton motive force
dependent transporter (Bolhuis et al., 1995). Recent
work shows resistance to macrolides, lincosamides
and streptogramins, and tetracycline is associated
with LmrP expression (van Veen et al., 1999). Inter-
estingly, multidrug transporter LmrA in Lc. lactis is a
homologue of the human multidrug-resistance P-gly-
coprotein, another drug pump involved in drug resis-
tance of human cancer cells encoded by the MDR1
gene. It was demonstrated that they are functionally
interchangeable and that this type of multidrug-resis-
tance efflux pump is conserved from bacteria to man
(van Veen et al., 1998).
Plasmid encoding tetracycline resistance gene tet
(M) was detected in Lactobacillus isolates from fer-
mented dry sausages (Gevers et al., 2002). Most of
 S. Mathur, R. Singh / International Journal of Food Microbiology 105 (2005) 281–295
these R-plasmids had a size of approximately 10 kb,
and in few cases the R-plasmid was larger than 25 kb.
The two allele types of the plasmid encoded tet (M)
gene displayed high sequence similarities (N 99.6%)
with the tet (M) gene previously reported in Staphy-
lococcus aureus MRSA101 and in Neisseria menin-
gitides, respectively. In another study by Danielsen
(2002), the 10877 bp tetracycline resistance plasmid
pMD5057 from Lb. plantarum 5057 was completely
sequenced. The sequence revealed a composite struc-
ture containing DNA from up to four different
sources. The tetracycline resistant region containing
a tet (M) gene had high homology to sequences from
Clostridium perfringens and S. aureus. Recently a
19.3 kb plasmid, encoding a new erythromycin deter-
minant erm (LF) in erythromycin resistant Lb. fer-
mentum strains, was reported; however, in the same
study tet (M) gene for ribosomal protection protein
Tet (M) found in six different Lactobacillus strains
were not encoded by plasmids, but probably located
on the chromosome (Gfeller, 2003). The above-
reported tet (M) gene showed high homology to the
nucleotide sequence of the tet (M) gene from the E.
faecalis transposon Tn916 (accession no. M85225; Su
et al., 1992).
3.4. Conjugative transposons encoding AR genes in
LAB
Conjugative transposons are a main type of vehi-
cle regarding antibiotic resistance transport in Gram-
positive bacteria. They have been discovered in E.
faecalis (Tn916, Tn918, Tn920, Tn925, Tn2702), E.
faecium (Tn5233), Streptococcus pyogenes (Tn3701),
Streptococcus agalactiae (Tn93951) and Lc. lactis
(Tn5276, Tn5301). In enterococci and streptococci,
they determine resistances to tetracycline (tet (M)),
erythromycin (ermAM, erm), chloramphenicol (cat)
and kanamycin (aphA-3). In lactococci, they code for
nisin (nis) production and sucrose fermentation (sac).
These transposons vary in size between 16 and 70 kb
and may be inserted into plasmids or the chromosome
in one or multiple copies. They may mobilize plasmids
or chromosomal genes. The most remarkable observa-
tion, however, is the extreme host range, which is the
property of the Tn916/Tn1545 family. Resistance
transfer rates are in the order of 10 9 to 10 6 per
donor filter matings.
289
3.5. Conjugative transfer among LAB
Indigenous conjugation systems in lactococci are
very common, reflecting the abundance of plasmid
DNA (Neve et al., 1987; Gasson and Fitzgard, 1994).
In contrast reports of the literature on the conjugal
transfer of native Lactobacillus plasmids is limited:
one plasmid-associated transfer of lactose fermenting
ability in Lb. casei and bacteriocin production and
immunity in Lb. johnsonni. A similar situation is
evident in Leuconostoc and Pediococcus which, how-
ever, can accept broad host range antibiotic resistant
plasmids like pAMh1, pVA797DTn917, and pIP501
by high frequency conjugation from Lactococcus har-
bouring these plasmids (Dessart and Steenson, 1991).
