Review

Antibiotic resistance among commercially available probiotics

Poonam Sharma
a
, Sudhir Kumar Tomar
a,
, Pawas Goswami
b
, Vikas Sangwan
a
, Rameshwar Singh
c
a
Dairy Microbiology Division, National Dairy Research Institute Karnal, 132001, India
b
Department of Microbiology, Bhaskaracharya College of Applied Sciences, New Delhi, 110075, India
c
Directorate of Knowledge Management, Indian Council of Agricultural Research, New Delhi 110012, India
a b s t r a c t a r t i c l e i n f o
Article history:
Received 5 November 2013
Accepted 9 January 2014
Keywords:
Probiotics
Lactic acid bacteria
Antibiotic resistance
Pathogens
Commensal microbiota
Probiotics are known to have a long history of safe use and can be formulated into many different types of
products, including foods, drugs, and dietary supplements. Several probiotic microorganisms like genus
Lactococcus, Lactobacillus, Streptococcus, Enterococcus, Bidobacterium, Pediococcus and Propionibacteria spp. are
these days used in fermented dairy products and drug formulations. Due to their human consumption, safety
of these organisms is of primary importance as their resistance towards antibiotics can be one of the possible
threat. The gravity of this issue is further compounded by the possibility of bacteria to transfer resistance
determinants horizontally to pathogens and commensal gut microbiota. Antibiotic resistance in these benecial
microbes, either intrinsic or due to any mutation does not manifest a safety concern in itself. Some probiotic
strains with intrinsic antibiotic resistance per se could be useful for restoring the gut microbiota after antibiotic
treatment. However, specic antibiotic resistance determinants carried on mobile genetic elements, such as
tetracycline resistance genes, have often been detected in the typical probiotic genera, and hence constitute a
reservoir of resistance for potential food or gut pathogens, thus representing a serious safety issue. Plasmid-
associated antibiotic resistance, which occasionally occurs, is a matter of concern as it can be detrimental to
use of probiotics owing the possibility of the resistance spreading to harmful microorganisms inhabiting the
same niche. Further, the presence of transferable antibiotic resistance genes even to a less innocuous member
of the gut microbial community poses a safety hazard and needs to be taken into account. Probiotic safety is
beleaguered with the scarcity of well designed and targeted studies and needs to be dealt in right perspective.
This reviewaims to deliberate on the presence of antibiotic resistance in commercially used probiotic organisms
and their potential transfer to pathogens and other commensal microbiota present in the gut.
2014 Elsevier Ltd. All rights reserved.
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177
2. Dening probiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177
3. Consumption and market potential of probiotic products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178
4. Probiotic strains in dairy and fermented food products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178
4.1. Probiotic strains in commercial use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178
4.2. Criteria for the selection of probiotic strain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
5. Antibiotic resistance in probiotics: a safety concern . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180
6. Mechanisms of antibiotic resistance in probiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180
6.1. Phenotypic/Intrinsic resistance (natural resistance) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180
6.1.1. Enzymatic inactivation or modication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180
6.1.2. Increased antibiotic removal/Efux pumps and outer membrane (OM) permeability changes . . . . . . . . . . . . . . . . . 180
6.1.3. Alteration of bacterial target sites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180
6.1.4. Intracellular metabolic rearrangement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
6.2. Genotypic/Extrinsic (acquired resistance) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
7. Antibiotic resistance proles of probiotic LAB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185
7.1. Intrinsic resistance in LAB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185
7.2. Acquired resistance/mobile genetic elements in LAB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 186
7.3. Plasmids encoding antibiotic resistance in LAB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187
7.4. Conjugative transposons encoding antibiotic resistance in LAB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187
Food Research International 57 (2014) 176195
Corresponding author. Tel.: +91 184 2259196; fax: +91 184 2250042.
E-mail address: sudhirndri@gmail.com (S.K. Tomar).
0963-9969/$ see front matter 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodres.2014.01.025
Contents lists available at ScienceDirect
Food Research International
j our nal homepage: www. el sevi er . com/ l ocat e/ f oodr es
8. Horizontal gene transfer of antibiotic resistance from probiotic LAB to other species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188
8.1. Horizontal gene transfer from different sources via food products to the gastrointestinal tract . . . . . . . . . . . . . . . . . . . . . 189
9. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190
1. Introduction
Antibiotic resistance as a phenomenon is, in itself, not surprising. Nor it
is new. It is, however, newly worrying, because it is accumulating and
accelerating, while the world's tools for combating it decrease in power.
[Joshua Lederberg]
Bacteria, the most foremost life forms to appear on Earth are found
in almost every habitat on the planet. Many of them are benecial for
mankind in a variety of ways. Lactic acid bacteria (LAB) constitute one
such important group of microorganisms used in food fermentations.
They contribute to the taste and texture of fermented products and
inhibit food spoilage bacteria by producing growth-inhibiting
substances. Nowadays, LAB-derived products from the industrial
manufacture of fermented food such as milk products, vegetables,
meat and wine (Konings, Kok, Kuipers, & Poolman, 2000; Rhee, Lee, &
Lee, 2011) are their most important applications from an economical
point of view. Besides, the last decades have witnessed the phenomenal
boost in their use as probiotic organisms (Forssten, Sindelar, &
Ouwehand, 2011; Gleinser, Grimm, Zhurina, Yuan, & Riedel, 2012;
Kumari, Catanzaro, & Marotta, 2011; Mills, Stanton, Fitzgerald, & Ross,
2011; Wells, 2011). In modern medicine, antibiotics are the mainstay
of our defense against bacterial infections. But nowbacteria are ghting
back and developing resistance to antibiotics at an alarming pace.
Antibiotic resistance has become a major public health concern and is
drawing the interest of health and research professionals all around
the world. The prime concern is the increase in antibiotic resistance
and the potential spread of resistance genes to pathogenic bacteria.
Recently, it has been shown that even short-termantibiotic administra-
tion can lead to stabilization of resistant bacterial populations in the
human intestine that persist for years (Jacobson et al., 2010; Jernberg,
Lfmark, Edlund, & Jansson, 2007; Lofmark, Jernberg, Jansson, &
Edlund, 2006). It is estimated that approximately 10 million tons of
antibiotics have been released into the biosphere over the last
60 years (European Commission, 2005). Antibiotics are widely used in
human and veterinary medicine, and have been essential for ensuring
human and animal health. The selective pressure of such an intensive
use of antibiotics has urged bacteria sensitive to antibiotics to become
resistant in order to survive (Andersson & Hughes, 2010). Much of the
concern has been about pathogenic bacteria and their antibiotic
resistances, since infections caused by these resistant microorganisms
are not only more complicated to treat, but the treatment is much
more costly due to the more intensive and time consuming care needed
in these cases. Subsequently, with the spread of antibiotic resistance in
microbial communities, concerns have been raised about the existence
of antibiotic resistance in benecial bacterial species which includes
probiotic strains.
Commercial probiotics are generally considered as safe for humans
and with the availability of the probiotic strains in a wide variety of
food products and pharmaceutical preparations and multiple claims
made regarding their benecial health effects. There is a need to put
sufcient safeguards to protect the consumers from any adverse effects
ensuring standards of commercial products and their efcacy. The
safety of these probiotic strains is becoming pre-requisite with anti-
biotic resistance as an emerging issue and their potential to transfer
antibiotic resistance genes to pathogenic/commensal bacteria cannot
be neglected. Antibiotic resistance (AR) and potential transferability to
pathogenic bacteria/commensal microbiota of humangut is detrimental
to the safety of probiotic strains used in such products. There are certain
pros and cons associated with the probiotic strains that they can be co-
administered with antibiotics in case of antibiotic treatment while on
the other side there may be a transfer of resistance to bacterial
pathogens directly/indirectly via commensal ora. In this case, they
may also acquire resistance from human commensal microbiota
(Courvalin, 2006).
These days increased attention is given to safety of bacteria used in
the food. Although, LAB consumed in enormous quantities, primarily
in fermented foods such as meat, dairy and probiotic products, which
interact with gut microbiota on ingestion (Ammor, Florez, & Mayo,
2007). The anticipated problem is that probiotic strains and starter
cultures might contain naturally occurring antibiotic resistance genes.
European Food Safety Authority (EFSA) recommends that bacterial
strains harboring transferable antibiotic resistance genes should not
be used in animal feeds, fermented and probiotic foods for human use
(EFSA, 2007). The transmission of antibiotic resistance genes to unrelat-
ed pathogenic or potentially pathogenic bacteria in the gut is a major
health concern related to the probiotic application. In Europe, according
to the Qualied Presumption of Safety (QPS) approach, established by
the EFSA (EFSA, 2008), the nature of any antibiotic resistance determi-
nant present in a candidate microorganism should be determined
prior to approval for QPS status. Further, this concern is amply reected
in the newly released Codex Guidelines for risk analysis of food
borne antimicrobial resistance where document uses the broad term
antimicrobial resistant microorganisms, beyond food borne pathogens
when discussing the risks associated with the food chain (Codex
Alimentarius Commission, 2011). Antibiotic resistant microorganisms
in food system continue in vogue to be a growing menace (Doyle et al.,
2013). In order to eliminate or contain it, Minimum Inhibitory Concen-
tration (MIC) of the most relevant antimicrobials for each strain used as
a probiotic organism, food or feed additives could be determined using
protocols given by EFSA and on rm genetic grounds. As, probiotic
organisms are considered to pool the resistant genes and transfer
these to pathogenic/commensal bacteria. Therefore, besides the
presence of antibiotic resistance genes, probiotics in commercial use
need to be well documented with respect to their ability to transfer
such genes to other organisms. Thus, it is essential to thoroughly inves-
tigate the safety of microorganisms used in probiotic products (Borriello
et al., 2003).
2. Dening probiotics
In modern times, the universal meaning of the termprobiotic was
established by the World Health Organization (WHO) and the Food and
Agriculture Organization (FAO) of the United States. These two organi-
zations dened probiotics as Live microorganisms which when
administered in adequate amounts confer a health benet on the host
organism(FAO/WHO, 2002). A daily recommended dosage of 10
6

10
9
viable organisms being the most effective for the benecial effect
of probiotic intake (Donnet-Hughes, Rochat, Serrant, Aeschlimann, &
Schiffrin, 1999; Hamilton-Miller, Shah, & Winkler, 1999; Ishibashi &
Shimamura, 1993; Reid, Jass, Sebulsky, & McCormick, 2003; Sanders,
Tompkins, Heimbach, & Kolida, 2004). These live microorganisms are
also used as supplements for restoring microbial balance in case of
177 P. Sharma et al. / Food Research International 57 (2014) 176195
intestinal dysfunction (Corcionivoschi et al., 2010). Commercial
probiotic cultures of human consumption marketed as probiotic
products are generally considered as safe for humans. In fact, some
LAB strains of animal and human intestinal microora microbiota
have been adopted as probiotic food supplements primarily because
of the perception that they are desirable members of the intestinal
microora microbiota (Berg, 1998; Goldin & Gorbach, 1992) and
possess GRAS status including Enterococcus faecium, Lactobacillus
plantarum, Lb. acidophilus, Lb. casei subsp. rhamnosus. Several of
these probiotic strains including Lactobacillus, Bidobacterium and
Propionibacterium species (Tannok, 2005) are used in fermented dairy
products and drug formulations while Escherichia coli (Kruis et al.,
1997) and Enterococcus strains used as probiotics in non-food
formats. In case of strains other than bacteria and of nonhuman origin
Saccharomyces boulardii is the only fungal probiotic used in the fermen-
tation of dairy products for many years (Ishibashi & Yamazaki, 2001).
