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8. Horizontal gene transfer of antibiotic resistance from probiotic LAB to other species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1888.1. Horizontal gene transfer from different sourcesviafood products to the gastrointestinal tract . . . . . . . . . . . . . . . . . . . . . 189

9. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 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 foundin almost every habitat on the planet. Many of them are benecial formankind 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 andinhibit food spoilage bacteria by producing growth-inhibitingsubstances. 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 economicalpoint 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 now bacteria areghtingback 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 aroundthe 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-term antibiotic administra-

tion can lead to stabilization of resistant bacterial populations in thehuman 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 last60 years (European Commission, 2005). Antibiotics are widely used inhuman and veterinary medicine, and have been essential for ensuringhuman and animal health. The selective pressure of such an intensive

use of antibiotics has urged bacteria sensitive to antibiotics to becomeresistant in order to survive (Andersson & Hughes, 2010). Much of theconcern has been about pathogenic bacteria and their antibioticresistances, since infections caused by these resistant microorganisms

are not only more complicated to treat, but the treatment is muchmore costly due to the more intensive and time consuming care neededin these cases. Subsequently, with the spread of antibiotic resistance inmicrobial communities, concerns have been raised about the existenceof 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 offood products and pharmaceutical preparations and multiple claimsmade regarding their benecial health effects. There is a need to put

sufcient safeguards to protect the consumers from any adverse effectsensuring standards of commercial products and their efcacy. Thesafety 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 topathogenic bacteria/commensal microbiota of human gut is detrimental

to thesafety of probiotic strains used in such products. Thereare certainpros 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 bacterialpathogens directly/indirectlyvia commensal ora. In this case, theymay also acquire resistance from human commensal microbiota(Courvalin, 2006).

These days increased attention is given to safety of bacteria used inthe food. Although, LAB consumed in enormous quantities, primarilyin fermented foods such as meat, dairy and probiotic products, whichinteract 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 bacterialstrains harboring transferable antibiotic resistance genes should not

be used in animal feeds, fermented and probiotic foods for human use(EFSA, 2007). The transmissionof antibiotic resistance genes to unrelat-ed pathogenic or potentially pathogenic bacteria in the gut is a majorhealth concern related to the probiotic application. In Europe, according

to the Qualied Presumption of Safety (QPS) approach, established bythe EFSA (EFSA, 2008), the nature of any antibiotic resistance determi-nant present in a candidate microorganism should be determinedprior to approval for QPS status. Further, this concern is amply reected

in the newly released Codex Guidelines for risk analysis of foodborne antimicrobial resistancewhere document uses the broad termantimicrobial resistant microorganisms, beyond food borne pathogenswhen discussing the risks associated with the food chain (Codex

Alimentarius Commission, 2011). Antibiotic resistant microorganismsin food system continuein vogueto 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 foreach strain used as

a probiotic organism, food or feed additives could be determined usingprotocols given by EFSA and on rm genetic grounds. As, probioticorganisms are considered to pool the resistant genes and transferthese to pathogenic/commensal bacteria. Therefore, besides the

presence of antibiotic resistance genes, probiotics in commercial useneed to be well documented with respect to their ability to transfersuch genes to other organisms. Thus, it is essential to thoroughly inves-

tigate the safety of microorganisms used in probiotic products(Borrielloet al., 2003).

2. Dening probiotics

In modern times, the universal meaning of the term probioticwas

established by the World Health Organization (WHO) and the Food andAgriculture Organization (FAO) of the United States. These two organi-zations dened probiotics as Live microorganisms which whenadministered in adequate amounts confer a health benet on the host

organism(FAO/WHO, 2002). A daily recommended dosage of 106109viable organisms being the most effective for the benecial effectof 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

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intestinal dysfunction (Corcionivoschi et al., 2010). Commercialprobiotic cultures of human consumption marketed as probioticproducts are generally considered as safe for humans. In fact, someLAB strains of animal and human intestinal microora microbiota

have been adopted as probioticfood supplements primarily becauseof the perception that they are desirable members of the intestinalmicroora microbiota (Berg, 1998; Goldin & Gorbach, 1992) and

possess GRAS status including Enterococcus faecium, Lactobacillus

plantarum, Lb. acidophilus, Lb. casei subsp. rhamnosus. Several ofthese 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-foodformats. In case of strains other than bacteria and of nonhuman origin

Saccharomyces boulardii is the only fungal probiotic used in thefermen-

tation of dairy products for many years (Ishibashi & Yamazaki, 2001).Unlike many of the probiotic bacteria, it was not originally isolatedfrom human feces, but rather from fruit. Species from other bacterialgenera such asStreptococcus,Bacillus, andEnterococcushave also beenused 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 probioticstrains selected for commercial use in foods and in therapeutics mustretain the characteristics for which they were originally selected

(Salminen et al., 1998). These include the characteristics of growthand survival during manufacture and, after consumption, during transitthrough the stomach and small intestine. Importantly, probiotic mustretain these characteristics to provide various health benets to theconsumers. 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 arealso reported to exert several other positive manifestations on hosthealthviz. 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 reductionin obesity (DiBaise et al., 2008). It is therefore essential that the probiot-ic strains used are tested and meet safetystandards setby theEuropean

Union (EU).

