International Food Research Journal 18(3): 837-853 (2011) Antibiotic resistance of probiotic organisms and safety
of probiotic dairy products
Ashraf, R. and *Shah, N. P.
School of Biomedical and Health Sciences, Faculty of Health, Engineering and Science, Victoria University, Werribee Campus, P.O. Box 14428, Melbourne, Victoria 8001, Australia Abstract: Intrinsic resistances to tetracycline, vancomycin and erythromycin are common in Lactobacillus
species; however, resistance to streptomycin, clindamycin, gentamicin, oxacillin and lincosamide is also reported in these species. Resistant markers tet(W), tet(M) and erm(B) have been frequently detected in the resistant strains while van(A), lnu(A) and tet(L) have also been found in some strains of Lactobacillus. Bifidobacteria are commonly resistant to tetracycline, streptomycin, erythromycin, gentamicin and clindamycin. Resistance genes van(A), tet(L) and tet(M) are often detected in Enterococcus. Reports suggest enterococci to transfer tet(M) to E. faecalis or Listeria strains and van(A) to commercial strain of Lactobacillus acidophilus. Streptococcus species are highly resistant to tetracycline, ciprofloxacin and aztreonam and tet(M) was detected in strains of dairy origin. Clinical cases of endocarditis, septicemia, bacteremia and septic arthritis due to the species of Lactobacillus, Saccharomyces, Leuconostoc, Pediococcus and Bifidobacterium have been reported in patients with some underlying medical conditions.
Keywords: Antibiotic resistance, probiotics, minimum inhibitory concentration
According to the scheme, the members of the Lactococcus and Lactobacillus are most commonly The overwhelming use of antibiotics has played given "generally regarded as safe" (GRAS) status, a significant role in the outspread/emergence of whilst members of the genera Streptococcus and antibiotic resistance bacteria. Antibiotics added Enterococcus and some other genera of LAB contain to animal-feed and given to livestock that are used some opportunistic pathogens. Microorganisms as human food contribute to additional resistance. used in animal feed in the European Union (EU) are Reports suggest that commensal bacteria may act mainly strains of Bacillus (B. cereus var. toyoi, B. as potential reservoirs for antimicrobial resistance licheniformis, B. subtilis), Enterococcus (E. faecium), genes, hence bacteria used as probiotics for humans Lactobacillus (L. acidophilus, L. casei, L. farciminis, or animals should not carry any transferable L. plantarum, L. rhamnosus), Pediococcus (P. antimicrobial resistance genes (von Wright, 2005; acidilactici), Streptococcus (S. infantarius), and yeast European Food Safety Authority-EFSA, 2008; The of Saccharomyces cerevisiae and Kluyveromyces panel on additives and products or substances used species (Anadón et al., 2006).
in animal feed-FEEDAP, 2008). According to World Health Organization (WHO) global strategy for the Table 1. Human activities that exacerbate resistance
containment of antimicrobial resistance (World Health (adapted from Okeke et al. (2005)) Organization-WHO, 2001), the rate of emergence of antimicrobial resistance is expected to be increased Appropriate antimicrobial use in chemotherapy by misuse of antibacterial substances. The resistant Use of a narrow repertoire of antimicrobials on most micro-organisms present in food products originating Antimicrobial misuse and abuse in human beings from animal source may cause infections in humans Agricultural antimicrobial use and misuse Use of poor quality antimicrobials that are difficult to treat. A summary of risk factors Dissemination of resistant organisms
for antibiotic resistance particularly relevant to, but Inadequate infection control in health-care institutions not limited to, developing countries is outlined in Shortfalls in hygiene, sanitation, and public health Lack of surveillance and consequent late detection The European Food Safety Authority (2005) has outlined a scheme based on the qualified presumption The guidelines updated by the FEEDAP Panel of safety (QPS) that involves the individual in 2008 are expected to eliminate the possibility of assessment and evaluation of acquired antibiotic microorganisms from food chain to carry transmissible resistance determinants in lactic acid bacteria (LAB). resistances genes. However, no such guidelines exist *Corresponding author. All Rights Reserved Tel: (+61-3)-9919-8289; Fax: (+61-3)-99198284 838 Rabia, A. and Shah, N. P. concerning yeast resistance to antimycotics. As a Methods for determining antibiotic resistance
result, the use of antimicrobial growth promoters such as avoparcin, carbadox and alaquindox has Methods that are routinely used for testing been banned in the EU since 2006. The emergence antibiotic susceptibility of bacteria include Kirby- of vancomycin-resistant enterococci in food-animals Bauer (disc diffusion) method, Stokes method, E-test is correlated with the use of avoparcin. Avoparcin (based on antibiotic diffusion), agar and broth dilution is a glycopeptide that is used as a feed additive for or agar dilution methods for the determination of adding the growth of animals that can cause spread minimum inhibitory concentration (MIC). The E- of vancomycin-resistance from animals to humans test (Epsilometer Testprinzip, Ellipse gradient test- (Wegener, 2003). Since the resistance in many cases AB Biodisk) is a popular quantitative technique for is transmissible, non-pathogenic bacteria added into determining antimicrobial susceptibility. It is based the food chain could act as a reservoir of resistance on the combined concepts of in vitro dilution and and transfer this trait to pathogens.
diffusion tests. In the assay, ‘there is an immediate and effective release of the antimicrobials in a continuous Types of antibiotic resistance
exponential gradient when they are applied to an agar surface' (Ribeiro et al., 2005). The technique is There are three types of resistance: natural accurate and reproducible because of the stability of (intrinsic or innate), acquired and mutational. the antibiotics (Sader et al., 1994). According to FEEDAP (2008), strains carrying the These methods have been tested and compared acquired resistance due to acquisition of exogenous for different LAB and bifidobacteria. MICs can be resistance genes are unacceptable for use as animal determined by agar or broth dilution techniques by feed additives.
following the reference standards established by various authorities such as the Clinical and Laboratory Resistance gene reservoir hypothesis
Standards Institute (CLSI, USA), British Society for Antimicrobial Chemotherapy (BSAC, UK), Agence Colonic bacteria normally residing in colon act Francaise de Securite Sanitaire des Produits de Sante as reservoirs for resistance genes that can be acquired (AFFSAPS, France), Deutsches Institut für Normung from ingested bacteria (Figure 1). According to e.V. (DIN, Germany) & ISC/WHO. FEEDAP has reservoir hypothesis "commensal bacteria in the published guidelines regarding the testing procedures colon including those that could act as opportunistic since 2001. FEEDAP requires the determination of pathogens and those that are truly non-pathogenic, the MICs of the most relevant antimicrobials for exchange DNA with one another" (Salyers et each bacterial strain that is used as a feed additive al., 2008).The reservoir hypothesis suggests that in order to eliminate the possibility of transmissible antibiotic-resistant bacteria came into existence resistances. because of the selective pressures applied by antibiotic Mayrhofer et al. (2008) tested 104 strains of L. drugs (Table 1). ‘After antibiotic treatment, there is acidophilus using broth microdilution, disk diffusion, a decline in the populations of susceptible bacteria, and E-test. A good agreement was found between naturally resistant bacteria begin to thrive, creating a MICs from the broth microdilution method and the reservoir of antibiotic-resistant bacteria' (Salyers et E- test method. Agar based methods such as E- test al., 2004).