Unlike many of the probiotic bacteria, it was not originally isolated
from human feces, but rather from fruit. Species from other bacterial
genera such as Streptococcus, Bacillus, and Enterococcus have also been
used as probiotics, but there are concerns regarding the safety of such
probiotics because these genera contain many pathogenic species, pre-
dominantly Enterococcus (FAO/WHO, 2002). Many of the probiotic
strains selected for commercial use in foods and in therapeutics must
retain the characteristics for which they were originally selected
(Salminen et al., 1998). These include the characteristics of growth
and survival during manufacture and, after consumption, during transit
through the stomach and small intestine. Importantly, probiotic must
retain these characteristics to provide various health benets to the
consumers. Consumption of food containing probiotic bacteria are
known to provide a number of health benets which include an im-
provement in gut health (FAO/WHO, 2001; Homayouni & Ejtahed,
2009), modulation in host immune system (Galdeano, Leblanc,
Vinderola, Bonet, & Perdigon, 2007), and reduction of pathogen in the
gut (Soccol et al., 2010). Besides these health benets probiotics are
also reported to exert several other positive manifestations on host
health viz. reduction in the severity of infection (Shu et al., 2000), anti-
hypertensive effects (Yeo & Liong, 2010), reduction in cholesterol level
(Ejtahed et al., 2011), antioxidative effects (Songisepp et al., 2004), pro-
tection against cancer especially colon and bladder cancer (Mack,
Michail, Wei, McDougall, & Hollingsworth, 1999; Sanders, 2006), re-
duce symptoms of allergy in especially in infants (Ouwehand, 2007),
helps in the reduction dental carries (Haukioja, 2010) and reduction
in obesity (DiBaise et al., 2008). It is therefore essential that the probiot-
ic strains used are tested and meet safety standards set by the European
Union (EU).
3. Consumption and market potential of probiotic products
Probiotic products are sold either as capsules/tablets or fermented
dairy/food products and their established/claimed health effects
reinforcing the commercial development of products containing them.
Worldwide, the demand for consumption of functional foods is increas-
ing day by day due to the growing awareness of the consumers about
the impact of food on health. In the year 2000, the world-wide market
for functional foods generated US$ 33 billion, in 2005, and this total
was US$ 167 billion in 2010 (Granato, Branco, Cruz, Faria, & Shah,
2010; Granato, Branco, Nazzaro, Cruz, & Faria, 2010). Probiotic food
products are classied in the category of functional foods and represent
a signicant part of this market andcomprise between60and70%of the
total functional food market (Holzapfel, 2006). Food applications for
probiotics are found mostly in dairy products, with yogurts, ker,
and cultured drinks representing the major categories. The scientic
development of such products had shown the high sensory acceptance
with yogurt accounted to have the largest share of 36.6% (Almeida et al.,
2008, 2009; Zoellner et al., 2009). Besides, emerging food applications
include probiotic cheese (Albenzio, Santillo, Caroprese, Braghieri, et al.,
2013; Albenzio, Santillo, Caroprese, Ruggieri, et al., 2013; Escobar
et al., 2012; Esmerino et al., 2013; Gomes et al., 2011; Karimi,
Mortazavian, & Karami, 2012; Minervini et al., 2012), probiotic ice
cream (Cruz, Antunes, Sousa, Faria, & Saad, 2009; Cruz, Buriti, de
Souza, Faria, & Saad, 2009; Di Criscio et al., 2010; Granato, Branco,
Cruz, et al., 2010; Granato, Branco, Nazzaro, et al., 2010), dairy beverages
(Castro et al., 2013; Pimentel, Cruz, & Prudencio, 2013), ricotta cream
(Fritzen-Freire et al., 2013), and known probiotic fermented milk/or
yogurt (Cruz, Castro, Faria, Bogusz, et al., 2012; Cruz, Castro, Faria,
Lollo, et al., 2012; Cruz et al., 2010, 2013; Ibarra, Acha, Calleja, Chiralt-
Boix, & Wittig, 2012; Marafon et al., 2011; Pimentel et al., 2013),
nutrition bars, breakfast cereal, infant formula, and many others
(Cruz, Antunes, et al., 2009; Cruz, Buriti, et al., 2009; Granato, Branco,
Cruz, et al., 2010; Granato, Branco, Nazzaro, et al., 2010). Fruit juices,
desserts, and cereal-based products may be other suitable media for
delivering probiotics (Cargill, 2009). In the era of functional food,
yogurts and fermented milks are still the main vehicles for incorpora-
tion of probiotic cultures. Following food and beverages, the market
for dietary supplements has also perceived a magnicent growth.
Probiotic products (Dietary Supplements, Animal Feed, Foods & Bever-
ages) are the dominant segments of the global market and are likely
to grow at a CAGR (compound annual growth rate) of 6.8% from 2013
to 2018 and expected to reach USD 37.9 billion in 2018. In Asia-Pacic,
China and Japan dominate the market revenue for probiotics, with India
and other regions also showing signicant growth. Probiotics of the
Lactobacillus genus are having the largest share, representing 61.9% of
total sales in 2007 (Food Processing, 2009). Several probiotic
bacteria of human origin are now being exploited commercially e.g.,
Lb. rhamnosus GG (Saxelin, 1997), Lb. casei strain Shirota (Aso & Akazan,
1992 and Spanhaak, Havenaar, & Schaafsma, 1998), and Lb. acidophilus
LA-1 (Bernet, Brassart, Neeser, & Servin, 1994), many consumers,
consumer organizations, and members of the scientic community are
skeptical of such products and their publicized probiotic claims (Sanders
& Huis in't Veld, 1999). However, species belonging to the genera
Lactococcus, Enterococcus, Saccharomyces and Propionibacterium are also
considered as probiotic due to their health-promoting effects
(Blandino, Al-Aseeri, Pandiella, Cantero, & Webb, 2003; Rivera-
Espinoza & Gallardo-Navarro, 2010; Vinderola & Reinheimer, 2003).
Therefore, due to the awareness of the benecial effects of probiotic
strains in promoting gut and general health, development and con-
sumption of probiotic products have increased worldwide.
4. Probiotic strains in dairy and fermented food products
4.1. Probiotic strains in commercial use
Commercially available probiotic strains added to the variety of
dairy or non dairy products can either be bacteria, molds or yeast
(Chapman, Gibson, & Rowland, 2011). Most commercially available
probiotic products are sold in combination with Lactobacillus sp.
or Bidobacterium sp. (Weese, 2002, 2003; Weese & Martin, 2011).
Nowadays, multistrains or multispecies probiotic mixtures are
becoming increasingly popular compared with single strain probiotics
as they may have additive or even synergistic effects which can result
in higher efcacy (Chapman et al., 2011; Temmerman, Koning,
Mulder, Rombouts, & Beynen, 2004). Some of them are listed in
Table 1. Organisms other than lactic acid bacteria, which are currently
used in probiotic preparations, include Bacillus sp., yeasts (S. boulardii,
S. cerevisiae), lamentous fungi (Aspergillus oryzae) and E. coli Nissle
1917. These probiotic preparations may consist of a single bacterial
strain or it may be a consortium in commercial probiotic products and
available either in powders, liquid form, gel, paste, granules or in the
form of capsules, tablets or sachets (Mombelli & Gismondo, 2000;
Parvez, Malik, Kang, & Kim, 2006; Raja & Arunachlam, 2011; Wolfson,
1999). Probiotic microorganisms are primarily available in three
different types for direct or indirect human consumption: 1) culture
178 P. Sharma et al. / Food Research International 57 (2014) 176195
concentrate added to food (dried or in the deep freeze form) 2) food
products (fermented or non-fermented), and 3) dietary supplements
(drug products in powder, capsule or tablet forms) (Tannis, 2008).
4.2. Criteria for the selection of probiotic strain
In the development of probiotic foods intended for human
consumption, a consortium of LAB have been most commonly used as
it constitutes a major part of the normal microbiota of the human
intestine and when present in sufcient numbers creates a healthy
equilibrium between benecial and potentially harmful microbiota in
the gastrointestinal tract (Dunne et al., 2001). For some positive effects
on human health, a probiotic strain has to reach the large intestine at a
concentrationof about 10
7
viable cells/g (Stantonet al., 2001). The basic
requirement for probiotics is that products should contain sufcient
numbers of microorganisms up to the expiry date (Fasoli et al., 2003).
However, to have functional probiotic strains with predictable and
measurable health benets, a rigorous effort for strain selection is
required. The selection criteria for lactic acid bacteria to be used as
probiotic include the following: (a) exert a benecial effect on the
host (b) endure into a food product at high cell counts, and remain
viable throughout the shelf-life of the product (c) survive during
passage through the GIT (d) adhere to the intestinal epithelium
(e) produce antimicrobial substances and have antagonistic activity
against carcinogenic and pathogenic bacteria (f) stabilize the intestinal
microora and provide various health benets (g) genera of human
origin (h) stable against bile, acid, enzyme and oxygen (i) safety assess-
ment include nonpathogenic, nontoxic, nonallergic, nonmutagenic and
should not carry transmissible antibiotic resistance (Mattila & Saarela,
2000; Parvez et al., 2006; Tumola, Crittenden, Playne, Isolauri, &
Salminen, 2001). On the basis of these criteria, probiotic strain should
be selected to maintain the viability and stability of the strain during
commercial production as well as identifying the compatibility while
added to the end product (Godward et al., 2000; Mattila & Saarela,
2000; Talwalkar & Kailasapathy, 2004). In addition, these strains should
be metabolically active within the GIT and biologically effective against
the identied target (Korbekandi, Mortazavian, & Iravani, 2011).
Therefore, selection of resistant probiotic strains against production,
storage and gastrointestinal tract condition is of prime importance.
Most current probiotics have been selected using these criteria. There-
fore, it is important to note that each such strain has its own specic
properties and ideal strains must have established health and safety
Table 1
Commercially available probiotic strains in the market.