3. Consumption and market potential of probiotic products

Probiotic products are sold either as capsules/tablets or fermenteddairy/food products and their established/claimed health effectsreinforcing 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 aboutthe impact of food on health. In the year 2000, the world-wide marketfor 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 foodproducts are classied in the category of functional foods and representa signicantpartof this marketand comprise between60 and 70% of the

total functional food market (Holzapfel, 2006). Food applications forprobiotics are found mostly in dairy products, with yogurts, ker,and cultured drinks representing the major categories. The scienticdevelopment of such products had shown the high sensory acceptance

with yogurt accounted to have the largestshare of 36.6% (Almeidaet 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; Escobaret al., 2012; Esmerino et al., 2013; Gomes et al., 2011; Karimi,Mortazavian, & Karami, 2012; Minervini et al., 2012), probiotic icecream (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 fordelivering 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 marketfor 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 2013to 2018 and expected to reach USD 37.9 billion in 2018. In Asia-Pacic,China and Japan dominate the market revenue for probiotics, with Indiaand other regions also showing signicant growth. Probiotics of the

Lactobacillusgenus are having the largest share, representing 61.9% oftotal sales in 2007 (Food Processing, 2009). Several probioticbacteria of human origin are now being exploited commerciallye.g.,

Lb. rhamnosusGG (Saxelin, 1997),Lb. caseistrain Shirota (Aso & Akazan,

1992andSpanhaak, Havenaar, & Schaafsma, 1998), andLb. acidophilusLA-1 (Bernet, Brassart, Neeser, & Servin, 1994), many consumers,consumer organizations, and members of the scientic community areskeptical of such products and their publicized probiotic claims (Sanders

& Huis in't Veld, 1999). However, species belonging to the genera

Lactococcus,Enterococcus,SaccharomycesandPropionibacteriumare alsoconsidered 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 probioticstrains 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 availableprobiotic products are sold in combination with Lactobacillus sp.or Bidobacterium sp. (Weese, 2002, 2003; Weese & Martin, 2011).

Nowadays, multistrains or multispecies probiotic mixtures arebecoming increasingly popular compared with single strain probioticsas they may have additive or even synergistic effects which can resultin higher efcacy (Chapman et al., 2011; Temmerman, Koning,

Mulder, Rombouts, & Beynen, 2004). Some of them are listed inTable 1. Organisms other than lactic acid bacteria, which are currentlyused in probiotic preparations, includeBacillussp., yeasts (S. boulardii,

S. cerevisiae), lamentous fungi (Aspergillus oryzae) andE. coli Nissle

1917. These probiotic preparations may consist of a single bacterialstrain or it may be a consortium in commercial probiotic products andavailable either in powders, liquid form, gel, paste, granules or in theform 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

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concentrate added to food (dried or in the deep freeze form) 2) foodproducts (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 humanconsumption, a consortium of LAB have been most commonly used asit constitutes a major part of the normal microbiota of the humanintestine and when present in sufcient numbers creates a healthy

equilibrium between benecial and potentially harmful microbiota inthe gastrointestinal tract (Dunne et al., 2001). For some positive effectson human health, a probiotic strain has to reach the large intestine at aconcentration of about 107 viable cells/g(Stanton et al., 2001). The basic

requirement for probiotics is that products should contain sufcientnumbers of microorganisms up to the expiry date ( Fasoli et al., 2003).However, to have functional probiotic strains with predictable andmeasurable health benets, a rigorous effort for strain selection is

required. The selection criteria for lactic acid bacteria to be used asprobioticinclude 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 duringpassage 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 intestinalmicroora and provide various health benets (g) genera of humanorigin (h) stable against bile,acid,enzyme and oxygen(i) safety assess-

ment include nonpathogenic, nontoxic, nonallergic, nonmutagenic andshould 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 shouldbe selected to maintain the viability and stability of the strain during

commercial production as well as identifying the compatibility whileadded to the end product (Godward et al., 2000; Mattila & Saarela,2000; Talwalkar & Kailasapathy, 2004). In addition, these strains shouldbe 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 acidophilusNCFM Rhodia Inc. (Madison, Wis.) Yeung, Cano, Tong, and Sanders (1999)

Lb. acidophilusDDS-1 Nebraska cultures Yeung et al. (1999)

Lb. acidophilusSBT-2062 Snow Brand Milk Products Co., ltd.(Tokyo, Japan) Yeung et al. (1999)

Lb. acidophilusLA-1 (same strain LA-5 sold in Europe) Chr. Hansen, Inc. (Milwaukee, Wis.) Yeung et al. (1999)

Lb. caseistrain Shirota Yakult (Tokyo, Japan) Yeung et al. (1999)

Lb. caseiImmunitas Danone (Paris, France) Yeung et al. (1999)

Lb fermentasRC-14 Urex Biotech (London, Ontario, Canada) Yeung et al. (1999)Lb. johnsoniiLa1(same as Lj1) Nestle (Lansanne, Switzerland) Yeung et al. (1999)

Lb. plantarum299V Probio AB (Lund, Sweden) Yeung et al. (1999)

Lb. reuteriSD2112(same as MM2) Biogia (Raleigh, N.C.) Yeung et al. (1999)

Lb. rhamnosusGGa Valio Dairy (Helsinki, nland) Yeung et al. (1999)

Lb. rhamnosusGR-1 Urex Biotech (London, Ontario, Canada) Yeung et al. (1999)

Lb. rhamnosus271 Probio AB (Lund, Sweden) Yeung et al. (1999)

Lb. rhamnosusLB 21 Essum AB (Umea, Sweden) Yeung et al. (1999)

Lb. salivariusUCC 118 University College (Cork, Ireland) Yeung et al. (1999)

Lb. lactisL1A Essum AB (Umea, Sweden) Yeung et al. (1999)

Lb. acidophilusNCFMR Danisco (Madison WI) Sanders (2008)

Lb. rhamnosusGG (LGG) Valio Dairy (Helinsinki, Finland) Sanders (2008)

Lb. rhamnosusR0011,Lb. acidophilusR0052 Institute Rosell (Montreal, Canada) Sanders (2008)

Lb. caseiDN 114001 Danone (Paris, France) Sanders (2008)

Lb. acidophilusLB Lacteol Laboratory (Houdan, France) Sanders (2008)

Lb. paracaseiF19 Medi pharm (Des Moines, Iowa) Sanders (2008)

Lb. reuteriRC-14 Chr. Hansen (Milwaukee WI) Sanders (2008)

Bidobacterium lactisBb-12 Chr. Hansen (Milwaukee WI) Sanders (2008)Bidobacterium longumBB536a Morianga Milk Industry Co. Ltd. (Zama city, Japan) Sanders (2008)

Bidobacterium longumSBT-2928a Snow Brand Milk Products Co. Ltd. (Tokyo, Japan) Sanders (2008)

B. brevestrain Yakult Yakult (Tokyo, Japan) Sanders (2008)

Bidobacterium animalisDN173010 Activia, Danone Guarner et al. (2008)