and agar disk diffusion were suggested as valid methods compared to the broth microdilution method. Blandino et al. (2008) found MICs as identical to those obtained with the E-test. Danielsen and Wind (2003) suggested that MICs can be used as a microbiological breakpoint when screening Lactobacillus strains for transferable resistance genes. For antimicrobial susceptibility testing of bifidobacteria, Mättö et al. (2007) suggested that the E-test on LAB susceptibility test medium supplemented with cysteine was useful. The swab and agar overlay gradient diffusion method was found to be reliable by Charteris et al. (2001) for antibiotic susceptibility testing of rapidly growing, Figure1. The reservoir gene hypothesis. Bacteria residing in human
facultative anaerobic lactobacilli, using MRS agar as colon can act as reservoir of resistant genes that can be acquired from ingested bacteria (adapted from Salyers et al. (2008) International Food Research Journal 18(3): 837-853 Antibiotic resistance of probiotic organisms and safety of probiotic dairy products Egervärn et al. (2007) found that results obtained and products or substances used in animal feed- with the E-test or the broth microdilution method for FEEDAP, 2008), the MICs for relevant antimicrobials the assessment of antibiotic susceptibility of L. reuteri have been set for the following genera (and in some and L. fermentum strains (56 each) corresponded cases individual species): Lactobacillus, Lactococcus, well with each other. This is supported by the study Streptococcus thermophilus, Pediococcus, of Brown and Brown (1991) that showed a good Leuconostoc, Enterococcus, Propionibacterium, correlation between MICs by the agar dilution and Bifidobacterium and Bacillus. These genera also cover E-test methods. Turnidge and Paterson (2007) found the recent QPS lists for bacteria, and consequently that the distribution of MICs for wildtype strains of a the FEEDAP approach can be directly applied. single species was log-normal.
LAB are intrinsically resistant to many antibiotics. In many cases, resistance is not always transmissible, Acquisition and spread of resistances
and the species are also sensitive to many clinically used antibiotics in the case of a LAB-associated The antibiotic resistance gene can be transferred opportunistic infection. Therefore no particular by conjugation, transduction or transformation safety concern is associated with intrinsic type of (Figure 1). At present, reports regarding the spread of resistance. Plasmid-associated antibiotic resistance, antibiotic resistance among LAB and bifidobacteria which occasionally occurs, may spread resistance to suggest that resistant strains from human and animal other more harmful species and genera. colons are rather common, that confirms the transfer Using the disc diffusion method, antibiotic of resistances between commensal organisms in the resistance among 187 isolates from 55 European complex ecosystem of gastro-intestinal tract (GIT) probiotic products showed that 79% of the isolates (Ammor et al., 2007). There is a general concern that were resistant against kanamycin and 65% of the such microbes may harbor genes that may contribute isolates were vancomycin resistant. Remaining to opportunistic infections (Tompkins et al., 2008). resistances were in the order of tetracycline (26%), Theoretical risks that have been raised with respect penicillin G (23%), erythromycin (16%) and to the use of probiotics in humans include the chloramphenicol (11%). Overall, 68.4% of the potential for transmigration and colonization and isolates showed resistance against multiple antibiotics an adverse immunological effect. There is also a including intrinsic resistance (Temmerman et al., potential for antibiotic resistance transfer within the 2003). In a study by Toomey et al. (2010), intrinsic gastrointestinal tract from commensal or probiotic streptomycin resistance was observed in lactobacilli, bacteria to other bacteria or potential pathogen streptococci, lactococci and Leuconostoc spp. (Snydman, 2008).
Several studies have been carried out to test the Starter cultures used in food products could antimicrobial susceptibility of different probiotic also be a source of spread of antibiotic resistance. and LAB in different food products but only some Hence, strains intended for use in feed and food of these have demonstrated the genetic basis of these systems should be systematically monitored for resistances. Also, the data is available regarding resistance in order to avoid their inclusion in starters antimicrobial resistance pattern in food-associated and probiotic preparations (Ammor et al., 2007). LAB such as lactobacilli but it is mostly based on Two genes namely, transposon-associated tet(M) non-standardized methodologies and/or has been gene and plasmid-carried tet(L) gene that mediate obtained for only a limited number of strains (Huys 2 different tetracycline resistance mechanisms have et al., 2008). Studies regarding antimicrobial testing been described in L. sakei Rits 9 strain isolated from of different LAB, bifidobacteria and Bacillus strains Italian Sola cheese made from raw milk (Ammor et have been summarized in Table 2 and discussed al., 2008). Tetracycline resistance gene tet(K) in 5 below.
Staphylococcus isolates used as meat starter cultures were detected by Kastner et al. (2006). In a recent Lactobacillus
report where the gene tet(M) of L. plantarum isolated from pork abattoir was transferred to Lc. lactis BU-2- Lactobacilli display a wide range of antibiotic 60 and to E. faecalis JH2-2 (Toomey et al., 2010). resistance naturally, but in most cases antibiotic resistance is not of the transmissible type. Antibiotic resistance in LAB, Bifidobacterium and Lactobacillus strains with non-transmissible
antibiotic resistance do not form a safety concern. In a study by Danielsen and Wind (2003), out of 62 In the EFSA guidelines (The panel on additives strains tested for antibiotic susceptibility, 6 strains International Food Research Journal 18(3): 837-853 840 Rabia, A. and Shah, N. P. B. lactis While some of the strains HN001 and HN067, HN017 and fective antibiotics. None of Implication (Safety) Incidences of erythromycin, chloramphenicol, tetracycline, or β-lactam resistance in this study were low (<7%). In contrast, aminoglycoside (gentamicin and streptomycin) and ciprofloxacin resistances were higher than 70%, indicating that these may constitute intrinsic resistance, which could not be transferred in filter experiments.
The observed resistance in tested strains seemed to be intrinsic, except for salivarius erythromycin resitance was present. Antibiotic resistance determinants are likely to be present or can be transferred. None of the 4 new probiotic strains (L. r acidophilus tested in this study contained clinically significant transmissible antibiotic resistance genes. were intrinsically vancomycin resistant, all strains were sensitive to a number of clinically ef these new probiotic strains were any more resistant than the present-day commercial probiotic strains GG and LA-1.
