Criteria for Selection of Probiotic Bacteria
Different in vitro and in vivo approaches have been used to select potentially probiotic strains of bifidobacteria and lactobacilli, as well as to measure their efficacy (Gibson and Fuller 2000). Criteria for the selection of probiotic bacteria have been defined in several reviews (Adams 1999; Bhadoria and Mahapatra 2011; Gibson and Fuller 2000; Saarela et al. 2000; Salminen et al. 1998). They indicate that many aspects, including safety and functional and technological characteristics, have to be taken into consideration in the selection process of probiotic microorganisms.
Safety of Probiotic Bacteria
The safety of probiotic strains is of prime importance. Although vigorous debates continue on what constitutes appropriate safety testing for novel probiotic strains proposed for human use, it generally includes such characteristics as origin, nonpathogenicity and antibiotic-resistance characteristics.
Strains for human use are preferably of human origin, isolated from healthy GIT (Saarela et al. 2000). Probiotic bacteria must be non-pathogenic, with no history of association with diseases such as infective endocarditis or gastrointestinal disorders. Knowledge on survival of the probiotics within the GIT, their translocation and colonisation properties, is also important for the evaluation of possible positive or negative effect of probiotic consumption (Marteau et al. 1995). From this point of view, lactic acid bacteria and bifidobacteria are widely used in fermented food and dairy products with no case of local or systemic infections occurred, which confirms their GRAS (“generally regarded as safe”) status (Sleator 2010). Many findings indicate that the general human population is not at risk from exposure to probiotic bacteria of Bifidobacterium and Lactobacillus genera. Although the rare cases of infection associated with probiotics have occurred in groups of people whose conditions predispose them to opportunistic infections, in many cases people with serious underlying diseases have benefited from probiotics (Benchimol and Mack 2005; Reid 2006; von Wright 2005).
Another aspect of safety consideration is antibiotic resistance of probiotic bacteria strains. The resistance of bacteria to antibiotics is an increasingly important public health problem worldwide. There is a pressing need to limit the spread of resistance genes, since these could be transferred to opportunistic and pathogenic bacteria (Ammor et al. 2008; Blazquez et al. 2002). Antibiotic resistance could be “intrinsic” and “acquired.” Intrinsic resistance is inherent to bacteria species and involves the absence of the target, presence of low-affinity target, low cell permeability or presence of efflux mechanisms. The acquisition of antibiotic resistance occurs through the mutation of pre-existing genes or by horizontal transmission, i.e. acquisition of foreign DNA from other bacteria. Therefore attention is currently being paid to probiotic LAB and bifidobacteria with respect to their potential role in the spread and transmission of antibiotic-resistance determinants (Ammor et al. 2008; Saarela et al. 2000).
Most bifidobacteria are intrinsically resistant to nalidixic acid, neomycin, polymyxin B, kanamycin, gentamicin, streptomycin and metronidazole (Charteris et al. 1998). Their resistance to other antibiotics differs depending on strain and in some cases may be due to the presence of genetic determinants. Indeed, microarray analysis revealed presence of tet(W) genes in B. longum and B. bifidum strains, as well as aph(E) and/or sat(3) genes in B. bifidum, B. longum, B. catenulatum and B. pseudocatenulatum strains (Ammor et al. 2008). Screening of 26 B. animalis subsp. lactis strains isolated from different sources revealed the presence of tet(W) in all isolates. Moreover, in all strains a transposase gene upstream of tet(W) gene was detected, which is cotranscribed in tandem. Transposases have been found to be involved in the horizontal gene transfer of genetic elements among bacteria, but to date there is no evidence that tet(W) in B. animalis subsp. lactis is transmissible (Gueimonde et al. 2010). Presence of the resistance determinant erm(X) was demonstrated in six erythromycinand clindamycin-resistant B. thermophilum strains during investigation of a large collection of bifidobacteria that could be potential probiotics (Mayrhofer et al. 2007). Analysis of additional bifidobacteria revealed that this antibiotic-resistance gene was also present in B. animalis subsp. lactis strains (Määttö et al. 2007). It was demonstrated that the erm(X) gene from erythromycinresistant Bifidobacterium strains was part of transposon Tn5432 and was nearly identical to erm(X) determinants present in several opportunistic pathogenic corynebacteria and propionibacteria (van Hoek et al. 2008). Although most of the antibiotic-resistance genes were located on bacterial chromosome, studies on the genetics of antibiotic resistance of bifidobacteria are guarantee their safe application.
Lactobacilli display a wide range of antibiotic resistance, and antibiotic susceptibility patterns vary greatly between different species of these microorganisms (Charteris et al. 1998). Thus, L. delbrueckii strains as components of yogurt cultures showed intrinsic resistance toward mycostatin, nalidixic acid, neomycin, polymyxin B, trimethoprim, colimycin and sulphonamides. Susceptibility to cloxacillin, dihydrostreptomycin, doxycycline, novobiocin, oleandomycin, oxacillin and streptomycin was prominent while resistance to kanamycin and streptomycin varied. Many lactobacilli carry intrinsic resistance toward vancomycin (Marthur and Singh 2005). In most cases antibiotic resistance of lactobacilli is not of the transmissible type (Saarela et al. 2000), and such strains do not usually form a safety concern. Although plasmid-linked antibiotic resistance is not very common among lactobacilli, they do occur (Rinckel and Savage 1990). R-plasmids encoding tetracycline, erythromycin, chloramphenicol or macrolide–lincomycin–streptomycin resistance have been reported in L. reuteri, L. fermentum, L. acidophilus and L. plantarum, isolated from raw meat, silage and faeces. Most of these R-plasmids had a size smaller than 10 kb (Marthur and Singh 2005). The presence of 5.7 kb plasmid carrying erm gene conferring high-level erythromycin resistance was demonstrated in L. fermentum isolated from pig faeces (Fons et al. 1997). Plasmid-encoding tetracycline-resistance gene tet(M) was detected in Lactobacillus isolates from fermented dry sausages (Gevers et al. 2002). The 10,877 bp tetracycline-resistance plasmid pMD5057 from L. plantarum 5057 was completely sequenced and the sequence revealed that tetracycline-resistant region contains a tet(M) gene with high homology to sequences of this gene from Clostridium perfringens and Staphylococcus aureus (Danielsen 2002). Since transfer of antibiotic-resistance genes may occur between phylogenetically distant bacteria, Lactobacillus strains that harbour mobile elements carrying resistance genes should not be used either as human or animal probiotics (Saarela et al. 2000).