Commercial probiotic strains Manufacturer References
Lactobacillus acidophilus NCFM Rhodia Inc. (Madison, Wis.) Yeung, Cano, Tong, and Sanders (1999)
Lb. acidophilus DDS-1 Nebraska cultures Yeung et al. (1999)
Lb. acidophilus SBT-2062 Snow Brand Milk Products Co., ltd.(Tokyo, Japan) Yeung et al. (1999)
Lb. acidophilus LA-1 (same strain LA-5 sold in Europe) Chr. Hansen, Inc. (Milwaukee, Wis.) Yeung et al. (1999)
Lb. casei strain Shirota Yakult (Tokyo, Japan) Yeung et al. (1999)
Lb. casei Immunitas Danone (Paris, France) Yeung et al. (1999)
Lb fermentas RC-14 Urex Biotech (London, Ontario, Canada) Yeung et al. (1999)
Lb. johnsonii La1(same as Lj1) Nestle (Lansanne, Switzerland) Yeung et al. (1999)
Lb. plantarum 299V Probio AB (Lund, Sweden) Yeung et al. (1999)
Lb. reuteri SD2112(same as MM2) Biogia (Raleigh, N.C.) Yeung et al. (1999)
Lb. rhamnosus GG
a
Valio Dairy (Helsinki, nland) Yeung et al. (1999)
Lb. rhamnosus GR-1 Urex Biotech (London, Ontario, Canada) Yeung et al. (1999)
Lb. rhamnosus 271 Probio AB (Lund, Sweden) Yeung et al. (1999)
Lb. rhamnosus LB 21 Essum AB (Umea, Sweden) Yeung et al. (1999)
Lb. salivarius UCC 118 University College (Cork, Ireland) Yeung et al. (1999)
Lb. lactis L1A Essum AB (Umea, Sweden) Yeung et al. (1999)
Lb. acidophilus NCFM
R
Danisco (Madison WI) Sanders (2008)
Lb. rhamnosus GG (LGG) Valio Dairy (Helinsinki, Finland) Sanders (2008)
Lb. rhamnosus R0011, Lb. acidophilus R0052 Institute Rosell (Montreal, Canada) Sanders (2008)
Lb. casei DN 114001 Danone (Paris, France) Sanders (2008)
Lb. acidophilus LB Lacteol Laboratory (Houdan, France) Sanders (2008)
Lb. paracasei F19 Medi pharm (Des Moines, Iowa) Sanders (2008)
Lb. reuteri RC-14 Chr. Hansen (Milwaukee WI) Sanders (2008)
Bidobacterium lactis Bb-12 Chr. Hansen (Milwaukee WI) Sanders (2008)
Bidobacterium longum BB536
a
Morianga Milk Industry Co. Ltd. (Zama city, Japan) Sanders (2008)
Bidobacterium longum SBT-2928
a
Snow Brand Milk Products Co. Ltd. (Tokyo, Japan) Sanders (2008)
B. breve strain Yakult Yakult (Tokyo, Japan) Sanders (2008)
Bidobacterium animalis DN173010 Activia, Danone Guarner et al. (2008)
Bidobacterium animalis subsp. lactis Bb-12 Chr. Hansen Guarner et al. (2008)
B. infantis 35624 Align, Procter & Gamble Guarner et al. (2008)
B. lactis HN019 (DR10) Howaru Bido, Danisco Guarner et al. (2008)
Enterococus LAB SF 68 Bioorin, Cerbios-Pharma Guarner et al. (2008)
Escherichia coli Nissile 1917 Mutaor, Ardeypharm Guarner et al. (2008)
Lactococcus lactis L1A Norrmejerier Guarner et al. (2008)
Lb. reuteri ATCC 55730 Biogia Biologics Guarner et al. (2008)
Lb. rhamnosus ATCC 53013 (LGG) Vit, Valio Guarner et al. (2008)
B. lactis HN019 branded as DR10 , and strain Lb. rhamnosus HN001 as DR20 New Zealand Guarner et al. (2008)
Lactobacillus casei DN 014001 France Danone Raja and Arunachlam (2011)
Lactobacillus delbruekii Japan Meiji Milk Products, Tokyo Raja and Arunachlam (2011)
Saccharomyces boulardii USA Biocodex, Seattle Raja and Arunachlam (2011)
Bidobacterium longum BB536 Japan Morinaga Milk Industry Raja and Arunachlam (2011)
Sacchromyces boulardii Biocodex (Creswell OR) Tiwari, Tiwari, Pandey, and Pandey (2012)
Lb. fermentum VR1003(PCC) Probiomics (Eveleigh, Australia) Tiwari et al. (2012)
Lb. acidophilus LA-5 Chr. Hansen (Milwaukee, WI) Tiwari et al. (2012)
Lb. paracasei CRL 431 Chr. Hansen (Milwaukee, WI) Tiwari et al. (2012)
B. lactis Bb-12 Nestle (Glandale, CA) Tiwari et al. (2012)
179 P. Sharma et al. / Food Research International 57 (2014) 176195
data from randomized, controlled clinical trials (Lee & Salminen, 2009;
Ventura & Perozzi, 2011).
5. Antibiotic resistance in probiotics: a safety concern
Probiotics are considered to be safe yet there are certain concerns
associated with the safety of probiotics. There are chiey three theoret-
ical concerns regarding the safety of probiotics: (1) the occurrence of
disease, such as bacteremia or endocarditis; (2) toxic or metabolic
effects on the gastrointestinal tract; and (3) the transfer of antibiotic
resistance in the gastrointestinal ora (Snydman, 2008). The available
epidemiological data, clinical trials and acute toxicity studies recom-
mended that the LAB commonly occurring in fermented foods and
used in current probiotics are considered to be safe. Organisms that
are generally regarded as safe involve Lactobacilli, Lactococci,
Bidobacterium, and yeast. Though, there are other probiotic organisms,
such as Enterococcus, Bacillus, Streptococcus and spore-forming bacteria
which are not regarded as safe for human consumption yet are being
used as probiotics. Many strains of these species are used in the food
industry either as starter cultures or as probiotics so before they intend
to market for the target population's manufacturers effectively demon-
strate their safety. Probiotics must be safe under the intended condi-
tions of use (Boyle, Robins-Browne, & Tang, 2006; Vankerckhoven
et al., 2008). Factors to consider include the inherent properties of the
microbe, the physiologic status of the consumer, the dose administered,
and the possibility that probiotic bacteria could be a potential source of
antibiotic resistance transfer within the gastrointestinal tract (Salyers,
Gupta, & Wang, 2004; Senok, Ismaeel, & Botta, 2005). Thus, the spread
of antibiotic resistance among the intestinal microbiota may be possible
and care should be taken while selecting the strains to avoid carrying
transferable resistance determinants and their ability to facilitate
plasmid transfer (Salminen, von Wright, Ouwehand, & Holtzapfel,
2000). To prevent the undesirable transfer of resistance to endogenous
bacteria, probiotics should not carry resistance other than that required.
A safety goal for probiotics should not increase the already existing risks
of antibiotic transfer associated with the normal gut or food microbiota.
There are some methods for assessing the safety of lactic acid bacteria
through the use of in vitro studies, animal studies, and human clinical
studies indicated that some current probiotic strains are required to
full the safety standards (Donohue, Salminen, & Marteau, 1998). A
number of initiatives have been recently launched in order to check
the transferability of antibiotic resistance in starter cultures and
probiotic microorganisms. EFSA has proposed microbiological
breakpoints for several genera of LAB (EFSA, 2008). In this regard, the
presence of antibiotic resistance determinants, and their potential
mobility, requires special attention. Therefore, antibiotic resistance as
such is not a safety issue; it only becomes a threat when resistance is
transferable. Those probiotics belonging to species included in the
EFSA and QPS list (EFSA, 2012) have excellent safety records, and
detrimental effects produced as a consequence of their ingestion are
very scarce (Gouriet, Million, Henri, Fournier, & Raoult, 2012). Most
probiotics are ingested in large amounts in functional foods, and the
presence of antibiotic resistance determinants in their genome must
be systematically screened. Thus, it is of great interest to investigate
whether these determinants can be transferred into the food and the
gut environment (Lahtinen, Boyle, Margolles, Fras, & Gueimonde,
2009). In the European Union there has been an active policy to
eliminate transmissible resistances in these probiotic food products.
Such concern must be also expressed regarding consumption of
human probiotics. Epidemiological data on the safety of probiotics
suggests no evidence of probiotics being involved with human infec-
tions (Snydman, 2008). However, there always remains the possibility
that probiotic consumption can cause infection and those individuals
will respond in different ways to a specic strain. So, the food industries
need to carefully assess the safety and efcacy of all new species and
strains of probiotics before incorporating them into the food products.
6. Mechanisms of antibiotic resistance in probiotics
The global spread of antibiotic resistance is gradually more
important clinical and public health problem worldwide. Due to the
overwhelming use of antibiotics over the past fewyears in both animals
and humans play a signicant role in the outspread/emergence of anti-
biotic resistant bacteria. Antibiotic exposure allows bacteria to develop
novel mechanisms for overcoming the effects of antimicrobials. A single
bacterial strain may possess several types of resistance mechanisms.
But, there are mainly two mechanisms by which bacteria become resis-
tant to antibiotics. Bacterial resistance to antibiotics can be attained
either through intrinsic or acquired mechanisms (Fig. 1A,B). Among
these, whichmechanismprevails in the specic bacterial strain depends
on the nature of the antibiotic, its target site, the bacterial species itself
and whether it is related with plasmid or chromosomal mutation. There
are three types of resistance observed in LAB: intrinsic or innate,
acquired and mutational.
6.1. Phenotypic/Intrinsic resistance (natural resistance)
Intrinsic resistance to a bacterial genus or species results in an
organism's ability to survive in the presence of an antimicrobial agent
due to an inherent characteristic of an organism (Mathur & Singh,
2005). Intrinsic resistance is mainly due to changes in the bacterial
physiological state, such as stationary phase, antibiotic persisters, and
the dormant state (Zhang, 2007). Intrinsic resistance having a minimal
potential for horizontal spread and pose no risk in non-pathogenic
bacteria, although any gene responsible for intrinsic resistance may
spread provided that it is anked by insertion sequences (European
Commission, 2003). As a result, bacteria rstly adopt intrinsic mecha-
nisms to protect themselves from the effect of antibiotics. There are
mainly four mechanisms by which bacteria become intrinsically
resistant to a given antibiotic.
6.1.1. Enzymatic inactivation or modication
Some bacteria produce enzymes that are able to inactivate or
destroy a particular antibiotic molecule as bacteria modify its target
sites to prevent itself from the attack of antibiotic. In bacterial species
there are number of plasmids in their genome which are responsible
for transferring the antibiotic resistance genes (Deasy, 2009). Plasmids
carrying the antibiotic resistance genes degrade the antibiotic and
bacteria become ineffective against antibiotic. For example, beta-
lactamases are bacterial enzymes that split the beta-lactam ring of
penicillin antibiotics, cephalosporins, carbapenems, and monobactams.
Narrow-active enzymes are active against only a specic antibiotic;
while extended-spectrum beta-lactamases are resistant to multiple
antibiotics (Belletti et al., 2009).
6.1.2. Increased antibiotic removal/Efux pumps and outer membrane
(OM) permeability changes
Active drug efux pumps transport antibiotics away from bacteria
by reducing the concentration of the drug. Plasmid carrying resistance
genes has active drug efux pumps that transport antibiotic away
from the bacteria. Decrease in drug permeability and/or increase in
active efux (pumping out) of the drugs across the cell surface, causes
the development of resistance (Li & Nikadio, 2009). Multidrug efux
pumps prohibit the effectiveness of tetracycline and the macrolides
(Belletti et al., 2009).
6.1.3. Alteration of bacterial target sites
Some antibiotics have binding proteins on the cell walls of sensitive
microorganism e.g. Penicillin binding protein. Alteration in such
proteins will bring about development of resistance to such micro-
organisms. This mechanism is used for resistance to a wide range of
antimicrobial agents including beta-lactams, macrolides, tetracyclines,
180 P. Sharma et al. / Food Research International 57 (2014) 176195
uoroquinolones, aminoglycosides, sulfonamide, and vancomycin
(Belletti et al., 2009).
6.1.4. Intracellular metabolic rearrangement
One of the possible mechanisms by which bacteria resort to develop
resistance against the antibiotics is a modication of its targeted
metabolic pathways resulting in internal changes. Bacteria make some
phenotypic changes so that antibiotics may not reach its target site
and making them ineffective against the bacterial species (Madhavan
& Sowmiya, 2011).
6.2. Genotypic/Extrinsic (acquired resistance)
Bacteria may acquire antibiotic resistance through Horizontal Gene
Transfer (HGT), and it is a process where genetic material can be
transferred between individual bacteria of the same species or even
Fig. 1. Mechanisms of antibiotic resistance in probiotics. (A) Intrinsic antibiotic resistance a) efux pumps, b) antibiotic degrading enzyme, c) antibiotic altering enzyme and d) Inner
change adapted from Levy (1998). (B) Acquired antibiotic resistance (a) transformation, (b) conjugation, (c) and transduction adapted from Levy (1998).
181 P. Sharma et al. / Food Research International 57 (2014) 176195
Table 2
Overview of antibiotic resistances reported in probiotic LAB.