Chromosomal mutation of the for ribosomal protein S12 cause resistance so it is unlikely to be transferred to other microor Kiwaki and Sato (2009) Method used for antibiotic resistance analysis E-test, PCR, Southern hybridization, filter mating experiment E-test, broth microdilution test Broth microdilution method, PCR and sequencing experiment ferent LAB, bifidobacteria and Origin/ source of probiotic (Country of study conducted) 3 strains were newly developed and 2 from commercial probiotic product. (Germany) Probiotic product -dried food supplements and fermented functional foods (Italy) Fonterra Research Centre Culture Collection (New Zealand, USA) Commercial strain, Fonterra Research Centre Culture Collection (New Zealand, USA) Culture Collection Research Laboratory of Institute for Microbiological Research (T Acquisition or spread of resistance/ Intrinsic, inactive cat Intrinsic (V Intrinsic (V Intrinsic (V Atypical (Ery) Intrinsic Intrinsic (contain plasmids but antibiotic resistance is not linked) Antibiotic resistance and safety implication of dif Antibiotics found to be resistant* Fus, Gen, Kan, Nal, Neo, Pol, Str Fus, Kan, Nal, Neo, Pol, Clo, Gen, Kan, Nal, Neo, Pol, Str Fus, Kan, Nal, Pol, Str Clo, Fus, Gen, Kan, Nal, Neo, Pol, Str Clo, Fus, Gen, Kan, Nal, Neo, Pol, Str Fus, Gen, Kan, Nal, Neo, Pol, Str Clo, Fus, Gen, Kan, Nal, Neo, Pol, Str Clo, Gen, Kan, Nal, Neo, Pol, Str Clo, Fus, Gen, Kan, Nal, Neo, Pol, Str rhamnosus Probiotic studied L. far BFE 7438 L. salivarius BFE 7441 L. r BFE 7442 L. acidophilus BFE 7444 L. casei 7445 L. paracasei L. plantarum L. salivarius L. acidophilus S. thermophilus L. r HN001 (DR20 L. r HN067 B. lactis HN019 (DR10 L. acidophilus HN017 L. plantarum HN045 L. GG L. acidophilus LA-1 International Food Research Journal 18(3): 837-853 Antibiotic resistance of probiotic organisms and safety of probiotic dairy products strain considering antibiotic B. subtilis The reduce susceptibility to the ferent species since none of these Antibiotic resistance determinants are likely to be transferred or spread, so these strains should be tested for the presence of transferable resistance gene before being used as probiotics.
Both strains did not harbour plasmid. Certain risks may exist for the licheniformis resistance, complementary studies are necessary in order to exclude that chloramphenicol and clindamycin resistances are harboured in a transposable element. considered as non-pathogenic and safe for human consumption Positive amplicons were obtained for resistance genes encoding aminoglycoside (aph(3′)-III, aadA, aadE) and tetracycline (tet(S). Higher prevalence of phenotypic resistance for aminoglycoside was found. cephalosporin and cefpodoxime can be attributed to intrinsic resistance of the dif bacteria were resistant to penicillin (first generation β- lactam). could act as reservoir of antimicrobial resistant genes and can transfer the resistances to other bacteria.
Sorokulova al. Disk dif E-test, microarray and membrane hybridization techniques, PCR, partial sequencing methods and filter mating experiments Disk dif method, serial antibiotic dilution procedure Micro-broth and agar dilution, PCR and conjugation experiments ganisms (France, French yogurt (Switzerland) Human origin. One strain obtained from other from commercial tablet (Switzerland) Human (Switzerland) Ukrainian Collection of Microor Russia, UK) Human, adult biopsy (Denmark) Human, child feces, Europe (Denmark) Human, adult feces, Europe (Denmark) Semi-hard cheese, Europe (Denmark) Human, child biopsy (Denmark) (Cip), (Str) microchip vanE mef by hybridization) gyrA tet aadE aph(3′)-III (Kanamycin, Neo) aph(3′)-III (Kan, Neo) aadA, aadE (Str), (Spe) , Gen, Kan, Sul, , Cep, Cip, Col, Gen, , Cip, Nan, Neo, Spe, Tri, , Cep, Cip, Col, Gen, , Cep, Cip, Col, Gen, , Cip, Col, Gen, Kan, Nan, Neo , Cip, Col, Gen, Kan, Nan, , Cip, Col, Gen, Kan, Nan, Gen, Kan, Nal, Ofl, Str (confirmed by E-test) Cli, Ctx, Fus, Kan, Lin, Met, Nal, Nit, Ofl, Oxa, Pen, Str Van Fus, Kan, Nal, Nit, Va Amp, Cet, Cex, Chl, Cli, Ctx, Met, Oxa, Apr Neo, Spe, Sul, Amp, Cip, Col, Ery Nan, Neo, Spe, Str Va Amp, Kan, Nan, Neo, Sul, Amp, Sul, Apr Apr Neo, Apr Neo, Spe, Sul, Amp, Kan, Nan, Neo, Str Amp, Kan, Nan, Neo, Spe, Str Va B. lactis 10140 L. r 21 (A L. r strain GG (A Bacillus subtilis VKPM B2335 Bacillus licheniformis VKPM B2336 L. acidophilus L. plantarum L. paraplantarum B. longum B. bifidum L. paracasei International Food Research Journal 18(3): 837-853 842 Rabia, A. and Shah, N. P. vancomycin not gradient specific probes. (A) resistance genes (B) gene that was not , Erythromycin; Fus, Fusidic genes nor they hybridized The β-lactam resistance is probably , vanH or vanA 12 and 5 bands in strains nature swab proposed Resistant to fusidic acid was demonstrated in a high percentage (92%) of isolates. Two probiotic isolates were phenotypically resistant to erythromycin, one of which contained an transferable to enterococcal recipients. Neither none of the strains possessed vanA vanZ with Plasmids could be found in only two of the six strains. Six plasmid bands in SD21 were identified. Safety of these strains as probiotic with regard to vancomycin resistance has been reassured.
No known β-lactam resistance genes were found. caused by a number of alterations in the corresponding genes and can be regarded as not transferable. It harbor two plasmids carrying which were cured and daughter strains retained probiotic properties, confirmed after series of in vitro properties and human clinical trials.
All the The was antibiotic susceptibility testing. Vankerckhoven et al. Broth microdilution, filter mating experiments, PCR- based detection of resistant genes, PFGE and (GTG) PCR, multiplex PCR Microdilution, PCR for genes, Southern hybridization, probe and DNA/DNA hybridization for the detection of the van analysis Broth microdilution, E-test, draft genome sequence analysis, BLAST methods Agar overlay disc dif gradient dif test (E-test), Arla group, Stockholm Aberdeen, Scotland). 6 strains from Probiotic products, 2 strains as probiotics for human and animal consumption comes from fecal flora (Sweden; 1968) and dairy product, cheese (Italy), 2 strains used commercially as a probiotic for human consumption. (Belgium, Germany) Strain collection of BioGaia Biologics, Inc., Raleigh, NC, USA Biogaia (Sweden) Adult human feces. Prof. Range Fondén (Panove Partner AB, Sweden). (Ireland) Dairy product, NCFB(NCIMB Ltd., (Ireland) Trimethoprim; Tri, Phenotypic and genetic resistances Plasmid borne and intrinsic resistances (B), (pC194), (piP501), (E), (A) erm cat cat aad aph Tetracycline; Tet, , Apramycin; Ctx, Cefotaxime; Cex, Cefoxitin; Cep, Cefpodoxime; Cet, Ceftriaxon; Cep, Cephalthoin; Chl, Chloramphenicol; Cip, Ciprofloxacin; Cli, Clindamycin; Clo, Cloxacillin; Col, Colistin; Ery Cip, Gen, Oxa, Sul/T Cip, Gen, Oxa, Sul/ Cip, Gen, Oxa, Sul/T Amp, Cep, Cip, Gen, Oxa, Sul/ Tri, Cip, Gen, Oxa, Sul/T Cip, Gen, Oxa, Sul/T , Streptomycin; Sul, Sulphamethoxazole; euteri TCC 55149) euteri TCC 53608) euteri TCC55730) euteri TCC 55148) hamnosus E. faecium L. r 20016 L. r (A L. r (A L. r SD21 (A L. r (A L. r GG (A 53103) L. r ATCC 55730 (SD21 Lactobacillus strain * acid; Gen, Gentamicin; Kan, Kanamycin; Lin, Lincomycin; Met, Methicillin; Mez, Metronidazole; Nan, Nalidixan; Nal, Nalidixic acid; Neo, Neomycin; Nit, Nitrofurantoin; Ofl, Ofloxacin; Oxa, Oxacillin; Pen, Penicillin; Pol, Polymyxin B; Spe, Spectinomycin; Str International Food Research Journal 18(3): 837-853 Antibiotic resistance of probiotic organisms and safety of probiotic dairy products of lactobacilli showed transferable resistance genes lnu(A). Two plasmids carrying tet(W) tetracycline, on the basis of their resistance to chloramphenicol, and lnu(A) lincosamide resistance genes were also erythromycin/clindamycin, and tetracycline. One identified by Rosander et al. (2008) in a commercial strain of L. rhamnosus exhibited an elevated MIC for strain of L. reuteri ATCC55730. oxacillin. The genetic basis of this kind of resistance Both a transposon-associated tet(M) gene, and was proposed to be either due to mutations in the plasmid-carried tet(L) gene presenting 2 different penicillin-binding proteins or due to the presence of tetracycline resistance mechanisms have been a β-lactamase.