Some of the above-listed R-plasmids and transpo-
sons have been shown to be transferable to other LAB,
Gram-positive bacteria and even Gram-negative bac-
teria. Enterococci are known to be very well receptive
for conjugation (Clewell and Weaver, 1989), but are
also successful donor organisms for the transfer of
antibiotic resistance genes to unrelated enterococci
(Rice et al., 1998), lactobacilli (Shrago and Dobro-
gosz, 1988), other Gram-positives including Bacillus
subtilis (Christie et al., 1987), Staphylococcus and
Listeria spp. (Perreten et al., 1997b), and even
Gram-negative bacteria (Courvalin, 1994; Brisson-
Noel et al., 1988; Trieu-Cuot et al., 1988). Moreover,
the transfer of conjugative elements, including a plas-
mid-encoded kanamycin resistance (Doucet-Populaire
et al., 1992) and a transposon-encoded tetracycline and
erythromycin resistance (Doucet-Populaire et al.,
1991), were shown to be transferable from E. faecalis
to Escherichia coli and L. monocytogenes, respective-
ly, in the digestive tract of mice. In contrast, reports of
conjugative transfer of antibiotic resistance genes in
other LAB are rare. Two in vivo studies were per-
formed, to examine the possibility of conjugative
transfer between native Gram-positive members of
the gut. Therefore, the broad host range conjugative
plasmid pAMh1 was transferred in vitro to Lb. reuteri
(Morelli et al., 1988) and Lc. lactis (Igimi et al., 1996)
and administered orally or using gastric intubation to
mice. By analysis of faecal content, plasmid transfer to
E. faecalis was observed in both studies. To improve
existing properties or add new properties (e.g. bacte-
riocin production or lactose fermentation) to strains
with industrial applications, the transfer of plasmids
290 S. Mathur, R. Singh / International Journal of Food Microbiology 105 (2005) 281–295
between different lactococci was studied (Gasson and
de Vos, 1994; Neve et al., 1984, 1987).
A well-characterized broad host range conjugative
plasmid pAMh1 isolated from E. faecalis carries a
constitutive MLS resistance. Self-transfer at frequen-
cies of about 10 4 per donor has been observed in
filter mating experiments with Enterococcus, Staphy-
lococcus, Clostridium, Lactobacillus and Bacillus
species (Macrina and Archer, 1993). Elaborate studies
have shown that pAMh1 could move from E. faecalis
into the plasmid free strain Bu2-60 of Lc. lactis subsp.
lactis biovar diacetylactis during filter mating. From
there, it conjugated to 8 out of 18 commercial isolates
of S. thermophilus with frequencies of 3.5  10 5 to
7.6  10 9 per recipient. Back transfer into Lactococ-
cus Bu2-60 occurred at high frequencies of about
10 4 (Kleinshmidt et al., 1993). pAMh1 was quite
stably maintained in S. thermophilus at 37 8C, but lost
within 90 generations of growth at 42 8C. Out of 13
Lb. delbrueckii subsp. lactis, 44 Lb. acidophilus, 1
Lactobacillus helveticus, 1 Lactobacillus brevis, 5 Lb.
plantarum, 6 Lb. casei subsp. rhamnosus, 1 Lb. fer-
mentum and 1 Lb. salivarius subsp. salicinius strains
only two strains, i.e. Lb. brevis 3030/62 and Lb.
helveticus 3048b, accepted pAMh1 during filter mat-
ing experiment at about 10 7 frequencies: (Soedings
et al., 1993).
The chloramphenicol and erythromycin resistances
in E. faecalis RE25 are encoded on 49-kb conjugative
plasmid pRE25 which transfers these resistances to
Listeria inocua, E. faecalis JH-2, and Lc. lactis BU2-
60 at frequencies of 105 to 106. The nucleotide se-
quence of the cloned chloramphenicol acetyltransfer-
ase is 100% identical with that of the S. agalactiae
pIP501 gene (Perreten, Moschetti and Teuber Gen-
Bank accession no. X92945). The erythromycin resis-
tance region of this plasmid hybridized with the
specific erm nucleotide probe from pAMh1. The
complete nucleotide sequence of this plasmid is cur-
rently being investigated.
Transfer of the tetracycline resistance gene (at the
nucleotide level 99.8% identical to tet (M) of trans-
poson Tn916) from E. faecalis FO1 isolated from a
raw milk mountain cheese, to S. aureus (10 9), E.
faecalis JH2-2, L. inocua, Leuconostoc mesenter-
oides (10 7), and Lc. lactis Bu2-60 (10 6) was
achieved by filter mating. The mobile genetic element
was identified as the Tn916-type transposon TnFO1.
It resides on a 30 kb plasmid in this strain (Perreten et
al., 1997b).