Commercial probiotic products/strain Probiotic strain used Antibiotic resistance Location References
Raw milk soft cheese Lc. lactis strain K214 Streptomycin Tetracycline, Chloramphenicol Str-tet (S).-cat Perreten, Kolloffel, and Teuber
(1997)
Greek cheese Lb. acidophilus ACA-DC 243 Penicillin Charteris et al. (1998)
Probiotic strain (Biogia) Lb. rhamanosus GG ATCC 53103 Vancomycin, Gentamicin, Ciprooxacin Klein et al. (2000)
Probiotic strains Lb. reuteri strains (ATCC 55730, ATCC 55149,
ATCC 55148, ATCC 53608 and DSM 20016),
Lb. rhamnosus strain GG (ATCC 53103)
Vancomycin Chromosome Klein et al. (2000)
Norwegian dairy products Lactobacillus, Lactococcus, Leuconostoc and
Streptococcus
StreptomycinLactobacillus spp. Katla, Kruse, Johnsen, and
Herikstad (2001)
Probiotic strain Lb. brevis strains ATCC 8287 and ATCC 14869
T
Vancomycin Rnk et al. (2003)
Fermented dry sausage Lb. plantarum DG507 Erythromycin erm(B), Plasmid Gevers et al. (2003)
Fermented dry sausage Lb. alimentarius DG500, DG499, DG498,
Lb. curvatus DG524, DG484 DG142, DG143,
G048, Lb. plantarum DG533, DG522, DG520,
DG515, DG512, DG509, DG013, Lb. sakei
DG516, DG489, DG488, DG485, DG483,
DG165
Tetracycline tet(M) Gevers et al. (2003)
Dairy product Lc. lactis Erythromycin erm(B), Plasmid Devirgiliis et al. (2010)
European probiotic products Lb. acidophilus, Lb. rhamnosus, Lb. casei, Lb. reuteri,
Lb. johnsonnii, Lb. plantarum, Lb. delbreukii spp.
bulgaricus
Tetracycline 26% Penicillin g 23% Erythromycin 16%
Chloramphenicol 11%.
Temmerman et al. (2003)
Turkish yoghurt S. thermophilus Vancomycin 65% Aslim and Beyatli (2004)
Nigerian fermented foods and
beverages
Lb. pentosus, Lb. acidophilus, Lb. casei, Lb. brevis,
Lb. plantarum, Lb. jensenii
Tetracycline 42.5% Erythromycin 17.5% Ampicillin
47.5% Cloxacillin 80% Penicillin 77.5%.
Olukoya et al. (1993)
Human origin and dairy products Lb. gasseri, Lb. casei, Lb. acidophilus Tetracycline Erythromycin, tet(M), erm(B) Cataloluk and Gogebakan (2004)
Probiotic Lactic acid bacteria Lb. rhamnosus HN001(DR20), HN067,GG,
Lb. acidophilus HN017, B. lactis HN019 (DR10 )
Fusidic acid, nalidixic acid, polymyxin B,
aminoglycosides neomycin, gentamicin, kanamy-
cin,
streptomycin and vancomycin
Plasmid Zhou et al. (2005)
Commercial probiotic LAB strains Lb. rhamnosus HN001 (DR20k), HN067,
Lb. acidophilus HN017 and B. lactis HN019
(DR10k)
Vancomycin and cloxacillin Lb. rhamnosus strains (HN001, HN067 and GG)
were resistant to vancomycin and cloxacillin,
Lb. rhamnosus HN001 contained plasmids
Zhou et al. (2005)
Parmigiano Reggiano cheese Lactobacillus GG and Lb. rhamnosus DSM 20021 cexime, vancomycin, neomycin, enoxacin,
peoxacin and sulphamethoxazole plus
trimethoprim, cephalexin, bacitracin and
lincomycin
Coppola et al. (2005)
Humans, animals and probiotics B. longum B36 Tetracycline tet(W) Moubareck, Gavini, Vaugien, Butel,
and Doucet-Populaire (2005)
Starter culture and probiotic bacteria Lactobacillus, Staphylococcus, Bidobacterium,
Pediococcus, lactococci and Streptococci
Tetracycline tet(K)-Staphlococcus, tet(W) (plasmid)-B.
lactis
DSM 10140, lnu(A)-L. reuteri SD 2112
Kastner et al. (2006)
Commercial probiotic Lactobacillus
strains
Lb. acidophilus R052, Lb. casei R0256, Lb. casei R0215,
Lb. delbrueckii subsp. lactis R0187, Lb. plantarum
R1096, Lb. plantarum R1078, Lb. plantarum R0202,
Lb. rhamnosus R0011, Lb. rhamnosus R1039,
Lb. acidophilus P/N 601379, Lb. bulgaricus P/N 601383,
Lb. casei paracasei subsp. paracasei P/N 601385,
and Lb. plantarum P/N 601387
Vancomycin, kanamycin, neomycin, paromomycin,
streptomycin and nalidixic acid
Kheadr (2006)
Probiotic dairy Products, yoghurt,
cheese
and fermented food (tarhana)
Lb. bulgaricus and Lb. sakei Ampicillin, vancomycin, oxacillin, cephalothin,
cefodizime, tobramycin
Erdourul and Erbulur (2006)
Human, animal and probiotics Bidobacterium spp. Tetracycline tet(W) Masco, Van Hoorde, De Brandt,
Swings, and Huys (2006)
Probiotic isolate Lb. crispatus Erythromycin erm(B) Rojo-Bezares et al. (2006)
Dairy and pharmaceutical products Lb. acidophilus, Lb. paracasei and Bidobacterium spp. D'Aimmo et al. (2007)
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Commercial probiotic products/strain
Probiotic strain used Antibiotic resistance Location References
Nalidixic acid, Aztreonam, cycloserin, kanamycin,
nalidixic acid, polymyxin B, spectinomycin
Dairy products Lb. gasseri Erythromycin, Tetracycline erm(B), tet(M), erm (LF),vat (E-1),
mdt (A),cat, str
Ammor et al. (2007)
Animal, human, probiotics Bidobacterium spp. Tetracycline tet (W) Ammor et al. (2007)
Fermented dry sausage Lb. alimentarius, Lb. curvatus, Lb. plantarum Tetracycline, Erythromycin tet(M), erm (B) Ammor et al. (2007)
Commercial probiotic product Lb. salivarius BFE 7441 Fosomycin, Colistin, Rifamycin Hummel et al. (2007)
Cheese and pharmaceutical product E. faecium Erythromycin erm(B), msr(A/B) Hummel et al. (2007)
Commercial probiotic and fermented
food products
Lb. salivarius BFE 7441 Ciprooxacin, Erythromycin, Gentamicin,
Streptomycin
erm(B)-Chromosome Hummel et al. (2007)
Probiotic cultures Lactobacillus, Pediococcus and Lactococcus Streptomycin, erythromycin, clindamycin and
oxytetracycline
erm(B), tet(W), tet(M) Klare et al. (2007)
Lactic acid bacteria strains LAB spp. Tetracycline tet (M), tet (W), tet (O) Ammor, Gueimonde, Danielsen,
et al. (2008)
Italian sola cheese made raw milk Lb. sakei Rits 9 Tetracycline, erythromycin tet(M), -transposon, tet(L), -plasmid Ammor, Gueimonde, Danielsen,
et al. (2008)
Commercial probiotic strain Lb. reuteri SD2112, Lb. reuteri ATCC 55730 tet (W) and lnu (A) resistance genes Plasmid Rosander, Connolly, and Roos
(2008)
Commercial probiotic strain Lb. reuteri ATCC 55730 Lincosamide lnu(A)-Plasmid Rosander et al. (2008)
Probiotic food supplements Lactobacillus and Enterococcus Vancomycin and Kanamycin Blandino et al. (2008)
Italian probiotic products Lactobacillus spp., S. salivarius spp. thermophilus,
E. faecium and Bidobacterium spp.
Vancomycin, Erythromycin, ciprooxacin Blandino et al. (2008)
Probiotic dairy product Lb. plantarum, Lb. salivarius and Lb. casei Vancomycin Ouoba et al. (2008)
Probiotic product Enterococcus faecium Chloramphenicol, Erthromycin, Gentamicin,
Streptomycin
erm(B), cat(pC194), cat(piP501), aad(E),
aad(E)-aph(A)
Vankerckhoven et al. (2008)
Commercial strain Lb. reuteri ATCC55730 (SD2112) tet (W) and lnu (A) Plasmid Rosander et al. (2008)
Cheese Lb. paracasei tet (M) or tet (W) and erm(B) Plasmid Huys et al. (2008)
Raw milk, starter-free cheese Lc. lactis Tetracycline tet(M), plasmid Florez et al. (2008)
Commercial dairy and pharmaceutical
products
Lactobacilli, Lactococci, Enterococci, S. thermophilus
and Bidobacterium spp.
Vancomycin, rifampicin, streptomycin, bacitracin,
erythromycin
msrC (E. faecium), vanX (L. plantarum), and
dfrA (S. thermophilus and L. lactis)
Liu et al. (2009)
Probiotic strain Lb. brevis KB290 Ciprooxacin, tetracycline, vancomycin Fukao et al. (2009)
Whey starter cultures and ripened Grana
Padano and Parmigiano Reggiano
cheeses
Lb. helveticus, Lb. delbrueckii subsp. lactis, Lb. rhamnosus,
and Lb. casei
Gentamicin, penicillin G, oxacillin, vancomycin,
cephalotin, and co-trimoxazole
Belletti et al. (2009)
Fermented dry sausage Lb. reuteri Erythromycin erm(B) Zonenschain et al. (2009)
Fermented dry sausage Lb. plantarum Erythromycin erm(B), erm(C) Zonenschain et al. (2009)
Dairy products Lb. sakei Erythromycin erm (B) Zonenschain et al. (2009)
Fermented dry sausage Lb. reuteri CH2-2, Lb. curvatus, Lb. sakei, Lb. paracasei,
Lb. brevis, Lb. salivarius CHS1-E,CH7-1E
Erythromycin erm(B) Zonenschain et al. (2009) Thumu
and Halami (2012)
Yakult B. breve Streptomycin rpsL gene Kiwaki and Sato (2009)
Commercial strain B. bidum strain Yakult YIT 4007 Erythromycin, neomycin, and streptomycin Sato and Iino (2009)
Raw milk dairy product Lb. fermentum ROT1 Erythromycin, Novobiocin, Tetracycline and
dalfopristin
Egervrn, Lindmark, Olsson, and
Roos (2010)
Irish pork and beef abattoirs Lb. paracasei, Lb. reuteri and Lb. curvatus Erythromycin resistant erm(B) and msrA/B genes Toomey et al. (2010)
Irish pork and beef abattoirs Lb. plantarum, Leuconostoc mesenteroides spp. Tetracycline resistance tet(M) gene Toomey et al. (2010)
Irish pork and beef abattoirs Lactobacilli, Streptococci, lactococci and
Leuconostoc spp.
