Summary auto-generated
This study addresses the difficulty of distinguishing Lactobacillus hilgardii from Lactobacillus brevis using conventional phenotypic methods, which often leads to misidentification. The researchers developed molecular techniques—RAPD (random amplified polymorphic DNA) and REP-PCR (repetitive element PCR)—to create species-specific genomic fingerprints for accurate identification. Analysis of ribosomal operons confirmed taxonomic relationships between the species. Using RAPD and REP-PCR data, the researchers isolated L. hilgardii-specific DNA probes from amplified fragments. Notably, three L. brevis strains (IOEB 7903, IOEB 9647, IOEB 9649) that could ferment arabinose unexpectedly clustered genetically with L. hilgardii based on fingerprinting patterns and 16S rDNA analysis. The researchers concluded that these arabinose-fermenting strains should be reclassified as L. hilgardii despite their positive arabinose fermentation. The study demonstrates that molecular methods based on DNA sequences are more reliable than biochemical tests for species-level bacterial identification, with important implications for quality control in winemaking.
Key findings
- RAPD and REP-PCR molecular fingerprinting revealed that three L. brevis strains (IOEB 7903, IOEB 9647, IOEB 9649) cluster genetically with L. hilgardii despite their ability to ferment arabinose, indicating phenotypic traits can diverge from genetic identity.
- L. hilgardii-specific DNA probes were successfully isolated and shown to hybridize only with L. hilgardii strains and the three misclassified L. brevis strains, providing tools for accurate species identification.
- 16S rDNA sequence analysis confirmed that the three anomalous strains belong to L. hilgardii species genotypically, containing the species-specific H2 oligonucleotide primer binding site.
- Molecular fingerprinting methods (RAPD and REP-PCR) are more reliable than arabinose fermentation assays for distinguishing L. hilgardii from L. brevis, as biochemical phenotypes can be influenced by gene regulation.
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Abstract
Conventional phenotypic methods lead to misidentification of the lactic acid bacteria Lactobacillus hilgardii and Lactobacillus brevis. Random amplified polymorphic DNA (RAPD) and repetitive element PCR (REP-PCR) techniques were developed for a molecular study of these two species. The taxonomic relationships were confirmed by analysis of the ribosomal operon. Amplified DNA fragments were chosen to isolate L. hilgardii-specific probes. In addition to rapid molecular methods for identification of L. hilgardii, these results convincingly proved that some strains first identified as L. brevis must be reclassified as L. hilgardii. The data clearly showed that these molecular methods are more efficient than phenotypic or biochemical studies for bacterial identification at the species level.