characterized in L. sakei Rits 9 strain isolated from In the study of D'Aimmo et al. (2007), lactobacilli Italian Sola cheese made from raw milk (Ammor et were found resistant to nalidixic acid, aztreonam, al., 2008). The 2 resistance determinants conferred cycloserin, kanamycin, metronidazole, polymyxin different levels of resistance and their expression is B, spectinomycin and susceptible to rifampicin, induced by different tetracycline concentrations.
bacitracin, clindamycin, erythromycin, novobiocin In a recent double blind clinical study by and penicillin. High resistance to nalidixic acid Egervärn et al. (2010), the transferability of was found among all strains of L. acidophilus and tetracycline resistance gene tet(W) from L. reuteri L. casei whereas L. casei also demonstrated high to human gut flora was investigated particularly to resistance to aztreonam, cycloserine, polymyxin B fecal enterococci, bifidobacteria and lactobacilli. L. reuteri ATCC 55730 harboring a plasmid-encoded MICs of 16 antimicrobials for 473 isolates of LAB tet(W) gene was consumed by 7 subjects and an comprising of the genera Lactobacillus, Pediococcus equal number of subjects consumed L. reuteri DSM and Lactococcus were determined by Klare et al. 17938. No tet(W)-reuteri signal was produced from (2007). The results suggested that majority of LAB any of the DNA samples and thus evidence of gene were susceptible to penicillin, ampicillin, ampicillin/ transfer to entrococci, bifidobacteria and lactobacilli sulbactam, quinupristin/dalfopristin, chloramphenicol during intestinal passage of the probiotic strain was and linezolid. LAB exhibited a broad or partly species- not found under the conditions tested. dependent MIC profile of trimethoprim, trimethoprim/ In the study of Gfeller et al. (2003), L. fermentum sulfamthoxazole, vancomycin, teicoplanin and fusidic ROT1 isolated from a raw milk dairy product was found acid. Noticeably, 3 probiotic Lactobacillus strains resistant to novobiocin, tetracycline, erythromycin and were highly resistant to streptomycin. Although dalfopristin. A chromosomal tetracycline-resistance erythromycin, clindamycin, and oxytetracycline determinant tet(M) was identified in the strain and possessed high antimicrobial activities, 17 a 19,398-bp plasmid (pLME300), present in several Lactobacillus isolates were resistant to one or more of erythromycin-resistant strains of L. fermentum, was these antibiotics. Eight of them, including 6 probiotic isolated and completely sequenced. and nutritional cultures possessed erm(B) and/or Several species of Lactobacillus including L. tet(W), tet(M) or unidentified members of the tet(M) rhamnosus and L. casei are intrinsically resistant to group. High resistance against streptomycin has also vancomycin. There is an underlying possibility that been reported in 1 strain of Lactobacillus isolated vancomycin resistance could be transferred to other from Norwegian dairy product (Katla et al., 2001).
bacteria but there are no such reports to date. However, In the study of Huys et al. (2008), genotypically the transfer of vancomycin resistance (vanA) from unique 65 strains of L. paracasei and L. casei enterococci to a commercial L. acidophilus strain were assayed for antibiotic resistance with broth was observed in vitro and in vivo in mice (Mater microdilution and E-test assays using the LAB et al., 2008). In a study by Klein et al. (2000), all susceptibility test medium. In both methodologies, Lactobacillus strains namely 6 L. reuteri strains strains appeared uniformly susceptible to ampicillin (ATCC 55730, ATCC 55149, ATCC 55148, ATCC and clindamycin but exhibited natural resistance to 53608 and DSM 20016T) and 1 L. rhamnosus strain GG streptomycin and gentamicin. Three L. paracasei (ATCC 53103) were found resistant to vancomycin strains from cheese displayed acquired resistance but susceptible to a broad range of antibiotics. Four to tetracycline (MIC ≥ 32 μg per mL) and/or of the Lactobacillus strains (including L. rhamnosus erythromycin (MIC >16 μg per mL), which were strains) did not harbor any plasmid but 2 of them linked to the presence of a tet(M) or tet(W) gene and/or showed 5 and 6 plasmid bands, respectively. None an erm(B) gene, respectively. In the study of Kastner of the strains possessed the vanA, vanB or vanC et al. (2006), L. reuteri SD 2112 has been shown to gene. The findings established the safety of the harbor tetracycline resistance gene tet(W) (residing Lactobacillus strains for use as probiotics concerning on a plasmid) and the lincosamide resistance gene their vancomycin resistance (Klein et al., 2000). Zhou International Food Research Journal 18(3): 837-853 844 Rabia, A. and Shah, N. P. et al. (2005) found 3 L. rhamnosus strains (HN001, and trimethoprim. L. reuteri strains with high MICs HN067 and GG) resistant to vancomycin and of the 4 for both ampicillin, and tetracycline exhibited genetic new probiotic strains namely, L. rhamnosus HN001, relatedness and 6 strains with high MICs for both HN067, L. acidophilus HN017 and B. lactis HN019, erythromycin and clindamycin were also closely only L. rhamnosus HN001 contained plasmids. A related.
plasmid-free derivative of the strain had the same antibiotic susceptibility profile as the parent strain.