Streptomycin Chromosome Toomey et al. (2010)
Chinese fermented foods Lb. fermentum NWL24 and Lb. salivarius NWL33;
Lb. plantarum NWL22 and Lb. brevis NWL59,
Lb. brevis and Lb. keri
Erythromycin 11% Tetracycline 17% Gentamicin 6%
Ciprooxacin 85%
erm (B), tet (M), tet(S) Nawaz et al. (2011)
Italian fermented products Lb. paracasei tetracycline and erythromycin tet (M) and erm (B) genes Comunian et al. (2010)
French yoghurt Bidobacterium lactis DSM 10140 Streptomycin, Kanamycin and Gentamicin,
Tetracycline tet(W)
Plasmid Ashraf and Shah (2011)
Chinese fermented foods-pickles, sausages Lb. plantarum, Lb. fermentum, Lb. helveticus, E. faecium Tetracycline, erythromycin, chloramphenicol,
kanamycin
tet (M) and erm(B) on plasmid and
chromosome, gene aph A3, plasmid,
mef A,-chromosome
Pan, Hu, and Wang (2011)
Probiotic strain B. animalis, B. pseudolongum and B. thermophilum chloramphenicol, linezolid, quinupristin/dalfopristin,
kanamycin, neomycin
Mayrhofer et al. (2011)
(continued on next page)
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Table 2 (continued)
Commercial probiotic products/strain Probiotic strain used Antibiotic resistance Location References
Chinese fermented foods Lactobacillus and S. thermophilus Intrinsicresistant to nalidixic acid, kanamycin, and
vancomycin Acquired resistance for penicillin,
erythromycin, clindamycin, and tetracycline while
resistance to gentamicin, ciprooxacin,
streptomycin, and chloramphenicol
erm(B) and tet(S) Nawaz et al. (2011)
Fermented milk culture Lb. pentosum (L08), Lb. jungurthi (L10), Lb. reuteri
(L16), Lb. fermentum (L18), Lb. plantarum (L29),
Lb. brevis (L43), and Lb. casei (L47)
Kanamycin, trimetroprim, rifampicin, kanamycin,
amphicilin and penicillin
Lavanya, Sowmiya, Balaji, and
Muthuvelan (2011)
Dairy product Lb. vaginalis NWL35 Erythromycin erm(B) Nawaz et al. (2011)
Traditional Ethiopian fermented food Lactic acid bacteria Methicillin, Noroxacin Beyan et al. (2011)
Probiotic strain Bidobacterium spp. Chloramphenicol, linezolid, and
quinupristin/dalfopristin
Mayrhofer et al. (2011)
Fermented sausages Leuco. mesenteroides subsp. mesenteroides,
Lb. curvatus, Lb. brevis, Lb. fermentum,
Lb. paracasei subsp. paracasei
Nitrofurantoin, trimethoprim, oxacillin,
streptomycin, enrooxacine, sulphonamides,
polymyxin B, neomycin
Zdolec et al. (2011)
Chinese yoghurts S. thermophilus and Lb. delbruekii ssp. bulgaricus Ampicillin, kanamycin, chloramphenicol,
chlortetracycline, tetracyclines, neomycin and
gentamicin
tet (M), ant 6, aph 3-IIIa Zhou et al. (2012)
Commercial probiotic product B. longum JDM301 Ciprooxacin, amikacin, gentamicin, streptomycin Wei, Zhang, Liu, Malakar, and
Guo (2012)
Traditional fermented foods and curd E. faecium, E. durans, P. pentosaceus Erythromycin erm(B), msr(C) Thumu and Halami (2012)
Indian vegetables and fermented foods Lb. plantarum, Lb. fermentum, Weissella spp. P. parvulus Gentamicin, vancomycin, noroxacin, kanamycin Patel, Lindstrm, Patel, Prajapati,
and Holst (in press)
Gene names and abbreviations: cat: chloramphenicol acetylase gene; erm: erythromycin resistance gene; tet: tetracycline resistance gene; str: streptomycin adenylase gene; lnu: lincosamide resistance gene; msrA/B: erythromycin resistance gene;
vat: acetyltranferase gene; rpsL: streptomycin resistance gene. Lb: Lactobacillus; Lc: Lactococcus; Leuco: Leuconosstoc; S: Streptococcus; P: Pediococcus; E: Enterococcus.
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different species (Madhavan & Sowmiya, 2011). Acquired resistance
is present in some strains within a species usually susceptible to the
antibiotic under consideration, and might be horizontally spread
among other bacterial species (Mathur & Singh, 2005). Acquired
resistance to antimicrobial agents can arise either from mutations in
the bacterial genome or through acquisition of additional genes coding
for a resistance mechanism. Mutations, whichmay cause genetic chang-
es in multiple regions of the genome, play only a minor role in the
development of resistance (Albert et al., 2005; Howden, Johnson, &
Ward, 2006). The evolutionof antibiotic resistance inmicrobial commu-
nities is enhanced by the horizontal transfer of resistance genes over
species and genus borders by (i) conjugative plasmids, (ii) transposons,
(iii) the possession of integrons and insertion elements; and (iv) lytic
and temperate bacteriophages (Davies, 1994; Devirgiliis, Barile, &
Perozzi, 2011). Thus, intestinal bacteria can acquire resistance either
by mutation or by horizontal transfer of resistance genes from other
intestinal species or any species that passes through the colon
(Liu, Zhang, Dong, Yuan, & Guop, 2009). Irrespective of the resistance
mechanism and the microbial taxon involved, the actual possibility of
spreading an antibiotic resistance through horizontal transfer relies on
its genetic basis (Radulovic, Petrovic, & Bulajic, 2012). The dissemina-
tion of antibiotic resistance genes necessitates the physical transfer or
acquisition of the genetic element encoding antibiotic resistance
(Sommer & Dantas, 2011). While, the gene transfer among organisms
within the same genus is common, this process has also been observed
between different genera, including transfer between such evolution-
arily distant organisms as gram-positive and gram-negative bacteria
(Courvalin, Goussard, & Grillot-Courvalin, 1995). Among the three
well known mechanisms for horizontal gene exchange between
bacteria, the relative contribution of these mechanisms is known but
conjugation is thought to be the main mechanism of HGT (Salyers,
1995).
Most of these mechanisms have been observed and studied in
various bacteria, however, there have not been specic studies dealing
with these mechanisms in probiotics (LAB or Bidobacteria).
7. Antibiotic resistance proles of probiotic LAB
Antibiotics have playeda signicant role inthe treatment of bacterial
infections, but misuse has contributed to a rise in bacterial resistance.
Emergence of resistant microorganisms has become a major threat to
public health and has led to the manifestation of bacteria resistant to
many modern antibiotic formulations (Mazel & Davies, 1999). The
food chain could be regarded as one of the main pathways for the trans-
mission of antibiotic resistant bacteria from animals to humans (Singer
et al., 2003). Use of probiotics containing antibiotic resistance strains
for commercial purpose may also have negative consequences, when
resistance is transferred to the intestinal pathogens. Even though,
antibiotic resistance is well studied and documented in human patho-
genic species (Mathur & Singh, 2005). However, since the last decade,
researchers have also focused on characterizing antibiotic resistance in
LAB (Belletti et al., 2009). Since, Lactobacillus sp. intentionally added to
our diet, concerns have been raised about the antibiotic resistance in
these benecial bacterial species. In addition, LAB can also acquire
resistances fromother bacteria inthe environment. Once a LABbecomes
resistant, the determinant is amplied and may be transmitted to
another host and when the right circumstances are present; these
resistance genes might be transferred to the indigenous ora. Hence,
the possibility of the transmission of genes coding for antibiotic
resistance also from benecial lactic acid bacteria, in the food chain via
animals to humans, has been thoroughly investigated. These aspects
consider food chain as a fundamental reservoir for the transmission of
antibiotic resistance and as a source of contamination in a variety of
food products (Witte, 1997). Hence bacteria used as probiotics for
humans or animals should not carry any transferable antibiotic resis-
tance genes (EFSA, 2008; von Wright, 2005). The large numbers of
LAB and Bidobacteria in fermented products and in the GIT help in
the appearance of different resistance mechanisms via mutation while
in case of horizontal evolution; genes pass from a resistant to a non-
resistant strain, conferring resistance on the latter. Therefore, checking
for signs of transferable antibiotic resistance in starter strains and
bacteria used as probiotics is essential. The knowledge of intrinsic,
chromosomally coded resistance of LAB to common antibiotics is
necessary to recognize acquired resistance traits (Mathur & Singh,
2005). Distinguishing between intrinsic (non-specic, nontransferable)
and acquired resistance is also necessary, a process that may require the
comparison of antimicrobial resistance patterns in many LAB and
Bidobacteria species from different sources (Teuber, Meile, &
Schwarz, 1999). Continuous attention should be paid to the selection
of the probiotic strains free of transferable antibiotic-resistance deter-
minants (Radulovic et al., 2012). While antibiotic resistance in itself is
not pathogenic, and the possibility that genes encoding antibiotic
resistance may be transferred between starter cultures, probiotics,
commensal bacteria and pathogens in the gut, are of great concerns
(Ammor et al., 2007; Salyers et al., 2004; Zhou, Pillidge, Gopal, & Gill,
2005). To address this aspect, the safety of LAB should be veried
with respect of their ability to acquire and disseminate resistance
determinants (Kastner et al., 2006).
7.1. Intrinsic resistance in LAB
LAB strains potentially serve as a source of antibiotic resistance. The
determination and comparison of antibiotic susceptibility patterns of a
representative number of strains of each species is an essential criterion
for potentially starter or probiotic Lactobacillus cultures (Charteris &
Kelly, 1993). Microbiological breakpoints have been dened by
European Commission (2008) for studying the MIC distribution in the
bacterial population and the part of the population that clearly deviates
from a susceptible majority is considered resistant (Olsson-Liljequist,
Larsson, Walder, & Miorner, 2007). The species of LAB were assessed
for antibiotic susceptibility using an E-test kit and a broth microdilution
method (Egervrn, Danielsen, Roos, Lindmark, & Lindgren, 2007).
In addition to phenotypic antibiotic resistance determinations, also
genotypic detection of particular gene causing resistance may be
performed. However, a phenotypically resistant strain may be geno-
typically susceptible. In contrast, a susceptible phenotype may also
carry silent genes, which are observed with genotyping. Antibiotic
susceptibility of different bacterial species and analysis of MIC in
dened species/antibiotic combinations helps to differentiate these
two resistance mechanisms. Antibiotic susceptibility proles have
recently been reported for several LAB in the EU-projects ACE-ART
(Florez et al., 2008; Korhonen et al., 2008) and PRO-SAFE (Klare et al.,
2007).
Antibiotic resistance in LAB has been carried out both at the physio-
logical and molecular level. Lactobacillus sp. is reported to have a high
natural resistance to bacitracin, cefoxitin, ciprooxacin, fusidic acid,
kanamycin, gentamicin, metronidazole, streptomycin, sulfadiazine,
nalidixic acid and vancomycin (Blandino, Milazzo, & Fazio, 2008;
Kastner et al., 2006; Temmerman, Pot, Huys, & Swings, 2003). Intrinsic
resistance to vancomycin was conrmed for L. paracasei, L. salivarius,
and L. plantarum and resistance to erythromycin was detected in one
strain of L. salivarius according to FEEDAP and CLSI breakpoints
(Blandino et al., 2008). Naturally high resistance to a number of
antibiotics, especially vancomycin, is characteristic of lactobacilli
(Bernardeau, Vernoux, Henri-Dubernet, & Guguen, 2008). The
Lactobacillus species have found susceptible to many cell wall synthesis
inhibitors, like penicillin and ampicillin (Coppola et al., 2005; Danielsen
& Wind, 2003). Most species proved susceptible to the antibiotics
tested, but resistance to tetracycline and/or erythromycin was detected
occasionally (Klare et al., 2007; van Hoek, Mayrhofer, Domig, & Aarts,
2008; van Hoek et al., 2008; van Hoek et al., 2008). Most of the studies
represent erythromycin, tetracycline, chloramphenicol and vancomycin
185 P. Sharma et al. / Food Research International 57 (2014) 176195
resistance of lactobacilli from different dairy products such as cheese
(Ammor, Gueimonde, Danielsen, et al., 2008; Comunian et al., 2010;
Devirgiliis et al., 2011; Perreten, Kolloffel, & Teuber, 1997), yoghurt
(Aslim & Beyatli, 2004) and fermented beverages (Olukoya, Ebigwei,
Adebawo, & Osiyemi, 1993).
A majority of LAB species is resistant to metronidazole as they are all
intrinsically resistant to sulfonamides and trimethoprim, while they are
usually susceptible to piperacillin and piperacillin plus tazobactam.