Bidobacterium animalissubsp. lactis Bb-12 Chr. Hansen Guarner et al. (2008)

B. infantis35624 Align, Procter & Gamble Guarner et al. (2008)

B. lactisHN019 (DR10) Howaru Bido, Danisco Guarner et al. (2008)

Enterococus LAB SF 68 Bioorin, Cerbios-Pharma Guarner et al. (2008)

Escherichia coliNissile 1917 Mutaor, Ardeypharm Guarner et al. (2008)Lactococcus lactis L1A Norrmejerier Guarner et al. (2008)

Lb. reuteriATCC 55730 Biogia Biologics Guarner et al. (2008)

Lb. rhamnosusATCC 53013 (LGG) Vit, Valio Guarner et al. (2008)

B. lactisHN019 branded as DR10, and strainLb. rhamnosusHN001 as DR20 New Zealand Guarner et al. (2008)

Lactobacillus caseiDN 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 longumBB536 Japan Morinaga Milk Industry Raja and Arunachlam (2011)

Sacchromyces boulardii Biocodex (Creswell OR) Tiwari, Tiwari, Pandey, and Pandey (2012)Lb. fermentumVR1003(PCC) Probiomics (Eveleigh, Australia) Tiwari et al. (2012)

Lb. acidophilusLA-5 Chr. Hansen (Milwaukee, WI) Tiwari et al. (2012)

Lb. paracaseiCRL 431 Chr. Hansen (Milwaukee, WI) Tiwari et al. (2012)

B. lactisBb-12 Nestle (Glandale, CA) Tiwari et al. (2012)



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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 concernsassociated 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 metaboliceffects on the gastrointestinal tract; and (3) the transfer of antibioticresistance in the gastrointestinalora (Snydman, 2008). The available

epidemiological data, clinical trials and acute toxicity studies recom-mended that the LAB commonly occurring in fermented foods andused in current probiotics are considered to be safe. Organisms thatare generally regarded as safe involve Lactobacilli, Lactococci,

Bidobacterium, and yeast. Though, there are other probiotic organisms,

such asEnterococcus,Bacillus,Streptococcusand spore-forming bacteriawhich are not regarded as safe for human consumption yet are beingused as probiotics. Many strains of these species are used in the foodindustry 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; Vankerckhovenet 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 ofantibiotic 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 possibleand care should be taken while selecting the strains to avoid carryingtransferable resistance determinants and their ability to facilitateplasmid transfer (Salminen, von Wright, Ouwehand, & Holtzapfel,

2000). To prevent the undesirable transfer of resistance to endogenousbacteria, probiotics should not carry resistance other than that required.A safety goalfor probiotics should not increase the already existingrisks

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 ofin vitrostudies, animal studies, and human clinicalstudies indicated that some current probiotic strains are required to

full the safety standards (Donohue, Salminen, & Marteau, 1998). Anumber of initiatives have been recently launched in order to checkthe transferability of antibiotic resistance in starter cultures andprobiotic microorganisms. EFSA has proposed microbiological

breakpointsfor several genera of LAB (EFSA, 2008). In this regard, thepresence of antibiotic resistance determinants, and their potentialmobility, requires special attention. Therefore, antibiotic resistance assuch is not a safety issue; it only becomes a threat when resistance is

transferable. Those probiotics belonging to species included in theEFSA and QPS list (EFSA, 2012) have excellent safety records, anddetrimental effects produced as a consequence of their ingestion arevery scarce (Gouriet, Million, Henri, Fournier, & Raoult, 2012). Most

probiotics are ingested in large amounts in functional foods, and thepresence of antibiotic resistance determinants in their genome mustbe systematically screened. Thus, it is of great interest to investigatewhether 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 toeliminate transmissible resistances in these probiotic food products.Such concern must be also expressed regarding consumption of

human probiotics. Epidemiological data on the safety of probioticssuggests no evidence of probiotics being involved with human infec-tions (Snydman, 2008). However, there always remains the possibilitythat probiotic consumption can cause infection and those individuals

will respondin differentways to a specic strain. So,the food industriesneed 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 moreimportant clinical and public health problem worldwide. Due to the

overwhelming use of antibiotics over the past few years in both animalsand 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 attainedeither through intrinsic or acquired mechanisms (Fig. 1A,B). Among

these, which mechanism prevails in the specic bacterial strain dependson the nature of the antibiotic, its target site, the bacterial species itselfand whether it is relatedwith plasmidor chromosomal mutation. Thereare 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, andthe dormant state (Zhang, 2007). Intrinsic resistance having a minimalpotential for horizontal spread and pose no risk in non-pathogenicbacteria, although any gene responsible for intrinsic resistance may

spread provided that it is anked by insertion sequences (EuropeanCommission, 2003). As a result, bacteria rstly adopt intrinsic mecha-nisms to protect themselves from the effect of antibiotics. There aremainly 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 targetsites to prevent itself from the attack of antibiotic. In bacterial speciesthere are number of plasmids in their genome which are responsiblefor transferring the antibiotic resistance genes (Deasy, 2009). Plasmidscarrying 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 ofpenicillin antibiotics, cephalosporins, carbapenems, and monobactams.Narrow-active enzymes are active against only a specic antibiotic;

while extended-spectrum beta-lactamases are resistant to multipleantibiotics (Belletti et al., 2009).

6.1.2. Increased antibiotic removal/Efux pumps and outer membrane

(OM) permeability changes

Active drug ef

ux pumps transport antibiotics away from bacteriaby reducing the concentration of the drug. Plasmid carrying resistance

genes has active drug efux pumps that transport antibiotic awayfrom the bacteria. Decrease in drug permeability and/or increase inactive efux (pumping out) of the drugs across the cell surface, causesthe development of resistance (Li & Nikadio, 2009). Multidrug efuxpumps 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 sensitivemicroorganism 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,



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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 targetedmetabolic 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 GeneTransfer (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 fromLevy (1998). (B) Acquired antibiotic resistance (a) transformation, (b) conjugation, (c) and transduction adapted fromLevy (1998).



