Genotypic methods, based on molecular techniques, which are powerful to fingerprint specific DNA patterns that are characteristics for a single strain, form the mainstay of strain typing of LAB. The introduction of molecular biology techniques has yielded a variety of DNA-based typing methods, which can even discriminate between isolates of a given species. Depending on the technical aspects, genetic typing methods currently used can be divided into different categories with different taxonomic resolution: restriction fragment length polymorphism (RFLP) analysis of genomic DNA, PCR-based technologies and a miscellaneous of other methods, such as plasmid profiling or DNA sequencing. In the following sections, the most applied methods to type food-associated LAB will be discussed.
4.2.1 Methods Based on Restriction Fragment Length Polymorphism (RFLP) of DNA
Restriction fragment length polymorphism (RFLP)-based methods of total chromosomal DNA were among the first of the DNA-based typing schemes. Restriction endonuclease analysis (REA) includes whole-genome DNA extraction, its digestion with restriction endonucleases and separation of the resulting array of DNA fragments by gel electrophoresis. Using REA, discrimination down to strain level can be reached, although the high number of fragments makes the interpretation of the profiles difficult. However, over time, many RFLP-based approaches, such as ribosomal RNA gene restriction analysis (ribotyping) and REA-pulsed field gel electrophoresis (REA-PFGE), have been introduced to reduce the number of DNA fragments that are analyzed.
Ribotyping utilizes the similarities and differences found in rRNA genes. These genes are highly conserved, yet vary in number, size, and position within the same chromosome. After digestion and electrophoretic separation of whole chromosomal DNA, the separated DNA fragments are transferred to a membrane, fixed, and hybridized with a chemiluminescent rRNA gene probe. The resulting pattern of bands makes it possible to delineate species and strains on the basis of the difference in the RFLPs of ribosomal genes. Various species and individual strains of lactobacilli can be discriminated by ribotyping (Giraffa and Neviani 2000; Domig, et al. 2003). REA-PFGE, which is considered to be the gold standard among molecular typing methods, allows the comparison of 15 to 20 restriction DNA fragments generated after digestion of the whole chromosome by rare cutting restriction endonucleases. Bands are then separated by gel-electrophoresis under conditions that allow efficient resolution of high molecular size DNA fragments. REA-PFGE has been successfully used in the identification and subtyping of food-associated LAB and enterococci (Klein, et al. 1998; Giraffa and Neviani 2000; Domig, et al. 2003; Coppola, et al. 2006).
Both ribotyping and REA-PFGE are reproducible, easy to interpret, and highly discriminative; on the other hand, both techniques are difficult to apply in industry because they are cumbersome, difficult to use, and expensive (Olive and Bean 1999). This has led to an automation of methods. For example, the RiboPrinter system, which operates a completely automated ribotyping procedure, allows detection of the resulting hybridization pattern of bands on the membrane by a camera. The image is then transferred to a computer for analysis and is compared with a database containing 10,000 fingerprints of known bacteria. Using this system, which shows a high rate of inter-laboratory reproducibility, a bacterial isolate can be identified within eight hours (Dawson 2001). Strains of E. faecium resistant to vancomycin were successfully characterized by automated riboprinting (Brisse, et al. 2002).
PCR-based DNA fingerprinting methods using arbitrary primers, such as arbitrarily primed PCR (AP-PCR) and randomly amplified polymorphic DNA (RAPD)-PCR, have been developed for studying genomic DNA polymorphisms. Among PCR-based typing techniques, RAPD-PCR is the most popular typing technique applied to food ecosystems. RAPD-PCR is based on the use of short random sequence primers, nine or 10 bases in length, which hybridize with sufficient affinity to chromosomal DNA sequences at low annealing temperatures. If two RAPD-PCR primers anneal within a few kilobases of each other, a PCR product will result. As the number and location of sites vary for different strains, a pattern of band is then generated which, in theory, is characteristic of a given bacterial strain. In recent years, hundreds of articles reported the application of RAPD-PCR to identify the presence, succession, and persistence of microorganisms (both useful and pathogens) in both fermented food and industrial environments (Maukonen, et al. 2003; Carminati, et al. 2004; Giraffa 2004). The numerous applications of this technique to different foods (and the relative references) will be detailed in the specific chapters of this book.