Resistance against neomycin, kanamycin, streptomycin and gentamicin
(aminoglycosides) has been observed more frequently among lactobacilli
(Coppola et al., 2005; Danielsen, 2002; Zhou et al., 2005). A high
resistance to cefoxitin was acknowledged for Lactococcus sp., Leuconostoc
sp., andLactobacillus sp. whereas Leuconostoc sp., Pediococcus sp. andmost
lactobacilli species were intrinsically resistant to vancomycin. Resistance
of many species of lactobacilli except Lb. delbrueckii subsp. bulgaricus,
Lb. acidophilus, Lb. johnsonii, and Lb. crispatus to glycopeptides is also
considered intrinsic (Charteris, Kelly, Morelli, & Collins, 1998; Mathur &
Singh, 2005). In another study, the phenotypic and genotypic resistance
in LAB from fermented foods have been identied and all the strains
were susceptible to ampicillin, bacitracin, and cefsulodin and intrinsically
resistant to nalidixic acid, kanamycin, and vancomycin (Nawaz et al.,
2011).
Among LAB species, Bidobacterium sp. is rarely associated with
gastrointestinal infections. The strains of Bidobacterium were found
susceptible to ampicillin, cefotaxime and erythromycin. Bidobacterium
strains displayed intrinsic resistance with high MICs for streptomycin
and gentamicin (Mtt, Alakomi, Vaari, Virkajrvi, & Saarela, 2006). In
another study by D'Aimmo, Modesto, and Biavati (2007) found that
Bidobacteria were resistant to aminoglycosides, cycloserine, nalidixic
acid and strongly resistant to kanamycin, polymyxin B and aztreonam.
Bidobacteria are intrinsically resistant to mupirocin, an antibiotic
that is being used in selective media for this genus. The high level of
mupirocin resistance is a consequence of an atypical isoleucyl-tRNA
synthetase that contains key amino acid residues (Serani et al.,
2011). Furthermore, they are not susceptible to high concentrations
of aminoglycosides, most likely as a consequence of the lack of
cytochrome-mediated drug transport (Mayrhofer, Mair, Kneifel, &
Domig, 2011).
Lactococcus (Lc. lactis) strains were found to be resistant towards
tetracycline and erythromycin (Devirgiliis, Barile, Caravelli, Coppola, &
Perozzi, 2010) while some strains were sensitive to amikacin, ampicil-
lin, rst generation cephalosporin, chloramphenicol, erythromycin,
gentamicin, imipenem, oxacillin, penicillin, pipericillin, sulfonamides,
tetracycline, trimethoprim/sulfomethoxazole and vancomycin (De
Fabrizio, Parada, & Torriani, 1994). A slightly lowered susceptibility
was observed towards carbenicillin, ciprooxacin, dicloxacillin and
noroxacin. Intrinsic resistances were recorded towards colistin,
fosfomycin, pipemidic acid and rifamycin. Resistance to gentamicin,
kanamycin, lincomycin, neomycin, rifampin and streptomycin varied
(Mathur & Singh, 2005).
While the members of Enterococcus contain some opportunistic
pathogens, hence it is debated as to whether these organisms could be
used as probiotics. Regarding the prevalence of antimicrobial resistance
of enterococcal strains in different environments, the frequency of
various antimicrobial resistances was lower in food isolates in compar-
ison to clinical strains (Abriouel et al., 2008). Enterococcal food isolates
(mainly E. faecalis and E. faecium) were analyzed for resistances to
a broader range of different antibiotics using phenotypic susceptibility
testing, both in raw meat (Knudtson & Hartman, 1993; Quednau,
Ahrne, Petersson, & Molin, 1998) and fermented milk and meat
products (Franz et al., 2001; Teuber & Perreten, 2000). E. faecium
derived from probiotic product was susceptible to all the tested
antibiotics including vancomycin, ampicillin, cefaclor, cefotaxime,
erythromycin, ciprooxacin and gentamicin (Blandino et al., 2008).
Similarly, in another study, E. faecalis strains isolated from Italian
fermented dairy products were found to have high MIC values for
tetracycline (Devirgiliis et al., 2010). The enterococcal strains are
found to be intrinsically resistant to -lactams, cephalosporins,
lincosamides and polymyxins. The high prevalence of (multiple)
antibiotic resistant enterococci in foods, were most susceptible to the
clinically relevant antibiotics ampicillin and vancomycin. The suscepti-
bility to vancomycin is of great importance as this glycopeptide anti-
biotic is one of the last therapeutic options in clinical therapy. A
specic cause for concern and a factor contributing to the pathogenesis
of enterococci is the resistance they acquire to aminoglycosides,
tetracyclines, macrolides, chloramphenicol, penicillin, and ampicillin
(Gray, Stewart, & Pedler, 1991) and their capacity to exchange genetic
information by conjugation. The determination of antimicrobial suscep-
tibility of a bacterial strain is an important prerequisite for its approval
as probiotic. Determination of antibiotic resistances among LAB
is confounded by problems regarding the use of media and MIC
breakpoints for the genera or species (Franz, Hummel, & Holzapfel,
2005; Huys, D'Haene, & Swings, 2002). Furthermore, MIC breakpoint
values have been shown to be species specic and thus vary between
species of the same genera (Danielsen & Wind, 2003). An overview
of antibiotic resistance proles of different LAB in fermented and
commercial probiotic bacteria compiled in (Table 2).
7.2. Acquired resistance/mobile genetic elements in LAB
Emerging food application of probiotics is fraught with the danger of
being one of the potential sources for the spread of antibiotic resistance
genes. Bacteria use a complex array of mechanisms to share and spread
resistance determinants. The key mechanisms of horizontal transfer in
bacteria are supposed to be conjugation and transduction via bacterio-
phages (Kleinschmidt, Soeding, Teuber, & Neve, 1993). In conjugation,
plasmids and tranposons play an important role in dissemination of
antimicrobial resistance (Grillot-Courvalin, Goussard, & Courvalin,
2002; Kleinschmidt et al., 1993). Despite the fact that reports on the
presence of antibiotic resistance genes associated with mobile genetic
elements have been detected in lactobacilli. However, there are several
studies that have documented the presence and expression of antibiotic
resistance genes infood-associated LAB (Borriello et al., 2003; Danielsen
& Wind, 2003; Gevers, Danielson, Huys, & Swings, 2003; Perreten,
Kolloffel, & Teuber, 1997; Salminen et al., 1998; Teuber et al., 1999).
Lactobacilli that harbors antibiotic resistance determinants have been
found in fermented drinks and yoghurts (Temmerman et al., 2003),
cheese (Florez, Delgado, & Mayo, 2005 and Herrero, Mayo, Gonzalez, &
Suarez, 1996), and meat products (Gevers et al., 2000). The possibility
of exchange of resistance factors with other microorganisms, especially
those belonging to the gut microbiota is limited compared to that of
other LAB such as enterococci (Mathur & Singh, 2005; Temmerman
et al., 2003). The prevalence of acquired antibiotic resistance genes so
far been detected in Lactobacillus species are sensitive to antibiotics
which inhibit protein synthesis such as tetracycline, erythromycin,
and chloramphenicol. In various probiotic, food and research strains
tet(M) and erm(B) resistance genes have been detected (Bernardeau
et al., 2008; Huys et al., 2006; Klare et al., 2007; Mathur & Singh,
2005; Ouoba, Lei, & Jensen, 2008). However, the most common resis-
tance determinants found in lactobacilli are the tetracycline resistance
genes, which are sometimes found in combination (Ammor,
Gueimonde, Danielsen, et al., 2008). At least 11 different tetracycline re-
sistance genes have been detected in lactobacilli, these include genes
coding for ribosomal protection proteins (tet(W), tet(M), tet(S), tet(O),
tet(Q), tet(36), tet(Z), tet(O/W/32/O/W/O), tet(W/O) and efux pumps
(tet(K) and tet(L)) (Lahtinen et al., 2009). Chloramphenicol resistance
genes (cat; chloramphenicol acetyl transferases) have been identied
in the Lb. acidophilus, Lb. delbrueckii subsp. bulgaricus (Hummel,
Hertel, Holzapfel, & Franz, 2007), and Lb. johnsonii (Mayrhofer et al.,
2010) as well as in Lb. reuteri (Lin, Fung, Wu, & Chung, 1996) and Lb.
plantarum(Ahn, Collins-Thompson, Duncan, &Stiles, 1992). In addition,
erythromycin resistance genes, such as erm(A), erm(C), or erm(T)
186 P. Sharma et al. / Food Research International 57 (2014) 176195
responsible for the macrolides, lincosamides, and streptogramins (MLS)
resistance phenotype, while the erm(B) gene, most frequently found
among Lactobacillus sp. (Mayrhofer et al., 2010 and van Hoek,
Margolles, Damig, Korhonen, et al., 2008). The erm(B) gene was detect-
ed fromtwo strains of each of the Lb. fermentum and Lb. vaginalis, and
one strain each of Lb. plantarum, Lb. salivarius, Lb. acidophilus, Lb.
animalis, and S. thermophilus. Aminoglycoside acetyl transferase re-
sistance genes, such as aac(6)-aph(2), ant(6), and aph(3)-IIIa
(Rojo-Bezares et al., 2006), phosphotransferase encoding genes
aaa(60)-aph(200) (Ouoba et al., 2008) and the lincosamide resis-
tance gene lnu(A) was also found in Lactobacillus strains (Cataloluk
& Gogebakan, 2004; Hummel et al., 2007; Kastner et al., 2006). In
the PROSAFE (Product Safety Enforcement of Europe, is a non-
prot professional organization for market surveillance authorities
and ofcers to improve the safety of users of products and services
in Europe), project, probiotic lactobacilli were reported to possess
erm(B) and/or tet(W), tet(M) or unidentied members of the tet(M)
group (Klare et al., 2007). In probiotic commercial Lb. reuteri ATCC
55730, tet(W) and the lincosamide resistance gene lnu(A) were detect-
ed (Kastner et al., 2006). Hummel et al. (2007), determined the antibi-
otic resistance of probiotic Lb. salivarius BFE 7441 having an erm(B)
gene on chromosome. The probiotic strain Lb. plantarum CCUG 43738,
displayed phenotypic resistance to tetracycline and minocycline, was
found to have a tet(S) gene on plasmid of approximately 14 kb (Huys
et al., 2006).
While in case, of Bidobacterium (B. animalis subsp. lactis and
B. thermophilum), a gene coding for a ribosomal protection protein,
erm(X) and transposonTn5432 was identied (van Hoek, Mayrhofer,
Domig, & Aarts, 2008). Tetracycline resistance in Bidobacterium
sp. deserves special attention. The genes tet(W), tet(M), tet(O), tet
(W/32/O),and tet(O/W) have been detected in several Bidobacterium
species, including B. longum (subsp. infantis and subsp. longum),
B. breve, B. animalis subsp. lactis, B. bidum, B. pseudocatenulatum, and
B. thermophilum (Aires, Doucet-Populaire, & Butel, 2007; Aires,
Thouverez, Doucet-Populaire, & Butel, 2009; Ammor, Flrez, Alvarez-
Martn, et al., 2008; Flrez, Ammor, Alvarez-Martn, Margolles, &
Mayo, 2006; Gueimonde et al., 2010; Kazimierczak, Flint, & Scott,
2006; van Hoek, Mayrhofer, Domig, Flrez, et al., 2008). The tet(W)
gene has been identied at high frequency in B. longum strains and in
all B. animalis subsp. lactis strains (Aires et al., 2007; Ammor, Flrez,
Alvarez-Martn, et al., 2008; Gueimonde et al., 2010) paradoxically
this strain is extensively used in the functional food industry, especially
in fermented dairy products (Masco, Huys, DeBrandt, Temmerman, &
Swings, 2005). Many of the genetic determinants mentioned above
are sometimes found on potentially mobile elements, such as trans-
posons and plasmids, which could spread the antibiotic resistance
genes mainly by conjugation mechanisms. The ability to transfer anti-
biotic resistance genes must be considered as an important parameter
for the selection of the probiotic strains. According to European Union,
acquired antibiotic resistance genes have been found among LAB organ-
isms and there is no barrier between pathogenic (e.g., streptococci), po-
tentially pathogenic (e.g., enterococci), and commensal (e.g., lactobacilli
and lactococci) bacterial species (Mathur & Singh, 2005). The presence
of transmissible antibiotic resistance markers in LAB and some antibiot-
ic resistance determinants located on plasmids have been reported to
occur in Lactococcus lactis and Enterococcus species (Gevers et al.,
2003). It is important to authenticate that probiotic and nutritional
LAB strains consumed on a daily basis worldwide lack acquired antimi-
crobial resistance properties and before considering them safe for
human and animal consumption. Acquired resistance genes of probiotic
lactobacilli have been reported previously (Table 2).