Gevers et al. (2003) isolated Tcr Lactobacillus
isolates from fermented dry sausages and studied
their conjugative transfer to other Gram-positive bac-
teria. Seven out of 14 tetracycline resistant Lactoba-
cillus isolates were able to transfer in vitro this
resistance to E. faecalis at frequencies ranging from
10 4 to 10 6 transconjugants per recipient. Two of
these strains could also transfer their resistance to Lc.
lactis subsp. lactis, whereas no conjugal transfer to a
S. aureus recipient was found. Recently, Huys and
coworkers (2004) studied the phenotypic and geno-
typic assessment of tetracycline resistance in Entero-
coccus isolates from European cheeses. The
tetracycline resistance could be linked to the presence
of tet (M) genes in enterococcal isolates, all of which
also harbored a member of the Tn916–Tn1545 con-
jugative transposon family.
4. Conclusions
There have been very few systematic studies to
investigate acquired antibiotic resistance in LAB of
food origin. Most data exist on opportunistic patho-
genic enterococci, while the number of reports on
lactococci and lactobacilli is limited. However, it is
recently expanding due to increased interest in probio-
tic lactic acid bacteria and genetic modification of LAB
for different purposes. The following general observa-
tions can be made after examination of these data:
There may be intrinsic resistance traits, e.g. vanco-
mycin resistance in Leuconostoc, certain lactobacilli
and others, or resistance to nalidixic acid. Distinction
between intrinsic and acquired resistance is difficult as
it is not possible to trace an investigated strain into the
preantibiotic era. If LAB live in a biotope challenged
regularly with antibiotics (human intestine, animal
intestine, bovine udder) acquired antibiotic resistance
is found in LAB from such habitats including Entero-
coccus, Lactococcus and Lactobacillus species.
There is no barrier between pathogenic (e.g. strep-
tococci), potentially pathogenic (e.g. enterococci) and
commensal (e.g. enteric lactobacilli, lactococci) LAB
regarding acquired resistances. Identical genes respon-
sible for resistances are found, e.g. for tetracycline
(e.g. tet (M)), erythromycin (ermAM), chlorampheni-
 S. Mathur, R. Singh / International Journal of Food Microbiology 105 (2005) 281–295
col (cat), streptomycin (str), streptogramin (sat) in all
three cohorts. These data support the view that in
antibiotic challenged habitats lactic acid bacteria like
other bacteria participate on the communication sys-
tems, which transfer resistance traits over species and
genus borders.
LAB, like all other bacteria are prone to gene
exchange to enhance survival in antibiotic containing
environments. For the food microbiologist, there is no
doubt that it is necessary to avoid the distribution of
bacteria with mobilizable antibiotic resistances
(WHO, 1997). Measures include the use of proper
starter cultures and proper substrates for food fermen-
tations. For example, the transfer of antibiotic resis-
tant bacteria from animals into fermented and other
foods can be avoided if the raw substrate milk or
meat is pasteurized or heat-treated. In addition, gen-
eration of antibiotic resistant bacteria in food animals
and plants has to be minimized by prudent use of
antibiotics. To preserve the life saving potential of
antibiotics the spread of resistance genes at all levels
must be stopped. This includes the ban of antibiotics
with clinical application as growth promoters in an-
imal husbandry as recently established politically in
the European Union and Switzerland: antibiotics with
cross resistance to compounds used in human medi-
cine (tylosin, virginiamycin) or used as such in
human medicine like bacitracin have been banned.
Antibiotic resistance traits as selectable markers in
genetic modification of lactic acid bacteria for differ-
ent purposes are presently being replaced, e.g. by
metabolic traits to generate food grade vectors. To
prevent the undesirable transfer of resistance or con-
ferment of resistance to endogenous bacteria, probio-
tics should not carry resistance other than that
required. Although special purpose probiotics for
use in combination with antibiotics have been devel-
oped through the introduction of multiple resistance
to the bacteria, probiotics generally should not be
designed to carry more resistance than is required
for a specific purpose. Above all the biosafety of
the probiotic lactic acid bacteria (e.g. lactobacilli,
lactococci, enterococci, bifidobacteria) for human
consumption must be assessed by proposing criteria,
standards, guidelines and regulations on the one hand,
and standardizing methodologies of premarketing
biosafety testing and post marketing surveillance on
the other hand.
291
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