Charteris et al. (2001) found vancomycin resistance in all tested strains of Lactobacillus In the study of Mättö et al. (2007), human or strain GG and 11 closely related, rapidly growing, probiotic associated Bifidobacterium species (203 facultatively anaerobic, potentially probiotic L. strains) showed high MIC for tetracycline i.e. ≥16 rhamnosus strains. Moreover, these strains were mg per mL (prevalence of 4-18%) that was attributed also resistant to co-trimoxazole, metronidazole, to the presence of tet gene, where tet(W), and tet(O) gentamicin, and streptomycin but sensitive to were detected. Occasional erythromycin (2%) and/or pencillin G, ampicillin, rifampicin, tetracycline, clindamycin (5%) resistant strains were found, while chloramphenicol, and erythromycin. Antibiotic the strains were uniformly susceptible to ampicillin susceptibility pattern of the strains derived from 10 and vancomycin. MICs of tetracyclines were Italian probiotic products was determined by Blandino determined for 86 human Bifidobacterium isolates and et al. (2008). Intrinsic resistance to vancomycin was 3 environmental strains. The tet(O) gene was absent confirmed for L. paracasei, L. salivarius and L. in these isolates. tet(W), and tet(M) were found in 26, plantarum, and atypical resistance to erythromycin and 7%, respectively, of the Bifidobacterium isolates, was detected in 1 strain of L. salivarius according to and one isolate contained both genes. Chromosomal FEEDAP and CLSI breakpoints (MIC ≥8 mg per L) DNA hybridization showed that there was one (Blandino et al., 2008). chromosomal copy of tet(W), and/or tet(M) (Aires In the study of Toomey et al. (2010), all strains of et al., 2007). The tetracycline resistance gene tet(W) Lactobacillus spp. including L. paracasei, L. reuteri in the probiotic culture of B. lactis DSM 10140 was and L. curvatus, except L. plantarum were resistant detected by Kastner et al. (2006).
to erythromycin containing erm(B) and msrA/B Kiwaki and Sato (2009) determined the MICs genes. Tetracycline resistance was demonstrated by of 17 antimicrobials for 26 Bifidobacterium breve only L. plantarum determined by tet(M) gene and strains of various origins by broth microdilution. MIC Leuconostoc mesenteroides spp. containing tet(S) distributions for 17 antimicrobials were unimodal gene, respectively. L. plantarum was also intrinsically except streptomycin and tetracycline, in which it resistant to vancomycin, however no vancomycin was bimodal. The probiotic B. breve strain Yakult gene markers were found in Lactobacillus species. showed intrinsic susceptibility to all antimicrobials Intrinsic streptomycin resistance was observed in except streptomycin to which the strain showed lactobacilli besides streptococci, lactococci and an atypically higher MIC of >256 μg per mL. The Leuconostoc species. In another report, L. reuteri resistance of B. breve strain Yakult to streptomycin 12002 of African origin, isolated from pig feces and was caused by a chromosomal mutation of the rps(L) used as probiotic intervention studies was found to gene for ribosomal protein S12, and thus unlikely to harbor the erm(B) gene that could be transferred in be transferred to other microorganisms. vitro to enterococci. Twelve probiotic isolates of In another study by Blandino et al. (2008), the European origin demonstrated high prevalence of strains of Bifidobacterium were found susceptible phenotypic resistance for aminoglycosides (Ouoba et to ampicillin, cefotaxime and erythromycin. In the al., 2008).
study of Mättö et al. (2007), Bifidobacterium strains In a study by Egervärn et al. (2007), L. reuteri displayed generally high MICs for streptomycin and and L. fermentum (56 strains of each) were assessed gentamicin suggesting intrinsic resistance. D'Aimmo for antibiotic susceptibility using an E-test kit and et al. (2007) found that bifidobacteria were resistant a broth microdilution method. L. fermentum has to aminoglycosides, cycloserine, nalidixic acid and shown an uniform distribution for tested antibiotics strongly resistant to kanamycin, polymixin B, and including ampicillin, tetracycline, erythromycin, aztreonam (MIC90 = 1000 µg per mL).
clindamycin, streptomycin, and gentamicin, whereas L. reuteri strains displayed bimodal distribution Enterococcus
of MICs or above the test range for erythromycin, clindamycin, kanamycin, vancomycin, tetracycline, Members of Enterococcus contain some International Food Research Journal 18(3): 837-853 Antibiotic resistance of probiotic organisms and safety of probiotic dairy products opportunistic pathogens, hence, it is debated as to was demonstrated by the resistant strains that pose whether these organisms could be used as probiotics. a potential risk of horizontal transfer of the resistant Several studies have examined the antibiotic gene among other food borne commensal bacteria. resistance profile, and evaluated the transferability of E. faecalis strains isolated from Irish pork and the resistance determinants to other microorganisms. beef abattoirs were susceptible to vancomycin, Rizzotti et al. (2009) studied the diversity and however, 4 of 10 strains of E. faecium were resistant transferability of tetracycline gene tet(M) of 20 to vancomycin but no corresponding genetic enterococci belonging to species of E. faecalis (12 determinants for this phenotype were detected (Toomey strains), E. faecium (4), E. durans (2), E. hirae (1), et al., 2010). E. faecium isolated from an European and E. mundtii (1) originating from swine meat. The probiotic product was found resistant to vancomycin gene tet(L) was observed in the 50% of the strains and using disc diffusion method but later it was confirmed tet(M) was found correlated with a transposon of the by broth dilution and PCR that the isolates were Tn916-1545 family. Moreover 50% of enterococcal vancomycin sensitive (Temmerman et al., 2003). strains showed the ability to transfer tet(M) gene to Susceptibility of 128 isolates of E. faecium used as E. faecalis or Listeria innocua strains, which affirms probiotic cultures was tested for 16 antimicrobial the spread of tetracycline resistance in enterococci agents using broth microdilution. Two isolates were to potentially pathogenic bacteria occurring in food phenotypically resistant to erythromycin, 1 of which contained an erm(B) gene that was not transferable to Mater et al. (2008) observed the transfer of enterococcal recipients (Vankerckhoven et al., 2008). vancomycin resistance (vanA) from enterococci to In the study of Tompkins et al. (2008), MIC values a commercial strain of L. acidophilus in vitro and for E. faecium R0026 for 17 antimicrobials were in vivo in mice. The transconjugants were obtained below the break-point values published by EFSA. in high ferquency and were capable of persisting The strain used in different commercial probiotic in the digestive environment of mice. Since the products was susceptible to gentamicin, streptomycin same transfer is expected to occur in human and vancomycin.