RAPD-PCR typing can be done quickly, especially in cases where fingerprinting is carried out with DNA from single-colonies growing on an agar plate. Therefore, RAPD-PCR is best suited for studies where specific bacterial strains are sought among a large number of isolates. Due to the low stringency of the PCR amplification, variability of RAPD-PCR fingerprints can sometimes be observed. The use of more than one primer and/or annealing temperatures (with increasing stringency) may improve reproducibility, but make the technique more laborious. A higher reproducibility of RAPD-PCR can be more practically achieved by careful standardization of the experimental methodology and by more objective comparison of DNA fingerprinting data. The development of bioinformatics has enabled the implementation of fingerprint databases, thus improving the interpretation and elaboration of RAPD-PCR data (Rossetti and Giraffa 2005).
In the repetitive element sequence-based PCR (Rep-PCR), repetitive chromosomal elements, which are randomly distributed in bacterial genomes, are the target of the PCR amplification. In Rep-PCR, primers anneal to repetitive parts of the chromosome and amplification occurs when the distance between primer binding sites is short enough to enable this. In Rep-PCR, amplification yields DNA fragments of varying size, which are separated by agarose gel electrophoresis (Versalovic, et al. 1991). Rep-PCR has been applied to characterize LAB isolated from fresh Sausages (Cocolin, et al. 2004).
ITS-PCR, which is a species-specific identification method, shows some potential for use as a microbial typing system, especially when applied to infraspecies identification of E. gallinarum and E. faecium (Domig, et al. 2003). A variation of this technique is PCR-ribotyping, which takes the advantage of the heterogeneity that exists within the spacer regions located between all the ribosomal genes. Improved discrimination, with respect to ITS-PCR, is obtained by using primers flanking conserved regions of 16S, 23S, and 5S rRNA genes, so that the intergenic spacer regions between the three ribosomal genes will be amplified. This technique has been used to type organisms such as E. faecium, Escherichia coli, Enterobacter spp., and Listeria monocytogenes (Domig, et al. 2003).
As stated above, the main criticism of the PCR-based typing systems (such as RAPD-PCR) is the limited interlaboratory reproducibility. To overcome this problem and to obtain a more sensitive discrimination between related strains, AP (or RAPD)-PCR could be performed using a fluorescence-labeled primer and the amplified fragments separated electrophoretically and detected by an automatic DNA sequencer (Cancilla, et al. 1992). Alternatively, more reproducible PCR-based microbial fingerprinting techniques (such as adaptor fragment length polymorphism or AFLP) could be used. AFLP involves restriction of total bacterial DNA with two endonucleases of different cutting frequencies, followed by ligation of the fragments to oligonucleotide adapters complementary to the sequences of the restriction site. Selective PCR amplification of the subset of fragments is achieved using primers corresponding to the contiguous sequences in the adapter and restriction site, plus a few nucleotides in the target DNA. Amplified fragments are then analyzed by gel electrophoresis. Unlike RAPD that uses multiple, arbitrarily chosen DNA regions to be amplified, the AFLP technique allows only two genomic regions to be amplified by selective primers and gives more reproducible results (Vos, et al. 1995; Janssen, et al. 1996). AFLP has recently been automated by using fluorescently dye-labeled primers, followed by separation of the labeled fragments through capillary electro-phoresis under denaturing conditions and laser detection of the AFLP fragments using an automated analyzer (Gancheva, et al. 1999). A recent simplification of the AFLP method is the technique named SAU-PCR. Like the AFLP method, this technique uses primers based on the restriction enzyme recognition sequence, but it does not require the addition of linkers, and the products can be resolved on agarose gels. The proposed technique is based on the digestion of genomic DNA with the restriction endonuclease Sau3AI and subsequent amplification with primers whose core sequence is based on the Sau3AI recognition site (Corich, et al. 2005).
AFLP is a fairly new technique and, therefore, few data regarding its application to fingerprint food-associated microbes are available. AFLP proved a sensitive and reproducible technique for the typing of Clostridium perfringens, Listeria monocy-togenes, and vancomycin-resistant E. faecium (Aarts, et al. 1999; Antonishyn, et al. 2000; McLauchlin, et al. 2000). The phenotypically closely related species Lb. plantarum, Lb. pseudoplantarum, and Lb. pentosus were discriminated on the basis of RAPD and AFLP patterns, which also allowed an effective infraspecific differentiation of 30 silage and cheese isolates to be obtained (Torriani, et al. 2001a).
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