16S rRNA gene sequencing and MALDI TOF mass spectroscopy identification of Leuconostoc mesenteroides isolated from Algerian raw camel milk.

BACKGROUND: Eighty-three strains of Leuconostoc mesenteroides were isolated from Algerian raw camel milk. Based on morphological, biochemical, and physiological characters tests, strains were identified as Ln. mesenteroides subsp. mesenteroides. Seven strains had a remarkable antagonistic and probiotic characterization. The present study aims at identifying these strains by means of 16 s rRNA gene sequencing and Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), extending phenotypic and genotypic studies done previously. RESULTS: The phyloproteomic dendrograms of the studied strains based on MALDI-TOF MS provided the same identification with more intraspecific information from the 16S rRNA gene sequencing based on phylogenetic analysis. The latter were in agreement with the previous biochemical/physiological identification, the seven isolated strains were Ln. mesenteroides subsp. mesenteroides. CONCLUSIONS: Remarkably, MALDI-TOF MS fingerprinting was found to be effective enough as 16S rRNA gene sequencing identification, allowing faster and more reliable analysis than biochemical/physiological methods.

Genomic and proteomic characterization of bacteriocin-producing Leuconostoc mesenteroides strains isolated from raw camel milk in two southwest Algerian arid zones.

Information on the microbiology of camel milk is very limited. In this work, the genetic characterization and proteomic identification of 13 putative producing bacteriocin Leuconostoc strains exhibiting antilisterial activity and isolated from camel milk were performed. DNA sequencing of the 13 selected strains revealed high homology among the 16S rRNA genes for all strains. In addition, 99% homology with Leuconostoc mesenteroides was observed when these sequences were analysed by the BLAST tool against other sequences from reference strains deposited in the Genbank. Furthermore, the isolates were characterized by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDITOF MS) which allowed for the identification of 2 mass peaks 6242 m/z and 5118 m/z that resulted to be specific to the species L. mesenteroides. Remarkably, the phyloproteomic tree provided more intraspecific information of L. mesenteroides than phylogenetic analysis. Accordingly, phyloproteomic analysis grouped L. mesenteroides strains into different subbranches, while all L. mesenteroides isolates were grouped in the same branch according to phylogenetic analysis. This study represents, to our knowledge, the first report on the use of MALDI-TOF MS on the identification of LAB isolated from camel milk.

Recent patents on bacteriocins: food and biomedical applications.

Most types of bacteria produce bacteriocins, which are proteinaceous extracellular compounds that can inhibit the growth of other undesirable microorganisms. Bacteriocins are receiving increasing attention, due to their many applications, ranging from their initial application in strategies for food preservation to more recent proposed uses in biomedical strategies aimed at fighting certain bacterial infections. Thus, while nisin has a long history of use as a safe additive in certain food products for the purpose of food preservation, certain bacteriocin-producing lactic acid bacteria, which are generally recognised as safe microorganisms, or their extracellular extracts are receiving increased attention as protective cultures or antimicrobial extracts in minimally processed food products. More recently, a number of these bacteriocinproducing cultures have been proposed for use in other applications, such as in probiotics, for the inhibition of biofilms in the food industry, or even as coadjuvants of combined therapeutical strategies along with other antimicrobial agents in biomedical applications. This review aims to provide a brief overview of the most relevant recent patents in this field.

Technological aptitude and applications of Leuconostoc mesenteroides bioactive strains isolated from Algerian raw camel milk.

Two strains (B7 and Z8) of the Leuconostoc mesenteroides subspecies mesenteroides that were isolated from Algerian camel milk from an initial pool of 13 strains and demonstrated a high ability to inhibit the growth of Listeria spp. were selected and characterised at the phenotypic and genotypic levels. Probiotic profiling and inhibition spectra against food borne pathogens in mixed cultures were also investigated. The bacteriocin produced by L. mesenteroides strain B7 was identified as leucocin B by specific PCR. In vitro studies demonstrated that both Leuconostoc mesenteroides strains exhibited a marked probiotic profile, showing high survival at low pH (2-3 and 4) in the presence of 0.5%, 1%, and 2% of bile salts and at pH 3 in the presence of 3 mg/mL pepsin. Susceptibility testing against antimicrobial agents was also performed for both strains. When tested in a mixed culture with Listeria innocua, Listeria ivanovii, or Staphylococcus aureus, strain B7 reduced the numbers of these species by 1.87, 1.78, and 1.38 log units, respectively. Consequently, these two strains were found to possess good probiotic properties in vitro and a high capacity for Listeria spp. inhibition in mixed cultures. Therefore, these strains have a favourable technological aptitude and a potential application as novel probiotic starters.

Recognition of saccharides by the OpcA, OpaD, and OpaB outer membrane proteins from Neisseria meningitidis.

The adhesion of the pathogen Neisseria meningitidis to host cell surface proteoglycan, mediated by the integral outer membrane proteins OpcA and Opa, plays an important part in the processes of colonization and invasion by the bacterium. The precise specificities of the OpcA and Opa proteins are, however, unknown. Here we use a fluorescence-based binding assay to show that both proteins bind to mono- and disaccharides with high affinity. Binding of saccharides caused a quench in the intrinsic fluorescence emission of both proteins, and mutation of selected Tyr residues within the external loop regions caused a substantial decrease in fluorescence. We suggest that the intrinsic fluorescence arises from resonance energy transfer from Tyr to Trp residues in the beta-barrel portion of the structure. OpcA bound sialic acid with a Kd of 0.31 microM and was shown to be specific for pyranose saccharides. The binding specificities of two different Opa proteins were compared; unlike OpcA, neither protein bound to monosaccharides, but both bound to maltose, lactose, and sialic acid-containing oligosaccharides, with Kd values in the micromolar range. OpaB had a 10-fold higher affinity for sialic acid-containing ligands than OpaD as a result of the mutation Y165V, which was shown to restore this specificity to OpaD. Finally, the OpcA- and Opa-dependent adhesion of meningococci to epithelial cells was shown to be partially inhibited by exogenously added sialic acid and maltose. The results show that OpcA and the Opa proteins can be thought of as outer membrane lectins and that simple saccharides can modulate their recognition of complex proteoglycan receptors.