Polyphasic Approaches to Lactobacilli Identification: Comparison of Phenotypic, Genotypic and Spectroscopic Methods

Lactobacillus forms a large and diverse group of lactic acid bacteria (LAB). In raw milk and dairy products such as cheeses, yoghurts and fermented milks, lactobacilli are either naturally present or added intentionally for technological reasons or to generate a health benefit for the consumer. The Lactobacillus taxonomy has continued to evolve over the last twenty years, and currently consists of over 170 species.
We employed API 50CH (Apparatus and Procedure of Identification) and SDS-PAGE (sodium dodecyl sulfate-–-polyacrylamide gel electrophoresis) as phenotypic techniques to establish the identity of 52 wild lactobacilli isolated from artisanal dairy products. The identifications were confirmed using repetitive genomic element-PCR (Rep-PCR) fingerprinting and spectral methods including fluorescence spectroscopy. The SDS-PAGE results confirmed about 90% of API identification results. PCR using Boxair primers discriminated tested strains into Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus delbrueckii subsp. lactis, Lactobacillus fermentum, Lactobacillus paracasei subsp. paracasei, and Lactobacillus plantarum. This genetic method confirmed all the SDS-PAGE phenotyping results. Synchronous fluorescence spectra (SyFS) were recorded from 31 non-transparent bacterial colonies using a FluoroMax-2 spectrofluorometer linked to an optic fiber, and the results confirmed all the genotypic results, excluding certain strains: Lb. plantarum (217N and 1025RM) and Lb. paracasei subsp. paracasei 286KC as identified by the fluorescence method were genetically reclassified as Lb. delbrueckii subsp. lactis and Lb. plantarum, respectively.
Our study therefore highlights that spectral data provide real phenotypic fingerprints of the bacteria and can thus be used for taxonomic purposes. This study found that BOXPCR is a good tool for confirming most of the phenotypic identifications, making it possible to taxonomically