digestive tract, it raises a safety concern regarding Use of growth promoters creates a major food the use of probiotics comprising lactobacilli in animal reservoir of resistant bacteria, with a potential either immunocompromised individuals or during for spread to humans through food intake or by contact antibiotic therapy. In vancomycin resistant E. faecium with animal (Wegener, 2003). Butaye et al. (2000) isolates collected from Michigan hospitals, the tested 76 E. faecium strains originated from poultry location of vanA genes was found on both plasmid meat, cheese and raw pork for their susceptibility and and chromosome that suggests the possibility of resistance to growth-promoting antibacterials used transposon dissemination among these isolates (Thal in animals and antibiotics used therapeutically in et al., 1998). humans. High-level of streptomycin resistance was Regarding the prevalence of antimicrobial observed in strains of all origins, though infrequently resistance of enterococcal strains in different but the strains isolated from poultry meat showed environments, the frequency of various antimicrobial more resistances against bacitracin, virginiamycin, resistances was much lower in food isolates in narasin, tylosin (a macrolide antibiotic), ampicillin, comparison to clinical strains (Abriouel et al., 2008). glycopeptides avoparcin and vancomycin. Similar findings were reported by Blandino et al. Enterococcus species can be found in the same (2008) where E. faecium derived from probiotic habitat as of the Listeria species. Hence, these can be product from Italy was susceptible to all the tested important sources of transferring antibiotic resistance antibiotics including vancomycin, ampicillin, through mobile genetic elements such as transposons cefaclor, cefotaxime, erythromycin, ciprofloxacin and to Listeria. A horizontal spread of resistance to gentamicin. However, in the Moroccan food isolates Listeria spp. could be possible in some steps of the studied by Valenzuela et al. (2008), the frequency of food production (Rizzotti et al., 2009). antimicrobial resistance was remarkably high. The resistance profiles of E. faecalis were different from Streptococcus
those of E. faecium, tetracycline resistance being typical to the former and erythromycin resistance to A strain of S. thermophilus isolated from a the latter. Similarly, in the study of (Devirgiliis et al., probiotic product available in Italy was found 2010), high MIC values for tetracycline were found resistant only to ciprofloxacin among the tested among 16 strains of E. faecalis isolated from Italian antibiotics (Blandino et al., 2008). D'Aimmo et al. fermented dairy products. The presence of tet(M) (2007) reported that S. thermophilus was resistant International Food Research Journal 18(3): 837-853 846 Rabia, A. and Shah, N. P. to cycloserine, kanamycin, metronidazole, nalidixic for tetracycline were found for 26 strains while 17 acid, neomycin, paromomycin, polymyxin B, strains showed high MIC values for both tetracycline spectinomycin, and streptomycin (MIC ranging and erythromycin (Devirgiliis et al., 2010). from 64 to 500 µg per mL). It was found highly resistant to aztreonam having a MIC of 1000 µg per Safety of probiotic foods
mL.Antibiotic resistance of 39 srains of S. bovis Lactobacillus, Bifidobacterium, Pediococcus, representing the microflora of a typical Italian dairy and Lactococcus have long history of use in food product was found. It displayed high MIC values and extensively been used as probiotics (Shah, for tetracycline and the presence of tet(M) was 2007). It is estimated that per capita consumption of detected in these strains. This poses a potential risk fermented milk in Europe is 22 kg; this amounts to of horizontal transfer of antibiotic-resistance genes approximately 8.5 billion kg per year, a total of 8.5 among foodborne commensal bacteria (Devirgiliis et x 1020 LAB (assuming 108 cfu per g), and 3400 tones al., 2010). of LAB cells (assuming each cell weighs 4 x 10-12 g) (Shah, 2010). US sales of probiotics were estimated to be worth $764 million in 2005 and were projected to be worth $1.1 billion in 2010. Sales of probiotics Bacillus strains have been increasingly proposed used in the manufacture of food supplements were for prophylactic and therapeutic use against several projected to reach at $291.4 million in 2010, and food gastro-intestinal diseases (Sorokulova et al., 2008). applications are expected to dominate the market, Reports suggest higher MIC for Bacillus strains. with sales estimated at $700 million in 2010 which In the study of Luna et al. (2007), all B. anthracis include yogurts, kefir, and cultured drinks as major isolates (18) were found resistant to trimethoprim/ categories (Vanderhoof et al., 2008). sulfamethoxazole. Only B. thuringinesis (19) was The most common microorganisms used in resistant to β-lactams, 3 of 42 isolate of B. cereus, fermented products belong to the genera Lactococcus, 1 of 5 isolates of B. mycoides and all species of Leuconostoc, Pediococcus, and Lactobacillus. B. pseudomycoides (6 isolates) were resistant Lactobacilli and bifidobacteria are important to clindamycin. Of 7 erythromycin resistant/ indigenous microbiotia of man and animals, rarely intermediate B. cereus species, 3 were clindamycin being implicated as cause of infection with quite resistant and 1 was both clarithromycin and few exceptions and generally recognized as safe clindamycin resistant. Vancomycin-resistant B. cereus (GRAS). However B. dentium, a causative agent of was isolated from respiratory samples from patients dental caries, was found to be pathogenic. Similarly, in a paediatric intensive care unit of a hospital Kalpoe B. animalis naturally colonizes animal habitats, so its et al. (2008). B. licheniformis strain was reported to use in humans appears to be inappropriate because the be resistant to chloramphenicol and clindamycin criteria for a probiotic product consumed by humans (Sorokulova et al., 2008). must contain bacteria from human origin (D'Aimmo Presence of mobile plasmid-encoded tetracycline et al., 2007).
resistance in the B. cereus group was mentioned in Based on safety records, microorganisms can the EFSA opinion on QPS (European Food Safety be placed in 3 groups: safe strains (Lactococcus, Authority-EFSA, 2007). B. brevis and B. firmus Leuconostoc, Pediococcus, Lactobacillus, intended to be used as biomass for animal feed Oenococcus, S. thermophilus, Bifidobacterium, were inappropriate for QPS (European Food Safety Carnobacterium, E. saccharolyticus, and E. faecium), Authority-EFSA, 2008). doubtful strains (Enterococcus, L. rhamnosus, L. catenaforme, Vagococcus, and B. dentium) and risky strains (Peptostreptococcus, and Streptococcus) (Mogensen, 2003). There are 3 theoretical concerns Some potential risks are involved regarding the regarding the safety of probiotic organisms: (1) use of fermented foods that could act as potential the occurrence of disease, such as bacteremia or vehicles for the spread of antibiotic resistance to endocarditis; (2) toxic or metabolic effects on the consumers through the food chain. Tetracycline and gastrointestinal tract; and (3) the transfer of antibiotic erythromycin-resistance genes were found among resistance in the gastrointestinal flora (Snydman, the strains of Lc. lactis, representing the fermenting 2008).