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Table 2(continued)

Commercial probiotic products/strain Probiotic strain used Antibiotic resistance Location

Chinese fermented foods Lactobacillus andS. thermophilus Intrinsicresistant to nalidixic acid, kanamycin, andvancomycin Acquired resistance for penicillin,

erythromycin, clindamycin, and tetracycline while

resistance to gentamicin, ciprooxacin,

streptomycin, and chloramphenicol

erm(B) andtet(S)

Fermented milk culture Lb. pentosum(L08),Lb. jungurthi(L10),Lb. reuteri

(L16),Lb. fermentum(L18),Lb. plantarum(L29),

Lb. brevis(L43), andLb. casei(L47)

Kanamycin, trimetroprim, rifampicin, kanamycin,

amphicilin and penicillin

Dairy product Lb. vaginalisNWL35 Erythromycin erm(B)

Traditional Ethiopian fermented food Lactic acid bacteria Methicillin, Noroxacin

Probiotic strain Bidobacterium spp. Chloramphenicol, linezolid, and

quinupristin/dalfopristin

Fermented sausages Leuco. mesenteroidessubsp. mesenteroides,

Lb. curvatus,Lb. brevis,Lb. fermentum,

Lb. paracaseisubsp. paracasei

Nitrofurantoin, trimethoprim, oxacillin,

streptomycin, enrooxacine, sulphonamides,

polymyxin B, neomycin

Chinese yoghurts S. thermophilusandLb. delbruekiissp. bulgaricus Ampicillin, kanamycin, chloramphenicol,

chlortetracycline, tetracyclines, neomycin and

gentamicin

tet(M),ant6, aph 3-IIIa

Commercial probiotic product B. longumJDM301 Ciprooxacin, amikacin, gentamicin, streptomycin

Traditional fermented foods and curd E. faecium,E. durans,P. pentosaceus Erythromycin erm(B),msr(C)Indian vegetables and fermented foods Lb. plantarum,Lb. fermentum, Weissella spp.P. parvulus Gentamicin, vancomycin, noroxacin, kanamycin

Gene names and abbreviations: cat: chloramphenicol acetylase gene; erm: erythromycin resistance gene; tet: tetracycline resistance gene; str: streptomycin adenylase gene; lnu: lincosamvat: 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 resistanceis present in some strains within a species usually susceptible to theantibiotic under consideration, and might be horizontally spreadamong other bacterial species (Mathur & Singh, 2005). Acquired

resistance to antimicrobial agents can arise either from mutations inthe bacterial genome or through acquisition of additional genes codingfor a resistance mechanism. Mutations, which may 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 evolution of antibioticresistancein microbial 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) lyticand temperate bacteriophages (Davies, 1994; Devirgiliis, Barile, &Perozzi, 2011). Thus, intestinal bacteria can acquire resistance eitherby 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 resistancemechanism and the microbial taxon involved, the actual possibility ofspreading 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 oracquisition of the genetic element encoding antibiotic resistance(Sommer & Dantas, 2011). While, the gene transfer among organismswithin 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 threewell known mechanisms for horizontal gene exchange between

bacteria, the relative contribution of these mechanisms is known butconjugation 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 dealingwith these mechanisms in probiotics (LAB or Bidobacteria).

7. Antibiotic resistance proles of probiotic LAB

Antibiotics have played a signicantrole in the treatment of bacterial

infections, but misuse has contributed to a rise in bacterial resistance.Emergence of resistant microorganisms has become a major threat topublic health and has led to the manifestation of bacteria resistant tomany modern antibiotic formulations (Mazel & Davies, 1999). The

food chain could be regardedas oneof the main pathways forthe trans-mission of antibiotic resistant bacteria from animals to humans (Singeret al., 2003). Use of probiotics containing antibiotic resistance strainsfor 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,Lactobacillussp. intentionally added toour diet, concerns have been raised about the antibiotic resistance inthese benecial bacterial species. In addition, LAB can also acquireresistances from other bacteriain the environment. Once a LABbecomes

resistant, the determinant is amplied and may be transmitted toanother host and when the right circumstances are present; theseresistance 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 chainviaanimals to humans, has been thoroughly investigated. These aspectsconsider food chain as a fundamental reservoir for the transmission ofantibiotic resistance and as a source of contamination in a variety of

food products (Witte, 1997). Hence bacteria used as probiotics forhumans 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 inthe appearance of different resistance mechanismsviamutation whilein 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 andbacteria used as probiotics is essential. The knowledge of intrinsic,chromosomally coded resistance of LAB to common antibiotics isnecessary to recognize acquired resistance traits (Mathur & Singh,

2005). Distinguishing between intrinsic (non-speci

c, nontransferable)and acquiredresistance is also necessary, a process thatmay require thecomparison of antimicrobial resistance patterns in many LAB andBidobacteria 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 isnot pathogenic, and the possibility that genes encoding antibioticresistance 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 resistancedeterminants (Kastner et al., 2006).

7.1. Intrinsic resistance in LAB

LAB strains potentially serve as a source of antibiotic resistance. Thedetermination and comparison of antibiotic susceptibility patterns of arepresentative number of strains of each species is an essentialcriterion

for potentially starter or probiotic Lactobacilluscultures (Charteris &Kelly, 1993). Microbiological breakpoints have been dened byEuropean Commission (2008)for studying the MIC distribution in thebacterial 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 assessedfor 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 beperformed. However, a phenotypically resistant strain may be geno-

typically susceptible. In contrast, a susceptible phenotype may alsocarry silent genes, which are observed with genotyping. Antibioticsusceptibility of different bacterial species and analysis of MIC indened species/antibiotic combinations helps to differentiate these

two resistance mechanisms. Antibiotic susceptibility proles haverecently 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.Lactobacillussp. is reported to have a highnatural 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). Intrinsicresistance to vancomycin was conrmed forL. paracasei,L. salivarius,andL. plantarumand 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 ofantibiotics, especially vancomycin, is characteristic of lactobacilli(Bernardeau, Vernoux, Henri-Dubernet, & Guguen, 2008). The

