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ABSTRACT:The antimicrobial properties of three potential probiotic strains of lactobacilli isolated from a lamb (Lactobacillus murinus C, Lactobacillus mucosae D and Lactobacillus reuteri E) were studied using the streak line method and the agar well diffusion assay. The probiotic lactobacilli strains Lactobacillus rhamnosus ATCC 53103, Lactobacillus reuteri ATCC 55730, Lactobacillus reuteri ATCC 55845 and Lactobacillus plantarum DSM 9843 were used for comparison. Using the streak line method the inhibitory activity of lactobacilli products towards ten Gram-positive and Gram-negative potential pathogenic bacteria under different cultivation conditions (anaeorobic or microaerobic preincubation of lactobacilli for 24 h or 48 h) was tested. The strongest inhibitory activity was demonstrated by the Lactobacillus reuteri E strain. The most sensitive strains to the antimicrobial activity of lactobacilli were Yersinia enterocolitica clinical isolate (19.9 ± 6.8 mm) and Listeria monocytogenes ATCC 51774 (17.7 ± 6.0 mm) after microaerobic and anaerobic preincubation, respectively. Generally, microaerobic conditions and longer preincubation of lactobacilli resulted in stronger inhibition of target bacteria. The inhibitory activity of lactobacilli towards selected lactobacilli strains was also tested. Only low inhibition of growth was observed. In the agar well diffusion assay the inhibitory effect of natural and modified lactobacilli culture cell-free supernatants, obtained from MRS broth cultures, on Staphylococcus aureus ATCC 6538 growth was determined. Supernatants were modified by heat (10 min/60 °C; 60 min/100 °C) and protease treatment and neutralization of pH. Neutralization elicited the most significant impact on the activity of supernatants and resulted in total loss of activity. After all other modifications supernatants retained some residual activity. The highest inhibitory activity was observed for the cell-free supernatant produced by Lactobacillus mucosae D.

The lactic acid bacterium E isolated from the stomach mucus of breast-fed lamb was identified by sequencing of 16S rDNA fragment and species-specific PCR as Lactobacillus reuteri. Its potential antimicrobial activity and ability to modulate immune system in vitro and in vivo was determined.

Five new strains of lactobacilli isolated from goatling's stomach were identified by molecular-biological approaches. Profiles of fermentable saccharides, Gram staining, and cell morphology were also determined. They were identified as Lactobacillus reuteri (strains KO4b, KO4m, KO5) and as Lactobacillus plantarum (strains KG1z, KG4). In DNA samples of all newly isolated L. reuteri strains as well as in L. reuteri E (Lreu E; originated from lamb), the part of gldC gene, coding large subunit of glycerol dehydratase, that is necessary for 3-hydroxypropionaldehyde (3-HPA; reuterin) production, was amplified using two designed primer sets. However, the 3-HPA production was revealed only in the strain Lreu E. It produced five- or ten-fold lower amount of 3-HPA in comparison with probiotic L. reuteri ATCC 55730 in aerobic or anaerobic conditions, respectively. Moreover, Lreu E completely lost its production ability after ca. five passages in MRS medium. The co-incubation of Lreu E, but not other L. reuteri isolates, with Escherichia coli re-induced 3-HPA production. In the case of L. reuteri ATCC 55730, the 3-HPA production increased more than four times after co-incubation with E. coli.

In the process of selecting a new probiotic candidate, several bacteria were isolated from the stomach mucosa of a lamb. Among them, three lactobacilli strains were identified and partially characterised. The strain, Lactobacillus mucosae D, showed several characteristics appropriate to the probiotics. In this study, we have focused on the further characterisation of L. mucosae D and testing of its ability to modulate metabolic and immunomodulatory activities of human mononuclear cells in vitro. L. mucosae D is resistant to antibiotics, like penicillin G, oxacillin, vancomycin and chemotherapeutics ofloxacin and ciprofloxacin. In in vitro conditions, L. mucosae D caused a significant increase in phagocytic activity and index (relative activities 1.05 and 1.44, respectively) of human monocytes. It decreased bactericidal activities of monocytes against Escherichia coli (relative activity 0.73) and Staphylococcus aureus (relative activity 0.36), whereas, candidacidal activity was enhanced (relative activity 1.15). Metabolic activities, lysozyme and peroxidase activity, of mononuclear cells were not changed or increased, respectively. L. mucosae D displayed the ability to enhance production of pro-inflammatory cytokine, IL-1β, in monocytes in vitro (relative activity 2.60). Therefore, we state that lamb isolate, L. mucosae D, has the required attributes for being a potential probiotic candidate.

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