microflora of typical Italian traditional cheese According to Food and Agriculture Organisation Mozzarella di Bufala Campana. High MIC values (FAO)/WHO guidelines for the evaluation of International Food Research Journal 18(3): 837-853 Antibiotic resistance of probiotic organisms and safety of probiotic dairy products probiotics in food (2002), it is suggested that probiotic any harm and the organisms cleared in 2 to 3 weeks organisms may theoretically be responsible for side- (Mogensen, 2003). L. delbrueckii ssp. bulgaricus, L. effects including systemic infections, deleterious rhamnosus, and B. lactis did not translocate. Lara- metabolic activities, excessive immune stimulation in Villoslada et al. (2007) carried out safety assessment susceptible individuals and gene transfer. Regarding of two probiotic strains including L. coryniformis the safety assurance of probiotic organisms in CECT5711 and L. gasseri CECT5714 using 20 food, FAO/ WHO guidelines (2002) suggest testing Balb/c mice which were orally treated with L. probiotic strains for antibiotic resistance patterns, coryniformis CECT5711 or L. gasseri CECT5714 for certain metabolic (e.g., D-lactate production, bile 30 days and reported no treatment-associated bacterial salt deconjugation) and hemolytic potential, toxin translocation as these organisms were not present production, side-effects, and epidemiological in liver or spleen. In another study, L. fermentum surveillance of adverse incidents during human CECT5716, a probiotic strain isolated from human studies and infectivity deficit in immunocompromised milk, was orally administered for 28 days to half of 40 Balb/c mice with a dose of 1010 colony forming units (cfu) per mouse per day and observed no bacteremia and no treatment-associated bacterial translocation to liver or spleen (Lara-Villoslada et al., 2009). Liong The safety concerning the use of these bacteria has and Shah (2006; 2007) administered L. casei and B. not been doubted for many years. However, some of infantis to 24 rats and no probiotics were detected the members of genera Lactobacillus, Leuconostoc, in the spleen, liver, and kidney suggesting that the Pediococcus, Enterococcus, and Bifidobacterium organisms were not translocated to these organs. have been frequently reported to be the cause of (Tompkins et al., 2008) reported absence of both various infections in patients with clinical conditions strains in the liver, kidneys, spleen or heart after 28- such as endocarditis and bloodstream infections days repeated high-dose oral treatment of E. faecium (Gasser, 1994). There are many sources of exposure R0026, and Bacillus subtilis R0179 used in Asian to these bacteria including probiotic preparations, probiotic products, to 30 Sprague-Dawly albino rats. fermented food products as well as the host's own Intestinal microflora of a subject also plays microflora (Borriello et al., 2003). Since these an important role in the prevention of probiotic organisms can adhere to epithelial lining and can translocation to internal organs. In a recent study by survive gastric conditions, they may pose risks Gronbach et al. (2010), it was reported that if both of translocation. They can translocate from the intestinal microbiotia and adaptive immunity are gastrointestinal lining to extraintestinal sites. They defective, translocation across the intestinal epithelium can enter regional lymph nodes, spleen, liver, blood and dissemination of probiotic bacteria such as vessels, and other tissues (Shou et al., 1994) causing E. coli Nissle could occur with potentially severe systemic infections, bacteremia, septicemia and adverse effects. Although translocation of probiotic multiple organ faliure (Berg, 1992; Liong, 2008). bacteria to internal organs of immunodeficient mice Indigenous microorganisms are not normally was observed in the study of Wagner et al. (1997), found in mesenteric lymph nodes, spleen, liver, or there was no evidence of increased inflammation blood of healthy subjects. They are eliminated by the or other pathologic findings in tissue sections from host's immune system as they attempt to translocate mice. Zhou et al. (2000) administered L. acidophilus. across the mucosal epithelium. Thus translocation B. lactis, and L. rhamnosus to 78 mice at 3 levels of probiotic organism is not detected in most of the including 5 ×107, 5 ×109, 5 ×1010 cfu per day and studies, in which probiotic organisms are administered found that the organisms were safe, and no adverse even at high doses to healthy subjects (Liong, 2008). effects were observed. Lara-Villoslada et al. (2009) found that the strain Animal model could be useful in evaluating the L. fermentum CECT5716 orally administrated to safety of new probiotics in immunocompromised Balb/c mice was non-pathogenic for mice even in hosts (Borriello et al., 2003). In most of experiments doses 10,000 times higher (expressed per kg of body performed in mice, translocation of bacteria is weight) than those normally consumed by humans. usually observed in immuno-compromised subjects Bacterial translocation does not occur commonly only but the response may vary with age of the in healthy specific pathogen-free animals but it animal. Wagner et al. (1997) suggested that the use can be found for a long duration in germ-free mice of probiotic is likely to be safe for immunocompetent (Ishibashi et al., 2001). Tanslocation was observed in and immunodeficient adults, but they should be tested sterile born mice; however, lactobacilli did not cause for safety in immunodeficient neonates. International Food Research Journal 18(3): 837-853 848 Rabia, A. and Shah, N. P. In vitro and in vivo assessments of the safety of effect (Jeppsson et al., 2004). Administration of two species of Bacillus, including B. subtilis , and B. live lactobacilli including strains of L. reuteri, L. indicus as a food probiotic were carried out by Hong plantarum and L. fermentum to male Sprague-Dawley et al. (2008). The Natto strain of B. subtilis invaded rats reduced the bacterial translocation (Adawi et al., and lysed cells but neither species was able to adhere 1997). This is supported by another study that showed significantly to any cell line. The Natto strain formed probiotic supplementation containing B. bifidum, biofilms and none of strains produced any of the L. acidophilus, and L. bulgaricus (2 x 109 cfu per known Bacillus enterotoxins. Only B. indicus carried day) reduced bacterial translocation and decreased resistance to clindamycin at higher MIC than EFSA intestinal mucosal atrophy in male Sprague-Dawley breakpoints. In vivo assessments of acute and chronic rats with thermal injury (Gun et al., 2005). Moreover, dosing in guinea pigs and rabbits, no toxicity was in a rat model of small bowel syndrome, probiotic observed in animals under these conditions. The organisms decreased the bacterial translocation authors reported that B. indicus and B. subtilis were through mechanisms dependant on intestinal mucosal safe for oral use but further study is required regarding integrity (Mogilner et al., 2007).