Lactobacillusspecies have found susceptible to many cell wall synthesisinhibitors, like penicillin and ampicillin (Coppola et al., 2005; Danielsen& Wind, 2003). Most species proved susceptible to the antibioticstested, 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

representerythromycin, tetracycline, chloramphenicol and vancomycin



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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 majorityof 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 forLactococcussp.,Leuconostocsp.,and Lactobacillus sp. whereas Leuconostocsp., Pediococcus sp.and mostlactobacilli species were intrinsically resistant to vancomycin. Resistanceof many species of lactobacilli except Lb. delbrueckiisubsp. bulgaricus,

Lb. acidophilus, Lb. johnsonii, andLb. crispatus to glycopeptides is also

considered intrinsic (Charteris, Kelly, Morelli, & Collins, 1998; Mathur &Singh, 2005). In another study, the phenotypic and genotypic resistancein LAB from fermented foods have been identied and all the strainswere 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 withgastrointestinal infections. The strains ofBidobacteriumwere foundsusceptible to ampicillin, cefotaxime and erythromycin. Bidobacteriumstrains displayed intrinsic resistance with high MICs for streptomycinand gentamicin (Mtt, Alakomi, Vaari, Virkajrvi, & Saarela, 2006). Inanother study byD'Aimmo, Modesto, and Biavati (2007)found thatBidobacteria were resistant to aminoglycosides, cycloserine, nalidixic

acid and strongly resistant to kanamycin, polymyxin B and aztreonam.Bidobacteria are intrinsically resistant to mupirocin, an antibioticthat is being used in selective media for this genus. The high level ofmupirocin 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 concentrationsof aminoglycosides, most likely as a consequence of the lack ofcytochrome-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 susceptibilitywas observed towards carbenicillin, ciprooxacin, dicloxacillin andnoroxacin. 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 beused as probiotics. Regarding the prevalence of antimicrobial resistanceof enterococcal strains in different environments, the frequency ofvarious 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 toa broader range of different antibiotics using phenotypic susceptibilitytesting, 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. faeciumderived from probiotic product was susceptible to all the testedantibiotics 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 arefound 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. Aspecic 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 geneticinformation 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 LABis confounded by problems regarding the use of media and MICbreakpoints 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 betweenspecies of the same genera (Danielsen & Wind, 2003). An overviewof antibiotic resistance proles of different LAB in fermented andcommercial probiotic bacteria compiled in (Table 2).

7.2. Acquired resistance/mobile genetic elements in LAB

Emerging foodapplication of probiotics is fraught with the danger ofbeing one of the potential sources for the spread of antibiotic resistancegenes. Bacteria use a complex array of mechanisms to share and spreadresistance determinants. The key mechanisms of horizontal transfer in

bacteria are supposed to be conjugation and transductionviabacterio-phages (Kleinschmidt, Soeding, Teuber, & Neve, 1993). In conjugation,plasmids and tranposons play an important role in dissemination ofantimicrobial resistance (Grillot-Courvalin, Goussard, & Courvalin,

2002; Kleinschmidt et al., 1993). Despite the fact that reports on thepresence of antibiotic resistance genes associated with mobile geneticelements have been detected in lactobacilli. However, there are several

studies that have documented the presence and expression of antibioticresistance genes in food-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 beenfound in fermented drinks and yoghurts (Temmerman et al., 2003),cheese (Florez, Delgado, & Mayo, 2005andHerrero, Mayo, Gonzalez, &Suarez, 1996), and meat products (Gevers et al., 2000). The possibility

of exchange of resistance factors with other microorganisms, especiallythose belonging to the gut microbiota is limited compared to that ofother LAB such as enterococci (Mathur & Singh, 2005; Temmermanet al., 2003). The prevalence of acquired antibiotic resistance genes so

far been detected in Lactobacillusspecies are sensitive to antibioticswhich inhibit protein synthesis such as tetracycline, erythromycin,and chloramphenicol. In various probiotic, food and research strains

tet(M) anderm(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 resistancegenes, 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 genescoding 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) andtet(L)) (Lahtinen et al., 2009). Chloramphenicol resistancegenes (cat; chloramphenicol acetyl transferases) have been identiedin the Lb. acidophilus, Lb. delbrueckii subsp. bulgaricus (Hummel,Hertel, Holzapfel, & Franz, 2007), andLb. johnsonii(Mayrhofer et al.,

2010) as well as inLb. reuteri(Lin, Fung, Wu, & Chung, 1996) andLb.plantarum (Ahn, Collins-Thompson,Duncan, & Stiles, 1992). In addition,

erythromycin resistance genes, such as erm(A), erm(C), or erm(T)



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responsible for the macrolides, lincosamides, and streptogramins (MLS)resistance phenotype, while the erm(B) gene, most frequently foundamong Lactobacillus sp. (Mayrhofer et al., 2010 and van Hoek,Margolles, Damig, Korhonen, et al., 2008). Theerm(B) gene was detect-

ed from two strains of each of theLb. fermentumandLb. vaginalis, andone strain each ofLb. plantarum, Lb. salivarius, Lb. acidophilus, Lb .

animalis, andS. 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 genesaaa(60)-aph(200) (Ouoba et al., 2008) and the lincosamide resis-tance genelnu(A) was also found in Lactobacillusstrains (Cataloluk

& Gogebakan, 2004; Hummel et al., 2007; Kastner et al., 2006). Inthe PROSAFE (Product Safety Enforcement of Europe, is a non-prot professional organization for market surveillance authoritiesand ofcers to improve the safety of users of products and servicesin Europe), project, probiotic lactobacilli were reported to possess

erm(B) and/ortet(W),tet(M) or unidentied members of the tet(M)group (Klare et al., 2007). In probiotic commercial Lb. reuteri ATCC55730, tet(W) and the lincosamide resistance genelnu(A) were detect-ed (Kastner et al., 2006).Hummel et al. (2007), determined the antibi-

otic resistance of probioticLb. salivariusBFE 7441 having an erm(B)gene on chromosome. The probiotic strainLb. plantarumCCUG 43738,displayed phenotypic resistance to tetracycline and minocycline, wasfound to have atet(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 transposonTn5432was identied (van Hoek, Mayrhofer,Domig, & Aarts, 2008). Tetracycline resistance in Bidobacteriumsp. deserves special attention. The genes tet(W), tet(M), tet(O), tet(W/32/O),and tet(O/W) have been detected in severalBidobacteriumspecies, including B. longum (subsp. infantis and subsp. longum),