In LAB, conjugative plasmids and transposons are common
(Clewell, 1993; Davison, 1999) and various commensal bacteria, in-
cluding benecial bacteria, were identied to be the carriers of anti-
biotic resistance determinants to the consumer via the food chain
(Gevers et al., 2003; Pourshaban, Ferrini, Mannoni, Oliva, & Aureli,
2002; Teuber et al., 1999). Such evidence has raised questions re-
garding LAB's traditionally accepted safety status and initiated inves-
tigations in the biosafety of probiotic products (Vankerckhoven
et al., 2008).
7.3. Plasmids encoding antibiotic resistance in LAB
It is a matter of concern that plasmid associated resistance possibly
spread to other, more harmful species and genera. Plasmids are com-
mon in enterococci, lactococci, leuconostoc, pediococci, and present in
some strains of lactobacilli and bidobacteria (Teuber, 1995). Natural
resistance transfer mechanisms identied in LAB are very similar and
focused on conjugative plasmids (e.g. pAM1, pAD1 or pIP501 type)
and conjugative transposons (e.g. Tn916 type). Several mobilizable
plasmids have been identied, e.g. the tetracycline resistance plasmid
pMD5057 of L. plantarum 5057 and resistance plasmid pLME300 of
L. fermentum ROT1 (Danielsen & Wind, 2003; Gfeller, Roth, Meile, &
Teuber, 2003). At least 25 species of lactobacilli contain native plasmids
(Wang &Lee, 1997), andoften appear to contain multiple (from1 to 16)
different plasmids in a single strain. R-plasmids encoding tetracycline,
erythromycin, chloramphenicol, or macrolide lincomycinstreptogramin
resistance has been reported in the Lb. reuteri (Lin et al., 1996; Tannock
et al., 1994), Lb. fermentum (Fons et al., 1997; Ishiwa & Iwata, 1980),
Lb. acidophilus (Vescovo, Morelli, Bottazzi, & Gasson, 1983), and
Lb. plantarum (Danielsen, 2002) isolated from raw meat, silage and
feces. The reported prevalence of antibiotic resistance genes such as
erythromycin, vancomycin, tetracycline, chloramphenicol, and gentami-
cin resistance genes, on transferable genetic elements in enterococci is
more extensive, both on plasmids (Murray, An, & Clewell, 1988) and
transposons (Clewell, Flannagan, & Jaworski, 1995; Perreten et al., 1997;
Rice & Marshall, 1994).
7.4. Conjugative transposons encoding antibiotic resistance in LAB
Conjugative transposons are the major vehicle regarding antibiotic
resistance transport in LAB. They have been discovered in E. faecalis
(Tn916, Tn918, Tn920, Tn925, Tn2702), E. faecium(Tn5233) andLc. lactis
(Tn5276, Tn5301). In enterococci and streptococci, resistances to
tetracycline (tet(M)), erythromycin (erm(M)), chloramphenicol (cat)
and kanamycin (aphA-3) have been determined. These transposons
vary in size between 16 and 70 kb and may be inserted into plasmids
or the chromosome in one or multiple copies (Mathur & Singh, 2005).
The role that conjugative transposons play in the spread of antibiotic
resistance is indicated by similar resistance genes present in diverse
bacterial species (Scott, 2002). Localization of tet(M) and erm(B) on
conjugative transposons of the Tn916-Tn1545 family (Ammor et al.,
2007; Clewell et al., 1995; Rizzotti, La Gioia, Dellaglio, & Torriani,
2009) and of tet(K) on small plasmids (Oppegaard, Steinum, &
Wasteson, 2001) or in close association with insertion sequences
have been found (Yazdankhah, Srum, & Oppegaard, 2000). Several
mobile elements have been found in lactobacilli, including ISL2
(Insertion Sequences) in Lb. helveticus, ISL3 in Lb. delbrueckii, IS1223 in
Lb. johnsonii, IS1163, IS1520 in Lb. sakei and ISLp11 in Lb. plantarum
(Nicoloff & Bringel, 2003).
Many ARtraits are quite stable inboth the environment andthe host,
even in the absence of antibiotic selective pressure (Johnsen et al., 2005;
Srumet al., 2006), mostly due to the presence of various plasmid stabi-
lizationmechanisms. There are fewreports available in literature linking
conjugative transposons and antibiotic resistance in Lactobacillus sp.
(Ammor et al., 2007). The outcome of the EU-PROSAFE project was to
recommend that all future probiotics should not contain known antibi-
otic resistance traits (Vankerckhoven et al., 2008), and currently the
EFSA qualied presumption of safety proposals seem to be following
this recommendation (Barlow et al., 2007).
187 P. Sharma et al. / Food Research International 57 (2014) 176195
8. Horizontal gene transfer of antibiotic resistance from probiotic
LAB to other species
The human gastrointestinal tract (GIT) harbors 10
13
10
14
bacterial
cells in adults and this microbiota is often exposed to a variety of antibi-
otics, both directly and indirectly, due to their routine use in clinical
settings (de Vries et al., 2011). Therefore, the human GIT microbiota
may serve as an important reservoir of antibiotic resistant strains that
could act as opportunistic pathogens or as donors of resistance genes
to other bacteria (Salyers et al., 2004). Human intestinal bacteria not
only share resistance genes among themselves but can also acquire or
donate resistance genes to bacteria that are just passing through the
intestine (Simonsen et al., 1998; Teuber et al., 1999; van den Braak,
van Belkum, van Keulen, Verbrugh, & Endtz, 1998). Bacteria that
normally reside in the human colon can transfer resistance genes
among themselves when harmless commensal bacteria transform into
pathogens (Davison, 1999; Finlay & Falkow, 1997; Kidwell & Lisch,
2000; Manges et al., 2001; Ochman, Lawrence, & Groisman, 2000) and
once acquired, resistance genes are not easily lost. Instead, they become
a relatively stable part of a genome. The safety aspects of these bacteria
are of concern, including the presence of potentially transferable antibi-
otic resistances (Mathur & Singh, 2005; Salyers et al., 2004; Suskovic,
Brkic, Matosic, & Maric, 1997).
Traditionally, antibiotic resistance genes were not part of the
standard screening assays for starter cultures and probiotic bacteria
used in foods or as food supplements. Consuming products containing
Antibiotic Resistance Transferring (ART) starter cultures or probiotic
bacteria could be detrimental instead of benecial, to gut and public
health. It is important that the bacterial culture intended for use in
fermentation or probiotic applications be characterized at the strain
(isolate) level for not only the absence of AR genes but also for the
potential of both acquisition and dissemination of such genes via
Horizontal Gene Transfer mechanisms. However, benecial bacteria,
including starter cultures and probiotic bacteria, are also susceptible to
HGT mechanisms (Wang, Mc Entire, Zhang, Li, & Doyle, 2012). While
the horizontal transmission of AR genes can occur not only in pathogens
and commensal bacteria, but also in benecial bacteria, including those
used as starter cultures and probiotics. For example, a Bidobacterium
spp. strain once used to supplement in yogurt products carried a tet
r
-
encoding gene. Consequently, LAB spp. also prone to HGT mechanisms,
although this feature in lactic acid bacteria was considered to be bene-
cial for bioengineering strains with industrial applications. Besides
serving as an AR gene reservoir, commensal bacteria may also serve as
an AR gene transmission amplier (Wang et al., 2012).
Probiotics as an industry has grown rapidly in the last couple of
decades based on the belief that consumption of certain lactic acid
bacteria and bidobacteria is benecial for the maintenance of a healthy
gut microbiota. This is supported by two main observations: (i) a
relatively higher population of bidobacteria and lactobacilli is found
in infants and some long lived adults than in the general population,
and (ii) the dominance of these bacteria may competitively inhibit
other bacteria, particularly pathogens (Wang et al., 2006). For a long
time, it was assumed that probiotic bacteria do not transmit bad traits
suchas AR. However, results frommicrobial genome sequencing studies
exemplify that a number of AR genes are already integrated into the
chromosomes of several lactic acid bacteria (Ammor, Gueimonde,
Danielsen, et al., 2008; Makarova et al., 2006). It has been further
identied that Enterococcus spp., Lactococcus spp., Leuconostoc spp.,
Streptococcus thermophilus, and Carnobacterium spp. are among the
dominant AR gene carriers found in various food products and are
capable of transmitting AR genes (Li & Wang, 2008; Wang et al.,
2006). As mentioned above, high-frequency conjugation system is
present in several lactic acid bacteria and other gram-positive bacteria,
and this system likely facilitates the transmission of AR genes
(Luo, Wan, & Wang, 2005). However, certain strains of lactic acid
bacteria and perhaps other probiotic strains, a number of AR genes
have been identied in several potential probiotic strains, and some of
them are transferable to other bacteria by HGT (Ouoba et al., 2008).
However, since commensal bacteria have been shown to transfer
determinants of resistance to pathogens (Liu et al., 2009), studies on
development of resistance should also include lactobacilli. Regular
consumption of ART bacteria, coupled with occasional colonization
and HGT events, will probably inuence the AR of the human gut micro-
biota. Interspecies gene transfer to E. faecalis of mobilizable erm or tet
containing plasmids from food-related Lb. plantarum has been demon-
strated in vitro and in the intestinal tract of gnotobiotic rats (Feld
et al., 2008; Jacobsen et al., 2007). The recent detailed studies on the
antibiotic resistance proles of lactic acid bacteria have demonstrated
the occasional presence of acquired resistance genes in Lactobacillus
species (Morelli, 2008). The prevalence of acquired antibiotic resistance
genes includes the frequent occurrence of tet(M) and erm(B) genes
(conveying resistance to tetracycline and erythromycin, respectively)
in lactobacilli isolated from different fermented foods (Comunian
et al., 2010; Zonenschain, Rebecchi, & Morelli, 2009). Interspecies
conjugative transfer of tetracycline and erythromycin resistance
plasmids from LAB has been demonstrated previously in vitro (e.g.
Feld et al., 2008; Gevers et al., 2003; Ouoba et al., 2008). While there is
prevalence of tet(M) and erm(B) genes has been estimated to be higher
than 60% in lactobacilli of human and dairy origin (Temmerman et al.,
2003). On the basis of these premises, an increasing amount of research
has been focussed to study the transfer antibiotic resistance in LAB
(Florez et al., 2008; Toomey, Bolton, & Fanning, 2010).
Table 3
Horizontal gene transfer of antibiotic resistance from probiotic LAB to other species.
