Casitone-dependent transcriptional regulation of the prtP and prtM genes in the natural isolate Lactobacillus paracasei subsp. paracasei.

The prtP-prtM intergenic region of Lactobacillus paracasei subsp. paracasei BGHN 14 was cloned and sequenced. The nucleotide sequence of the prtP-prtM intergenic region in BGHN 14, containing divergently orientated P(prtP) and P(prtP) promoters, was shorter by 35 bp in comparison with that in lactococci. The nucleotide sequence involved in casitone-dependent transcriptional regulation of the lactococcal prt genes was not found in the BGHN14. The activity of P(prtM) in L. lactis NZ9000 was very low and insignificantly changed in the presence of casitone, whereas P(prtP) was completely inactive. When L. casei ATCC393(T) was used as host, both P(prtP) and P(prtM) were active and strongly regulated by casitone. The results strongly indicate that the mechanisms of the casitone-dependent regulation of the prt genes in BGHN14 and lactococci are different.

Gut-associated lymphoid tissue, gut microbes and susceptibility to experimental autoimmune encephalomyelitis.

Gut microbiota and gut-associated lymphoid tissue have been increasingly appreciated as important players in pathogenesis of various autoimmune diseases, including multiple sclerosis. Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis that can be induced with an injection of spinal cord homogenate emulsified in complete Freund's adjuvant in Dark Agouti (DA) rats, but not in Albino Oxford (AO) rats. In this study, mesenteric lymph nodes (MLN), Peyer's patches (PP) and gut microbiota were analysed in these two rat strains. There was higher proportion of CD4(+) T cells and regulatory T cells in non-immunised DA rats in comparison to AO rats. Also, DA rat MLN and PP cells were higher producers of pro-inflammatory cytokines interferon-γ and interleukin-17. Finally, microbial analyses showed that uncultivated species of Turicibacter and Atopostipes genus were exclusively present in AO rats, in faeces and intestinal tissue, respectively. Thus, it is clear that in comparison of an EAE-susceptible with an EAE-resistant strain of rats, various discrepancies at the level of gut associated lymphoid tissue, as well as at the level of gut microbiota can be observed. Future studies should determine if the differences have functional significance for EAE pathogenesis.

Molecular typing and antimicrobial resistance of Salmonella Enteritidis isolated from poultry, food, and humans in Serbia.

Molecular typing and resistotyping coupled with gyrA single nucleotide polymorphism (SNP) of 60 Salmonella Enteritidis (SE) isolates originated from poultry, food, and humans in Serbia is described. Molecular fingerprinting was performed by randomly amplified polymorphic DNA (RAPD) using four primers, and the diversity index (D) was 0.688. In combination with resistotyping and gyrA SNP, D increased to 0.828. A total of 23 genetic groups were obtained. When four RAPD primers were combined, epidemic isolates from a fast-food restaurant outbreak were clustered in a distinctive genetic group. Among 60 SE strains, three had multiple resistances to three or more antibiotics. Nine strains were resistant to nalidixic acid (NAL; a non-fluorinated quinolone). The mutations in quinolone resistance-determining region (QRDR) found in NAL-resistant strains were attributed to Asp(87) → Asn in six strains, Asp(87) → Gly in one strain, and Ser(83) → Phe in one strain. One NAL-resistant strain had no mutations in QRDR, suggesting another mechanism of resistance.

Variation in specificity of the PrtP extracellular proteinases in Lactococcus lactis and Lactobacillus paracasei subsp. paracasei.

Comparison of cell-wall-bound extracellular proteinases (CEPs) from Lactobacillus paracasei (LBP) ssp. paracasei natural isolates BGHN14, BGAR75 and BGAR76 with Lactococcus lactis (LCL) ssp. cremoris Wg2, in their action on alpha(S1)-, beta- and kappa-casein was done. The CEPs of LBP strains were able to degrade alpha(S1)- and beta-caseins and their caseinolytic specificity depended on the type of buffer used. These CEPs, compared with LCL Wg2, differ in four amino acid residues in small segments predicted to be involved in substrate binding. The most striking features of this comparison are the presence of Ala instead of Ser(329) and the presence of Thr instead of Asn(256) and Ala(299), in the subtilisin-like region of the CEP in LBP natural isolates. Additional conservative amino acid substitution Leu to Ile(364) was found.

Molecular and biochemical characterizations of human oral lactobacilli as putative probiotic candidates.

INTRODUCTION: The objective of this study was to characterize the lactobacilli from the human oral cavity as a potential source of probiotic strains. METHODS: Samples were collected from four different locations within the oral cavity: surface of healthy tooth, oral mucous membrane, surface of tooth decay and deep tooth decay. On the basis of morphological and biochemical properties eight categories were formed and 26 isolates were selected for further characterization. The isolates were determined as Lactobacillus sp. using primers specific for 16S rDNA. Sequencing of 16S rDNA genes and repetitive sequence-based polymerase chain reactions were used for determination to species and subspecies levels. RESULTS: Predominant species were Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus salivarius and Lactobacillus paracasei subsp. paracasei, while Lactobacillus acidophilus, Lactobacillus cellobiosus, Lactobacillus delbrueckii subsp. lactis and Lactobacillus gasseri were also present. The isolates Lactobacillus salivarius BGHO1, Lactobacillus fermentum BGHO36 and BGHO64, Lactobacillus gasseri BGHO89 and Lactobacillus delbrueckii subsp. lactis BGHO99 exhibited antagonistic action on the growth of Staphylococcus aureus, Enterococcus faecalis, Micrococcus flavus, Salmonella enteritidis, Streptococcus pneumoniae and Streptococcus mutans, but not on growth of Candida albicans. Moreover, the isolates L. salivarius BGHO1 and L. gasseri BGHO89 were tolerant to low pH and high concentration of bile salts. CONCLUSION: Taken together, these findings imply that L. salivarius BGHO1 and L. gasseri BGHO89 might be subjects for additional investigation as potential probiotic strains.