B. breve,B. animalissubsp.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). Thetet(W)gene has been identied at high frequency inB. longumstrains and inallB. animalissubsp.lactisstrains (Aires et al., 2007; Ammor, Flrez,

Alvarez-Martn, et al., 2008; Gueimonde et al., 2010) paradoxicallythis strain is extensively used in the functional food industry, especiallyin 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 resistancegenes 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 antibioticresistance genes have beenfound among LABorgan-isms and thereis no barrier between pathogenic (e.g., streptococci), po-

tentially pathogenic (e.g., enterococci), and commensal (e.g., lactobacilliand lactococci) bacterial species (Mathur & Singh, 2005). The presenceof transmissible antibiotic resistance markers in LAB and some antibiot-ic resistance determinants located on plasmids have been reported tooccur in Lactococcus lactis and Enterococcus species (Gevers et al.,

2003). It is important to authenticate that probiotic and nutritionalLAB strains consumed on a daily basis worldwide lack acquired antimi-crobial resistance properties and before considering them safe forhuman 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 viathe 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 (Vankerckhovenet al., 2008).

7.3. Plasmids encoding antibiotic resistance in LAB

It is a matter of concern that plasmid associated resistance possiblyspread to other, more harmful species and genera. Plasmids are com-mon in enterococci, lactococci, leuconostoc, pediococci, and present insome strains of lactobacilli and bidobacteria (Teuber, 1995). Natural

resistance transfer mechanisms identied in LAB are very similar andfocused on conjugative plasmids (e.g.pAM1, pAD1 or pIP501 type)and conjugative transposons (e.g. Tn 916type). Several mobilizableplasmids have been identied,e.g.the tetracycline resistance plasmidpMD5057 ofL. plantarum 5057 and resistance plasmid pLME300 of

L. fermentumROT1 (Danielsen & Wind, 2003; Gfeller, Roth, Meile, &Teuber, 2003). At least 25 species of lactobacilli contain native plasmids(Wang & Lee, 1997), and oftenappear to containmultiple (from 1 to 16)different plasmids in a single strain. R-plasmids encoding tetracycline,

erythromycin, chloramphenicol, or macrolide lincomycinstreptograminresistance has been reported in the Lb. reuteri(Lin et al., 1996; Tannocket 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 andfeces. The reported prevalence of antibiotic resistance genes such aserythromycin, vancomycin, tetracycline, chloramphenicol, and gentami-cin resistance genes, on transferable genetic elements in enterococci is

more extensive, both on plasmids (Murray, An, & Clewell, 1988) andtransposons (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 inE. faecalis(Tn916, Tn918, Tn920, Tn925, Tn2702), E. faecium(Tn5233)and Lc.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 transposonsvary in size between 16 and 70 kb and may be inserted into plasmidsor the chromosome in one or multiple copies (Mathur & Singh, 2005).

The role that conjugative transposons play in the spread of antibioticresistance is indicated by similar resistance genes present in diversebacterial species (Scott, 2002). Localization oftet(M) anderm(B) onconjugative 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). Severalmobile elements have been found in lactobacilli, including ISL2(Insertion Sequences) inLb. helveticus, ISL3 inLb. delbrueckii, IS1223 in

Lb. johnsonii, IS1163, IS1520 in Lb. sakei and ISLp11 in Lb. plantarum(Nicoloff & Bringel, 2003).

Many AR traits arequite stable in both the environment andthe host,even in the absence of antibiotic selective pressure (Johnsen et al., 2005;Srum et al., 2006), mostly due to the presence of various plasmid stabi-lization mechanisms.There arefew reports available in literaturelinking

conjugative transposons and antibiotic resistance in Lactobacillussp.(Ammor et al., 2007). The outcome of the EU-PROSAFE project was torecommend 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).



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8. Horizontal gene transfer of antibiotic resistance from probiotic

LAB to other species

The human gastrointestinal tract (GIT) harbors 10131014 bacterial

cells in adultsand this microbiota is oftenexposed to a varietyof antibi-otics, both directly and indirectly, due to their routine use in clinicalsettings (de Vries et al., 2011). Therefore, the human GIT microbiotamay serve as an important reservoir of antibiotic resistant strains thatcould act as opportunistic pathogens or as donors of resistance genes

to other bacteria (Salyers et al., 2004). Human intestinal bacteria notonly share resistance genes among themselves but can also acquire ordonate resistance genes to bacteria that are just passing through theintestine (Simonsen et al., 1998; Teuber et al., 1999; van den Braak,

van Belkum, van Keulen, Verbrugh, & Endtz, 1998). Bacteria thatnormally reside in the human colon can transfer resistance genesamong themselves when harmless commensal bacteria transform intopathogens (Davison, 1999; Finlay & Falkow, 1997; Kidwell & Lisch,

2000; Manges et al., 2001; Ochman, Lawrence, & Groisman, 2000) andonce acquired, resistance genes are not easily lost. Instead, they becomea 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 bacteriaused in foods or as food supplements. Consuming products containingAntibiotic Resistance Transferring (ART) starter cultures or probioticbacteria could be detrimental instead of benecial, to gut and public

health. It is important that the bacterial culture intended for use infermentation 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 viaHorizontal Gene Transfer mechanisms. However, benecial bacteria,including starter cultures and probiotic bacteria, are also susceptible toHGT mechanisms (Wang, Mc Entire, Zhang, Li, & Doyle, 2012). Whilethe horizontaltransmission of AR genes can occur not only in pathogens

and commensal bacteria, but also in benecial bacteria, including thoseused as starter cultures and probiotics. For example, a Bidobacteriumspp. strain once used to supplement in yogurt products carried a tetr-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. Besidesserving as an AR gene reservoir, commensal bacteria may also serve asan 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 bidobacteriais benecial forthe maintenance of a healthygut microbiota. This is supported by two main observations: (i) arelatively higher population of bidobacteria and lactobacilli is foundin 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 longtime, it wasassumed that probiotic bacteria do not transmit bad traitssuchas AR. However, results frommicrobial genome sequencing studiesexemplify 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 furtheridentied that Enterococcus spp., Lactococcus spp., Leuconostocspp.,

