Identification at Genus Species and Subspecies Level

Species identification can be achieved by statistical analysis of fingerprint data obtained from the above described approaches, even if they are commonly used for strain typing. Moreover, other techniques can be used to achieve identification at species level.

PCR-RFLP of the amplified 16S rRNA gene (ARDRA-PCR) or 16S-23S rRNA intergenic spacer region (ISR-RFLP-PCR) was also applied to identify or characterize dairy LAB. Villani, et al. (1997) analyzed Eco RI and Hind III ARDRA-PCR patterns of Leuconostoc spp. and showed that the technique is unreliable for species differentiation. By contrast, Aquilanti, et al. (2006) identified enterococci by analyzing MboII, MspI and RsaI PCR-ARDRA patterns. De Las Rivas, et al. (2006) found inconsistent differences on analyzing the ISR-RFLP-PCR of Lb. plantarum strains. Indeed, the latter approach was proposed to identify and differentiate Lactobacillus species (Moreira, et al. 2005). Baruzzi, et al. (2000) differentiated Lb. planatrum and Lb. paraplantarum after sequence analysis of the 16S-23S rDNA ISR. Flint and Angert (2006), on the basis of the 16S-23S rDNA ISR sequence, designed a strain-specific PCR primer to identify and monitor Lactobacillus spp. HOFG1 (closely related L. animalis or L. murinus species) in cattle feed.

16S-23S rDNA ISR pattern analysis allows differentiation of dairy streptococci, enterococci and lactococci (Moschetti, et al. 1998; Blaiotta, et al. 2002; Fortina, et al. 2003; Mora, et al. 2003) while it is unreliable for identification and differentiation of lactobacilli.

However, the continuously accumulating set of fingerprinting data and the construction of reliable databases require a high degree of standardization in experimental methodology. It is also important to have high-performing bioinformatic tools at one's disposal to get the best possible information from huge quantities of fingerprinting data. The development of bioinformatics has enabled improvement of the interpretation and elaboration of microbiological data. Many bioinformatic software programs or on-line tools, which are often commercially available, enable nucleic acid (or protein) sequences, fingerprinting profiles and phenotypic data to be analyzed and integrated. Some computerized databases of LAB fingerprints are also available, such as the RFLP database of total DNA patterns (Chan, et al. 2003), SDS-PAGE protein databases (Pot, et al. 1994; Leisner, et al. 1999) and the commercial RiboPrinter® system (Dawson 2001). Acquisition of specialized programs, which are expensive and demand a high level of technical skill for their efficient use, is necessary so that the most important international microbial collections can manage, compare and implement databases with information on genotypic and phenotypic data (Rossetti and Giraffa 2005).

For reliable identification of strains, partial or full 16S rRNA gene sequences have been extensively compared (see Tables 2.3 to 2.11). However, the 16S rRNA gene shows discrimination pitfalls in the identification of closely related LAB species. The genes present in only one copy, such as the Elongation factor Tu (tuf) gene (Chavagnat, et al. 2002; Ventura, et al. 2003), the DNA repair recombinase (recA) (Felis and Dellaglio 2005), the chaperonin Hsp60 (Cpn60) (Dobson, et al. 2004) and the RNA polymerase B subunit (rpoB) (Rantsiou, et al. 2004), have been exploited for the differentiation of Lactobacillus species. These genes have significant advantages over the 16S rRNA gene because of their species-discrimination power, indicated by published studies to be one order of magnitude higher than that of the 16S rRNA gene (Ventura, et al. 2003). Itoh, et al. (2006) performed sequence analysis of dnaJ (a member of the Hsp70 protein family) and gyrB (the B-subunit of DNA gyrase, topoisomerase type II) genes of streptococci and concluded that they are efficient alternative targets for the classification of the genus Streptococcus, and that dnaJ is suitable for phylogenetic analysis of closely related Streptococcus strains. Goh, et al. (2000) sequenced a 552-bp region of the chaperonin 60 gene (Cpn60) and demonstrated that clustering of the analyzed species is similar to the published Enterococcus trees based on 16S rRNA gene sequences. Poyart, et al. (2000) partially sequenced the gene encoding manganese-dependent superoxide dismutase (sodA) in 19 enterococcal-type strains. Their results confirm that the sodA gene constitutes a more discriminative target sequence than the 16S rRNA gene and allows differentiation among closely related bacterial species. Rossi, et al. (2006) suggest that the recA gene can be used as an alternative to the 16S rRNA gene as a target for detecting/identifying propionibacteria species, but it is less reliable as a molecular marker for their classification and intraspecies distinction.

Species-specific single or multiplex PCR assays were designed and used for rapid identification of LAB occurring in dairy products (Tables 2.3 to 2.11). As reported in Table 2.13, specific PCR systems are now available for the most important bacterial species occurring in dairy products. Moreover, PCR was also used to detect specific genes encoding for particular traits. Some of these systems are: detection of the prtP gene (coding for a cell envelope proteinase in LAB) (Klijn, et al. 1995); detection of virulence or resistance factors in enterococci (Khan, et al. 2005; Domig, et al. 2003); detection of genes involved in the production of biologically active amines such as histamine, tyramine and putrescine in LAB (Fernandez, et al. 2004; Marcobal, et al. 2005; Aymerich, et al. 2006).

Table 2.13 Some Available Genus- and Species-specific PCR Assays for the Rapid Identification and/or Differentiation Dairy Microorgamisms

Genera

Level of specificity

References

Lactobacillus (Lb.)

Lb. plantarum group species differentiation Lb. helveticus

Differentiation ofLb. casei group species Lb. plantarum, Lb. curvatus and Lb. sakei Lb. acidophilus, Lb. delbrueckii, Lb. casei, Lb. gasseri, Lb. plantarum, Lb. reuteri and Lb. rhamnosus Lb. delbrueckii subsp. lactis and

Lb. delbrueckii subsp. bulgaricus Lb. helveticus, Lb. paracasei and

Lb. rhamnosus Lb. fermentum, Lb. casei/paracasei, Lb.

plantarum, Lb. reuteri and Lb. salivarius Lb. acidophilus, Lb. casei, Lb. brevis Lb. brevis

Lb. casei, Lb. delbrueckii and

Lb. helveticus/ acidophilusgroups

Torriani, et al. 2001 Fortina, et al. 2001 Ward and Timmins 1999 Berthier and Ehrlich 1998 Kwon, et al. 2004

Torriani, et al. 1999

Tilsala-Timisjarvi and

Alatossava 1997 Chagnaud, et al. 2001

Massi, et al. 2004 Guarneri, et al. 2001 Drake, et al. 1996a-b

(continued)

Genera

Level of specificity

References

Streptococcus (S.)

S. thermophilus

Lick, et al. 1996

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