Lactobacillus species identification by amplified ribosomal 16S-23S rRNA restriction fragment length polymorphism analysis.

Lactic acid bacteria strains are commonly used for animal and human consumption due to their probiotic properties. One of the major genera used is Lactobacillus, a highly diverse genus comprised of several closely related species. The selection of new strains for probiotic use, especially strains of Lactobacillus, is the focus of several research groups. Accurate identification to species level is fundamental for research on new strains, as well as for safety assessment and quality assurance. The 16S-23S internal transcribed spacer (ITS-1) is a deeply homologous region among prokaryotes that is commonly used for identification to the species level because it is able to acquire and accumulate mutations without compromising general bacterial metabolism. In the present study, 16S-23S ITS regions of 45 Lactobacillus species (48 strains) were amplified and subjected to independent enzymatic digestions, using 12 restriction enzymes that recognise six-base sequences. Twenty-nine species showed unique restriction patterns, and could therefore be precisely identified solely by this assay (64%). This approach proved to be reproducible, allowing us to establish simplified restriction patterns for each evaluated species. The restriction patterns of each species were similar among homologous strains, and to a large extent reflected phylogenetic relationships based on 16S rRNA sequences, demonstrating the promising nature of this region for evolutionary studies.

In vitro assessment of functional properties of lactic acid bacteria isolated from faecal microbiota of healthy dogs for potential use as probiotics.

Lactic acid bacteria were isolated and identified in the faeces of Chinese Crested and Yorkshire terrier pups and their probiotic features were investigated in vitro. Thirty seven isolates were identified as Lactobacillus or Enterococcus. Out of these isolates, 31 were lactic acid bacteria (LAB) and belonged to the species Lactobacillus reuteri (16/37; 43.3%), Lactobacillus animalis (7/37; 18.9%), Lactobacillus acidophilus (3/37; 8.1%), Lactobacillus sanfranciscensis (2/37; 5.4%), Lactobacillus murinus (2/37; 5.4%), and Lactobacillus paraplantarum (1/37; 2.7%), while six other LAB isolates were Enterococcus spp. (6/37; 16.2%). Strains were tested for resistance to gastric acidity (pH 2.5 for 3 h) and bile salts (0.3% ox gall), cell surface hydrophobicity by microbial adhesion to solvents, antagonism against pathogenic bacteria (Staphylococcus aureus, Enterococcus faecalis, Bacillus cereus, Pseudomonas aeruginosa, Escherichia coli, Salmonella enterica serovar Typhimurium and Listeria monocytogenes), production of hydrogen peroxide, and antibiotic susceptibility. Thirty four strains were highly resistant to acidic conditions with slight (18 strains) to moderate (16 strains) growth inhibition by bile salts. Seven isolates had highly hydrophobic cellular surfaces and 28 strains exhibited strong antagonism against the bacterial pathogens tested, although 8 isolates tested against Leptospira interrogans had no effect on pathogen growth. All isolates produced low rates of hydrogen peroxide. Based on these results, two Lactobacillus strains showed promising probiotic-related features and merit investigation as probiotics for dogs.