Streptococcus thermophilus, and Carnobacterium spp. are among the

dominant AR gene carriers found in various food products and arecapable of transmitting AR genes (Li & Wang, 2008; Wang et al.,2006). As mentioned above, high-frequency conjugation system ispresent 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 acidbacteria and perhaps other probiotic strains, a number of AR genes

have been identied in several potential probiotic strains, and some ofthem are transferable to other bacteria by HGT (Ouoba et al., 2008).However, since commensal bacteria have been shown to transferdeterminants of resistance to pathogens (Liu et al., 2009), studies on

development of resistance should also include lactobacilli. Regularconsumption of ART bacteria, coupled with occasional colonizationand HGT events, will probably inuence theAR of the human gut micro-biota. Interspecies gene transfer to E. faecalisof mobilizableermortetcontaining plasmids from food-related Lb. plantarumhas been demon-strated in vitro and in the intestinal tract of gnotobiotic rats (Feldet al., 2008; Jacobsen et al., 2007). The recent detailed studies on the

antibiotic resistance proles of lactic acid bacteria have demonstratedthe occasional presence of acquired resistance genes in Lactobacillusspecies (Morelli, 2008). The prevalence of acquired antibiotic resistancegenes includes the frequent occurrence oftet(M) anderm(B) genes(conveying resistance to tetracycline and erythromycin, respectively)

in lactobacilli isolated from different fermented foods (Comunianet al., 2010; Zonenschain, Rebecchi, & Morelli, 2009 ). Interspeciesconjugative transfer of tetracycline and erythromycin resistanceplasmids from LAB has been demonstrated previously in vitro (e.g.Feld et al., 2008; Gevers et al., 2003; Ouoba et al., 2008). While there isprevalence oftet(M) anderm(B) genes has been estimated to be higherthan 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. plantarumDG 507 E. faecalis In vitro Plasmid erm(B) Gevers et al. (2003)

E. faecalisLMG20790 E. faecalisJH2-2 In vitro Tn916Tn1545 erm(B) Huys, D'Haene, Collard,

and Swings (2004)

E. faecalisLMG20927 E. faecalisJH2-2 In vitro Plasmid/Tn916Tn1545 erm(B) Huys et al. (2004)

Lb. plantarumDG 522,

Lb. plantarumDG 507

E. faecalis In vivo Plasmid tet(M), erm(B) Jacobsen et al. (2007)

Lb. reuteriL4:12002 E. faecalisJH2-2 In vitro Plasmid erm(B) Ouoba et al. (2008)Lb. plantarumpLFE1 E. faecalis In vitro erm (B) Feld et al. (2008)

Lc. lactisSH4174 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. lactisBU-2-60 E. faecalisJH2-2 In vitro tet(M) Toomey et al. (2010)

Lb. fermentumNWL24 and

Lb. salivariusNWL33

E. faecalis181 In vitro erm(B),tet(M) Nawaz et al. (2011)

E. faecalisCM5 V, CM6 V E. faecalisOG1RF In vitro Plasmid erm(B) Vignaroli, Zandri, Aquilanti,

Pasquaroli, and Biavasco (2011)

E. duransPF1 V E. faecalis64/3 In vitro Plasmid erm(B) Vignaroli et al. (2011)

E. duransPF3 V E. faecalisOG1RF,E. faecalis64/3 In vitro Plasmid erm(B) Vignaroli et al. (2011)



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Transfer of antibiotic resistance from LAB strains has beendocumented in vitro, but very fewstudies have conrmed this antibioticresistance transferin vivoand it was only observed in the dissociatedanimal 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 hasbeen transferred from Lb. reuteri to Enterococcus faecalis (Morelli,Sarra, & Bottazzi, 1998) and amongLc. lactisstrains, the transfer could

not be observed in conventional rats (Schlundt et al., 1994). A trans-poson Tn916 includingte t(M) was transferred to E. faecalisstrain

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 includingLb. keriNWL78 isolated from a probiotic yogurt and erm(B) gene from

Lb. fermentumNWL24 andLb. salivariusNWL33 andtet(M) gene fromLb. plantarumNWL22 andLb. brevisNWL59 were successfully trans-

ferred to E. faecalis 181 in lter mating experiments (Nawaz et al.,2011). In terms of virulence,in vivostudies mostly reported transferof antibiotic resistance, including resistance to vancomycin, tetracyclineand erythromycin.Mater, Langella, Corthier, and Flores (2008) reported

the transfer ofvan(A) resistance fromE. faeciumto a commercially avail-able probiotic strain ofLb. acidophiluswas observedin vivo andin vitro inmice. Probiotic products and starter strains rarely had acquired antibiotic

resistance suchresultsdepict theattention for a strict monitoringandreg-ulation. Therefore, besides the presence of AR genes, the genetic charac-teristics of bacteria carrying the resistance genes are considered asimportant parameters for assessing the potential for HGT both in probiot-

ic and starter cultures (Li, Li, Alvarez, Harper, & Wang, 2011). HGT from

probioticLactobacillito other sp. mentioned in (Table 3).