the transmissibility of clindamycin resistance of B. The safety assessment of two Bacillus strains including B. subtilis, and B. licheniformis incorporated Docummented correlations between systemic into a popular East European probiotic product was infections and probiotic consumptions are few and carried out. Both were non-hemolytic and did not all occurred in patients with underlying medical produce Hbl or Nhe enterotoxins. Similarly, no bceT conditions (Food and Agricultural Organization of and cytK toxin genes were found. Study of acute the United Nations/ World Health Organization- toxicity in BALB/c mice demonstrated no treatment- FAO/WHO, 2002; Bernardeau et al., 2008). Many related deaths. The oral LD for both strains was of the probiotic organisms have a safe history in more than 2 × 1011 cfu per g. Chronic toxicity studies patients receiving nutritional support, although some showed no signs of toxicity or histological changes probiotic products have shown to increase the risk of in either organs or tissues of experimental animals. complications in specific patient groups (Whelan et B. subtilis strain was sensitive to all antibiotics listed al., 2010). by the EFSA but B. licheniformis strain was resistant Aguirre and Collins (1993) and Gasser (1994) to chloramphenicol and clindamycin that enclosed have reviewed clinical cases involving LAB and safety risks of using B. licheniformis strain. However, bifidobacteria between 1938 and 1993, and the B. subtilis strain was found to be non-pathogenic results are summarized in Table 3. Analysis of and safe for human consumption (Sorokulova et al., cases of infections revealed that out of 155 cases of infections involving LAB or bifidobacteria, 95 Tompkins et al. (2008) carried out safety cases involved Lactobacillus spp., 33 of Leuconostoc evaluation of 2 probiotic strains namely, E. faecium spp., 18 of Pediococcus spp. and 9 cases involved R0026 and B. subtilis R0179 used in Asian probiotic Bifidobacterium spp. (Table 3) (Gasser, 1994). products and found absence of both diarrheal Endocarditis was the most frequent infection in and emetic toxins in the latter strain. The authors which Lactobacillus species have been involved, in established, on the basis of the results of this study in particular strains of species of L. rhamnosus/casei combination with the observations of clinical studies have been most often isolated. in both infants and adults, that these microbes were safe for use as pharmaceutical probiotics and pose Table 3. Clinical cases in which lactic acid bacteria
or bifidobacteria have been isolated (Adapted from low risk to the consumer. Mogensen et al., 2002) Some of the studies have proposed beneficial effects of probiotic organisms in translocation and they have been tested to prevent bacterial translocation L. acidophilus in animal model. The findings by Zareie et al. (2006) indicated that probiotic bacteria can prevent L. plantarum L. rhamnosus chronic stress induced intestinal abnormalities and, thereby, exert beneficial effects in the intestinal tract. Bacterial species such as enteric gram-negatives and gram-positive cocci are more prone to translocation, Only about 180 cases of septicemia in humans whereas lactobacilli appear to have a protective involving LAB have been reported. In only 1 of International Food Research Journal 18(3): 837-853 Antibiotic resistance of probiotic organisms and safety of probiotic dairy products these cases, the identified LAB was identical with a the overall mortality was reported nearly 30%. commercially available dairy strain. E. faecium and Patients of all ages and both gender were afftected. E. faecalis are more frequently involved in clinical The main underlying conditions were recognized infection. In most cases of infection, people were as cancer, diabetes, transplantation particularly reported to be infected by their own flora, however, in of liver, abscesses, and hypertension. Husni et a few cases consumption of probiotic organisms was al. (1997) reviewed 45 cases of Lactobacillus a potential source. About 30 cases of fungaemia have infections occuring over 15 years and the organisms been reported in patients treated with Saccharomyces causing infections were characterized. The common boulardii (Gasser, 1994), and 2 cases of infection were underlying conditions were cancer (40%), recent with food-borne L. rhamnosus (Mackay et al., 1999). surgery (38%), and diabetes mellitus (27%). One in In another report, 62 patients became colonized with 39 deaths was attributed to Lactobacillus bacteremia. B. cereus including 2 with non-fatal Bacillus sepsis Cannon et al. (2005) recognized a very small and a death due to pneumoniae associated with the percentage (1.7%) of cases associated with heavy organism (Bryce et al., 1993).
dairy consumption, where 3 cases were associated Saxelin et al. (1996) studied the prevalence with endocarditis and 1 with a liver abscess. A case of of bacteremia caused by Lactobacillus species in aortic valve endocarditis caused by L. casei in a 53- Southern Finland and compared the characteristics year-old immunocompetent patient with past history of the blood culture isolates with probiotic dairy of rheumatic fever was reported by Zé-Zé et al. strains. Lactobacillus was identified in eight of (2004). Noticeably clinical symptoms appeared after 3317 blood culture isolates; however, there was no a dental extraction and the patient's diet included isolate from dairy strain. In a 74-year-old woman several tubs of yogurts per day. Presterl et al. (2001) with several years history of hypertension and non- reported a young man having diet comprising large insulin dependant diabetes mellitus, liver abscess was quantities of probiotic yogurt developed endocarditis reported due to L. rhamnosus strain indistinguishable and septic arthritis caused by L. rhamnosus. However from L. rhamnosus strain GG (Rautio et al., 1999).
the contradictory findings were reported by Wallet In a study by Kalliomäki et al. (2001), L. et al. (2002), where a case of endocarditis due to L. rhamnosus GG was given to 132 women who casei subsp. rhamnosus was found in 73-year-old were at high risk of their babies developing atopic man without previous history of dental manipulation dermatitis. There was no report of adverse effects in or daily yogurt intake. In relation to a consumption mothers indicating that the probiotic organism was of about 20 million tons of fermented milk annually, safe. Reports by Salminen et al. (2002) suggest that the above numbers are negligible (Mogensen, 2003). L. rhamnosus GG has been used widely in Finland There is no foundation for safety concern in relation since late 1980s and despite the long term use of this to probiotic dairy products on the market today. probiotic organism, there has been only few cases of Probiotic organisms are generally considered safe. As bacteremia (0.05 cases per 100 000 cases). evidenced by epidemiologic studies, bacteremia or Whelan and Myers (2010) reviewed of total sepsis from lactobacilli is extremely rare. Numerous of 1966 articles, of which they found 72 to fulfil probiotic organisms have a long history of safe use the inclusion criteria. There were 20 case reports and no health concerns have been observed. A long of adverse events in 32 patients, all of which were history of safe use is still the most credible safety infections due to L. rhamnosus GG or Saccharomyces test.
boulardii. The risk factors included central venous catheters and disorders associated with increased Conclusions
bacterial translocation. There were 52 articles reporting 53 trials in which 4131 patients received Selective pressure of using antibiotic in both probiotic organisms. Most trials showed either no human and animal treatment, and dissemination of effect or a positive effect on outcomes related to antibiotic resistance bacteria has the possibility to safety (e.g., mortality and infections). Only 3 trials aggravate acquisition and spread of resistant genes. showed increased complications, which were largely In this context, probiotic organisms are considered to non-infectious in nature and in specific patient groups pool the resistant genes and transfer these to pathogenic (e.g., transplant and pancreatitis). bacteria. In order to eliminate this possibility, MIC of Cannon et al. (2005) reviewed 241 clinical cases the most relevant antimicrobials for each strain used of Lactobacillus infections and found 129 cases of as a probiotic organism, food or feed additives could bacteremia and 73 cases of endocarditis. L. casei be determined using protocols given by EFSA and and L. rhamnosus were most common species and on firm genetic grounds. Several studies regarding International Food Research Journal 18(3): 837-853 850 Rabia, A. and Shah, N. P. the antibiotic susceptibilities of LAB, bifidobacteria tetracycline resistance mechanisms, plasmid-carried have been reviewed but only few have determined tet(L) and chromosomally located transposon- the genetic basis of these resistances. Majority of associated tet(M), coexist in Lactobacillus sakei rits resistance found in the species of Lactobacillus, 9. Applied and Environmental Microbiology 74 (5): Bifidobacterium, Enterococcus, Streptococcus and Bacillus were of intrinsic type. Resistances to Anadón, A., Rosa Martínez-Larrañaga, M. and Aranzazu Martínez, M. 2006. Probiotics for animal nutrition in tetracycline, vancomycin and erythromycin were the European Union. Regulation and safety assessment. frequent in these species and some showed to harbour Regulatory Toxicology and Pharmacology 45 (1): 91- genes tet(W), tet(M), van(A) and erm(B) mostly on chromosome with only few on plasmid or transposon. Berg, R.D. 1992. Translocation and the indigenous gut Intrinsic resistance, and resistance due to mutation of flora In: Fuller, R. (eds.): Probiotics: The Scientific chromosomal genes present a low risk of horizontal Basis. Chapman and Hall, London: 55-85.
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Medicines control council

Registration of Medicines PI standardised texts MEDICINES CONTROL COUNCIL PACKAGE INSERTS FOR HUMAN MEDICINES STANDARDISED TEXTS This guideline is intended to provide recommendations to applicants wishing to submit applications for the registration of medicines. It represents the Medicines Control Council's current thinking on the safety, quality and efficacy of medicines. It is