Donor strain Recipient strain Conjugal mating method Mode of transmission Resistance gene References
Lb. plantarum DG 507 E. faecalis In vitro Plasmid erm(B) Gevers et al. (2003)
E. faecalis LMG20790 E. faecalis JH2-2 In vitro Tn916Tn1545 erm(B) Huys, D'Haene, Collard,
and Swings (2004)
E. faecalis LMG20927 E. faecalis JH2-2 In vitro Plasmid/Tn916Tn1545 erm(B) Huys et al. (2004)
Lb. plantarum DG 522,
Lb. plantarum DG 507
E. faecalis In vivo Plasmid tet(M), erm(B) Jacobsen et al. (2007)
Lb. reuteri L4:12002 E. faecalis JH2-2 In vitro Plasmid erm(B) Ouoba et al. (2008)
Lb. plantarum pLFE1 E. faecalis In vitro erm (B) Feld et al. (2008)
Lc. lactis SH4174 Listeria monocytogenes (H7) In vitro pAM-1 Plasmid erm(B) Toomey, Monaghan, Fanning,
and Bolton (2009)
S. thermophilus Listeria monocytogenes (H7) In vitro Plasmid erm(B) Toomey et al. (2009)
Lc. lactis BU-2-60 E. faecalis JH2-2 In vitro tet(M) Toomey et al. (2010)
Lb. fermentum NWL24 and
Lb. salivarius NWL33
E. faecalis 181 In vitro erm(B), tet(M) Nawaz et al. (2011)
E. faecalis CM5 V, CM6 V E. faecalis OG1RF In vitro Plasmid erm(B) Vignaroli, Zandri, Aquilanti,
Pasquaroli, and Biavasco (2011)
E. durans PF1 V E. faecalis 64/3 In vitro Plasmid erm(B) Vignaroli et al. (2011)
E. durans PF3 V E. faecalis OG1RF, E. faecalis 64/3 In vitro Plasmid erm(B) Vignaroli et al. (2011)
188 P. Sharma et al. / Food Research International 57 (2014) 176195
Transfer of antibiotic resistance from LAB strains has been
documented in vitro, but very fewstudies have conrmed this antibiotic
resistance transfer in vivo and it was only observed in the dissociated
animal model (Feld et al., 2008; Gruzza, Fons, Ouriet, Duval-Iah, &
Ducluzeau, 1994; Jacobsen et al., 2007; Schlundt, Saadbye, Lohmann,
Jacobsen, & Nielsen, 1994) and the conjugative plasmid pAM1 has
been transferred from Lb. reuteri to Enterococcus faecalis (Morelli,
Sarra, & Bottazzi, 1998) and among Lc. lactis strains, the transfer could
not be observed in conventional rats (Schlundt et al., 1994). A trans-
poson Tn916 including tet(M) was transferred to E. faecalis strain
JH2-2 in mating experiments, but only at a low conjugation frequency
(Devirgiliis, Coppola, Barile, Colonna, & Perozzi, 2009). In one of studies
tetracycline genes were identied from lactobacilli including Lb. keri
NWL78 isolated from a probiotic yogurt and erm(B) gene from
Lb. fermentum NWL24 and Lb. salivarius NWL33 and tet(M) gene from
Lb. plantarum NWL22 and Lb. brevis NWL59 were successfully trans-
ferred to E. faecalis 181 in lter mating experiments (Nawaz et al.,
2011). In terms of virulence, in vivo studies mostly reported transfer
of antibiotic resistance, including resistance to vancomycin, tetracycline
and erythromycin. Mater, Langella, Corthier, and Flores (2008) reported
the transfer of van(A) resistance fromE. faeciumto a commercially avail-
able probiotic strain of Lb. acidophilus was observed in vivo and in vitro in
mice. Probiotic products and starter strains rarely had acquired antibiotic
resistance suchresults depict the attentionfor a strict monitoring andreg-
ulation. Therefore, besides the presence of AR genes, the genetic charac-
teristics of bacteria carrying the resistance genes are considered as
important parameters for assessing the potential for HGT both in probiot-
ic and starter cultures (Li, Li, Alvarez, Harper, & Wang, 2011). HGT from
probiotic Lactobacilli to other sp. mentioned in (Table 3).
8.1. Horizontal gene transfer fromdifferent sources via food products to the
gastrointestinal tract
The HGT may widely occur due to antibiotic treatment and post
pasteurization contamination in the process or lack of an adequate
treatment during manufacturing of the product. Transmission of anti-
biotic resistance results in the dissemination of antibiotic resistance
genes to our commensal ora and opportunistic pathogens. The latter
may further transfer resistance genes to the pathogenic bacteria in the
GIT. Horizontal transmissions of AR genes between commensal and
pathogenic microorganisms in ecosystems are much more than direct
AR gene dissemination from one pathogen to another (Andremont,
2003). For antibiotic resistance to happen, the bacterium has to remain
in the food process, through, for example, fecal contamination in the
process or via a recontamination by improper handling, or lack of an
adequate temperature treatment in the process. Due to the selective
pressure of antibiotic treatment, antibiotic resistance genes may spread
to sewage/manure, and either directly or indirectly through food
products. Through food these genes may further transfer to the human
and colonize in the GIT and causing infections or disease in humans.
Antibiotic resistant bacteria have been found not only in various food
products and environmental samples but also in hosts even without a
history of direct exposure to antibiotics (Gueimonde, Salminen, &
Isolauri, 2006; Ready, Bedi, Spratt, Mullany, & Wilson, 2003; Villedieu
et al., 2004). A broad spectrum of commensal bacteria, including lactic
acid bacteria, has been identied as being carriers of AR genes and is
able to transmit those genes to other bacteria leading to increased
resistance in the recipient organisms (Feld, Bielak, Hammer, & Wilcks,
2009; Wang et al., 2006). Even without direct exposure to antibiotics,
Fig. 2. Horizontal gene transfer fromdifferent sources and via food products to the gastrointestinal tract adaptedfromKhachatourians (1998); Anonym(2004); Claycamp and Hooberman
(2004); Wang et al. (2012); Verraes et al. (2013).
189 P. Sharma et al. / Food Research International 57 (2014) 176195
animal and human wastes are one of the most signicant sources of ART
bacteria directly contributing to the amplication cycle of ARin the global
ecosystem. Antibiotic exposure further selectively enriches ART bacteria
in the host gut microbiota. Ultimately, a large number of ART bacteria in
sewage and manure directly contribute to the prevalence of AR in the
environment (soil, water, etc.), raw food materials (meat, milk, plant
materials), food processing environments and food handlers. The
isolation of AR genes in food-borne pathogens from retail products
exemplied the potential contribution of the food chain in transmitting
ART pathogens to humans (Charpentier & Courvalin, 1999; Luo et al.,
2005; Zhao et al., 2001). The studies on commensal bacteria, however,
are limited and primarily focused on the opportunistic pathogen entero-
cocci (Cocconcelli, Cattiveli, & Gazzola, 2003; Johnston & Jaykus, 2004).
The presence of several AR markers including erm(B), erm(C), tet(S/M)
and tet(A), encoding ribosomal modication and tet efux mechanisms,
in selected food isolates were identied. With the signicant exception
of the enterococci, antibiotic resistance plasmids andtransposable genetic
elements are relatively rare among LAB(Florez et al., 2003) andapparent-
ly no antibiotic transferable resistance determinants have been detected
among the Bidobacterium spp. (Devirgiliis, Caravelli, Coppola, Barile, &
Perozzi, 2008). Among the lactobacilli, certain strains of Lb. fermentum,
Lb. acidophilus, Lb. reuteri and Lb. plantarum has been shown to harbor
resistance plasmids against erythromycin (and related macrolides),
tetracycline, and chloramphenicol. Many strains of these species are
used in the food industry either as starter cultures or as probiotics. In
one probiotic, Lactobacillus strain GG, the vancomycin resistance genes
(van) of enterococci were studiedandveriedthat the presence of vanco-
mycin resistance determinant in the particular strain was not closely
related to enterococcal van genes (Comunian et al., 2010). In conclusion,
transfer of antibiotic resistance does not appear likely in the case of
dairy or probiotic lactic acid bacteria, with the exception of enterococci.
Genetic material, especially that represented by plasmids, can be
transferred between food microbes and the human intestinal microbio-
ta or pathogens. The mechanisms of HGT between bacteria may occur in
the soil, in water, or in the digestive system of humans and animals, as
well as in food. (Fig. 2) depicts the HGT in food products from different
sources and its transfer to the gastrointestinal tract. The frequency of
HGT largely depends on the properties of the mobile genetic element,
the characteristics of the donor and recipient populations, and the
environment. In addition to conjugation, transduction and transforma-
tion, and other less well recognized mechanisms of DNA transfer may
occur in nature (Keese, 2008).
Hence, the impact of AR dissemination fromART pathogens in foods
to humans is probably minimal. However, recent studies have revealed
a large AR gene pool in food borne commensal bacteria in retail foods
(Comunian et al., 2010; Li et al., 2011; Wang, Jaykus, Wang, &
Schesinger, 2009) and some food items carried as many as 10
8
copies
of AR genes per gram of food (Luo et al., 2005), and a number of food
borne bacteria identied as the AR gene carriers (Citak, Yucel, &
Orhan, 2004; Duran & Marshall, 2005; Li et al., 2011; Oh et al., 2011;
Stanton, Humphrey, & Stoffregen, 2011; Teuber, Schwarz, & Perreten,
2003; Wang et al., 2009). Antibiotic resistance genes from food borne
bacteria were transferred to human inhabitant and pathogenic bacteria
by natural HGT mechanisms, leading to acquired resistance in the recip-
ient strains (Feld et al., 2009; Li, Sun, Zhang, Li, & Wang, 2010; Toomey
et al., 2010; Wang et al., 2006). Aconstant supply of these foods without
further processing will probably inuence the AR of the human gut mi-
crobiota (Wang et al., 2006). The evolution of resistance in both patho-
gens and commensal bacteria is affected by the type of antibiotic
exposure, the local microbial population, and other host and environ-
mental factors. Since commensal bacteria dominate in both numbers
and genetic diversity in natural and host environments. They can poten-
tially be good indicators of the ARand could provide anearly warning of
the emergence of ARinpathogens (Wang et al., 2009). It is nowbelieved
that the foodchain may well play a very important role in disseminating
AR genes and ART bacteria to the human digestive microbiota (Duran &
Marshall, 2005; Wang et al., 2006). The colonizationby ART bacteria and
associated HGT events certainly contribute to the increasing resistance
to antibiotics in humans (Wang et al., 2006). These studies further iden-
tied that various commensal bacteria, including some benecial bacte-
ria are involved in the HGT events, as either the AR gene recipients or
potential donors. A safety goal for probiotics should not increase the
already existing risks of antibiotic transfer associated with the normal
gut or food microbiota yet the use of probiotic strains in various food
products is to provide various health benets to the consumers.
9. Conclusions
The extraordinary capacity of a number of bacterial organisms to
develop resistance to antibiotic compounds continues to remain a
major concern for researchers as well as health and industrial managers
for several years. Apart from their various health benets probiotics
have also been used to treat and/or prevent antibiotic resistance in the
host. However, recent studies have reportedthe occurrence of antibiotic
resistance in probiotics themselves. These have led to the need for
further assessment of probiotics so that their possible negative con-
sequences do not outweigh their benets. The spread of antibiotic
resistance to other organisms is currently one of the most important
safety issues regarding the use of bacteria in a variety of food products
as food chain is considered as one of the main routes for the transmis-
sion of antibiotic resistant bacteria. Bacteria naturally present in foods
or food supplements, or deliberately added to them, including probiotic
bacteria, constitute a potential source of antibiotic resistance determi-
nants. Since there has been a signicant rise in the consumption of
probiotic products, it is imperative that probiotics are well researched
and documented for their antibiotic resistance prole. The ability to
transfer antibiotic resistant determinants must be considered as an
important parameter for the selection of probiotic strains. Multiple
drug resistance has been found in different species of probiotic strains,
whichis detrimental to food safety. Evaluation of the safety of probiotics
for human consumption must be guided by established criteria,
guidelines and regulations, and standardized methodology for premarket
biosafety testing and post market surveillance should be in place.
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