8.1. Horizontal gene transfer from different sources via food products to thegastrointestinal tract

The HGT may widely occur due to antibiotic treatment and postpasteurization contamination in the process or lack of an adequatetreatment during manufacturing of the product. Transmission of anti-

biotic resistance results in the dissemination of antibiotic resistancegenes to our commensal ora and opportunistic pathogens. The lattermay further transfer resistance genes to the pathogenic bacteria in theGIT. Horizontal transmissions of AR genes between commensal and

pathogenic microorganisms in ecosystems are much more than directAR gene dissemination from one pathogen to another (Andremont,2003). For antibiotic resistance to happen, the bacterium has to remainin the food process, through, for example, fecal contamination in the

process orvia a recontamination by improper handling, or lack of anadequate temperature treatment in the process. Due to the selectivepressure of antibiotic treatment, antibiotic resistance genes may spreadto sewage/manure, and either directly or indirectly through food

products. Through food these genes may further transfer to the humanand 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 ahistory of direct exposure to antibiotics (Gueimonde, Salminen, &Isolauri, 2006; Ready, Bedi, Spratt, Mullany, & Wilson, 2003; Villedieuet al., 2004). A broad spectrum of commensal bacteria, including lactic

acid bacteria, has been identied as being carriers of AR genes and isable to transmit those genes to other bacteria leading to increasedresistance in the recipient organisms (Feld, Bielak, Hammer, & Wilcks,

2009; Wang et al., 2006). Even without direct exposure to antibiotics,

Fig. 2. Horizontalgene transfer fromdifferent sources and via foodproducts to the gastrointestinaltract adapted fromKhachatourians (1998); Anonym (2004); Claycamp and Hooberman

(2004);Wang et al. (2012);Verraes et al. (2013).

http://localhost/var/www/apps/conversion/tmp/scratch_2/image%20of%20Fig.%E0%B2%80


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animal and human wastes are one of the most signicant sources of ARTbacteriadirectly contributing to the amplication cycle of AR in the globalecosystem. Antibiotic exposure further selectively enriches ART bacteriain the host gut microbiota. Ultimately, a large number of ART bacteria in

sewage and manure directly contribute to the prevalence of AR in theenvironment (soil, water, etc.), raw food materials (meat, milk, plantmaterials), food processing environments and food handlers. The

isolation of AR genes in food-borne pathogens from retail products

exempli

ed the potential contribution of the food chain in transmittingART 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 includingerm(B),erm(C),tet(S/M)andtet(A), encoding ribosomal modication andtetefux mechanisms,in selected food isolates were identied. With the signicant exception

of the enterococci,antibioticresistance plasmidsand transposable geneticelements are relatively rare among LAB (Florezet al., 2003) and apparent-ly no antibiotic transferable resistance determinants have been detectedamong theBidobacteriumspp. (Devirgiliis, Caravelli, Coppola, Barile, &

Perozzi, 2008). Among the lactobacilli, certain strains ofLb. fermentum,

Lb. acidophilus,Lb. reuteriandLb. plantarumhas been shown to harborresistance plasmids against erythromycin (and related macrolides),tetracycline, and chloramphenicol. Many strains of these species areused in the food industry either as starter cultures or as probiotics. In

one probiotic,Lactobacillusstrain GG, the vancomycin resistance genes(van) of enterococci were studied and veried that thepresence of vanco-mycin resistance determinant in the particular strain was not closelyrelated to enterococcalvangenes (Comunian et al., 2010). In conclusion,

transfer of antibiotic resistance does not appear likely in the case ofdairy or probiotic lactic acid bacteria, with the exception of enterococci.

Genetic material, especially that represented by plasmids, can betransferred between food microbes and the human intestinal microbio-

ta or pathogens. The mechanismsof HGTbetweenbacteria mayoccur inthe soil, in water, or in the digestive system of humans and animals, aswell as in food. (Fig. 2) depicts the HGT in food products from differentsources 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 theenvironment. In addition to conjugation, transduction and transforma-

tion, and other less well recognized mechanisms of DNA transfer mayoccur in nature (Keese, 2008).

Hence, the impact of AR dissemination from ART pathogensin foodsto 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 108 copiesof 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 bornebacteria were transferred to human inhabitant and pathogenic bacteriaby natural HGT mechanisms, leading to acquired resistance in the recip-ient strains (Feld et al., 2009; Li, Sun, Zhang, Li, & Wang, 2010; Toomeyet al., 2010; Wang et al., 2006). A constant supply of thesefoods 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 antibioticexposure, the local microbial population, and other host and environ-

mental factors. Since commensal bacteria dominate in both numbersand genetic diversity in natural andhost environments.They can poten-tially be good indicators of theAR and could providean early warning oftheemergence of ARin pathogens (Wang et al., 2009). It isnow believed

that the foodchain may wellplay a very important role in disseminating

AR genes and ART bacteria to the humandigestive microbiota (Duran&

Marshall, 2005; Wang et al., 2006). The colonization by ART bacteria andassociated HGT events certainly contribute to the increasing resistanceto antibiotics in humans (Wang et al., 2006). These studies further iden-tied that various commensal bacteria, including some benecialbacte-

ria are involved in the HGT events, as either the AR gene recipients orpotential donors. A safety goal for probiotics should not increase thealready existing risks of antibiotic transfer associated with the normalgut or food microbiota yet the use of probiotic strains in various food

products is to provide various health bene

ts to the consumers.

9. Conclusions

The extraordinary capacity of a number of bacterial organisms todevelop resistance to antibiotic compounds continues to remain amajor concern for researchers as well as health and industrialmanagers

for several years. Apart from their various health benets probioticshave also been used to treat and/or prevent antibiotic resistance in thehost. However, recent studies have reportedthe occurrence of antibioticresistance 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 antibioticresistance to other organisms is currently one of the most important

safety issues regarding the use of bacteria in a variety of food productsas food chain is considered as one of the main routes for the transmis-sion of antibiotic resistant bacteria. Bacteria naturally present in foodsor food supplements, or deliberately added to them, including probioticbacteria, constitute a potential source of antibiotic resistance determi-

nants. Since there has been a signicant rise in the consumption ofprobiotic products, it is imperative that probiotics are well researchedand documented for their antibiotic resistance prole. The ability totransfer antibiotic resistant determinants must be considered as an

important parameter for the selection of probiotic strains. Multipledrug resistance has been found in different species of probiotic strains,which is detrimentalto food safety. Evaluation of the safety of probioticsfor human consumption must be guided by established criteria,

guidelines and regulations, and standardized methodology for premarketbiosafety testing and post market surveillance should be in place.

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