Microfluidic technology applied to cell-wall protein analysis of olive related lactic acid bacteria

A fully automated electrophoretic method for separating cell-wall proteins was applied to 102 isolates from olive phylloplane and brine previously phenotypically identified as Lactobacillus plantarum (10), Leuconostoc mesenteroides subsp. mesenteroides (60), Leuconostoc spp. (12) and Enterococcus faecium (19). The high sensitivity of this technique allowed to discriminate isolates and to distinguish closely related strains within the same species. L. plantarum was characterized by proteins at 70.4 and 163.9 kDa, while 3 proteins (20.4, 52.9, 68. kDa) were typical for E. faecium. No characteristic proteins were found for Leuconostoc isolates except for that at 31.8 kDa mainly related to the ecological origin since only isolates from brine showed this protein. However, Leuc. mesenteroides subsp. mesenteroides and Leuconostoc spp. were differentiated by additional proteins. Sequence analysis of the 16S rRNA gene of selected strains was in agreement with the phenotypic identification and allowed to identify all Leuconostoc spp. isolates as Leuc. mesenteroides. Cell-wall protein fingerprinting performed by microfluidic technology completed the phenotypic characterization of olive-related lactic acid bacteria allowing their strain typing.