Protective Effect of Lactobacillus diolivorans 1Z, Isolated From Brazilian Kefir, Against Salmonella enterica Serovar Typhimurium in Experimental Murine Models

Kefir is a beverage obtained by fermentation of milk or sugar solution by lactic acid bacteria and yeasts, and several health benefits have been attributed to its ingestion, part of them being attributed to Lactobacillus species. The objective of the present study was to evaluate, in vivo, the probiotic potential of Lactobacillus diolivorans 1Z, isolated from Brazilian kefir grains. Initially, conventional mice were orally treated daily or not during 10 days with a suspension of L. diolivorans 1Z, and then orally challenged with Salmonella enterica serovar Typhimurium. Treatment with L. diolivorans 1Z resulted in higher survival (70%) of animals after the challenge with the pathogen than for not treated mice (0%). When germ-free mice were monoassociated (GN-PS group) or not (GN-CS group) with L. diolivorans 1Z and challenged after 7 days with S. Typhimurium, Salmonella fecal counts were significantly lower (P < 0.05) for the GN-PS group when compared to the GN-CS group. Histopathological analysis revealed less damage to the ileum mucosa, as demonstrated by smallest perimeter of major lesions for mice of the GN-PS group in comparison to the group GN-CS (P < 0.05). These findings were accompanied by a lower expression of IFN-γ and TNF-α in the intestinal tissue of GN-PS mice. Additionally, translocation of S. Typhimurium to liver was significantly lower in GN-PS than in GN-CS mice (P < 0.05), and IgA levels in intestinal content and number of Kupffer cells in liver were higher. No difference was observed for hepatic cellularity between GN-PS and GN-CS groups (P > 0.05), but the pattern of inflammatory cells present in the liver was predominantly of polymorphonuclear in GN-CS group and of mononuclear in the GN-PS group, and a higher hepatic expression of IL-10 and TGF-β was observed in GN-PS group. Concluding, L. diolivorans 1Z showed to be a potential probiotic strain that protected mice from death after challenge with S. Typhimurium, apparently by immunological modulation

Análise filogenética do minhocu­çu Rhinodrilus alatus, Righi 1971 (glossoscolecidae: annelida) baseada em sequências dos genes de rDNA 5.8S, do espaço interno transcrito (its1) e da subunidade i da citocromo C oxidase mitocondrial.

O minhocuçu Rhinodrilus alatus Righi, 1971 é uma espécie endêmica do cerrado de Minas Gerais e tem sido explorada como isca para pesca por >70 anos. O objetivo do trabalho foi validar marcadores moleculares para estudos genético-populacionais, de filogenia e de filogeografia do minhocuçu. Os genes rRNA 5.8S, espaço interno transcrito (ITS1) e subunidade I da citocromo oxidase (COI) mitocondrial foram estudados. Foram amostrados indivíduos da espécie R. alatus (n = 53) em diferentes hábitats de Minas Gerais e da espécie R. motucu Righi, 1971 (n = 3), coletados em brejos de Goiás, no Brasil. A análise filogenética do gene do rRNA 5.8S mostrou que todas as seqüências de R. alatus, R. motucu e Eisenia fetida (Savigny, 1826) agruparam-se no mesmo clado (bootstrap = 98), sugerindo que, nestes organismos, o gene é conservado. Os clados formados a partir de seqüências de rRNA 5.8S de invertebrados disponíveis na base de dados são inconsistentes do ponto de vista evolutivo, sugerindo taxas evolutivas distintas entre diferentes espécies. Para a região do ITS1, foram obtidos 11 sítios polimórficos, gerando nove haplótipos. Os exemplares de R. motucu apresentaram o haplótipo mais freqüente dentre os R. alatus de Minas Gerais, não havendo evidências moleculares de que se tratem de espécies diferentes. Para o gene COI, de um total de 634 pb, obteve-se 185 sítios polimórficos, gerando árvores filogenéticas com topologia mais adequada, separando R. motucu de R. alatus

Detecção de genes toxigênicos, susceptibilidade antimicrobiana e antagonismo in vitro de Staphylococcus spp. isolados de queijos artesanais

Staphylococcus spp. isolated from samples of Minas cheese traditionally manufactured following artisan procedures were identified using molecular techniques and further analyzed using PCR and specific primers for the detection of classic enterotoxins (SEA, SEB, SEC, SED, and SEE) and toxic shock syndrome toxin-1 (TSST-1). Specific sea, sec, sed, and see genes were identified using multiplex PCR, whereas seb and tst genes were detected by uniplex PCR. In vitro antagonism with Lactobacillus spp. was evaluated to assess antimicrobial susceptibility. Classic enterotoxins and TSST-1 genes were not detected. The antimicrobials sulfonamide, penicillin, ceftzadime, and oxacillin showed higher resistance rates in the antibiogram (100%, 80%, 60%, and 40%, respectively), whereas other antimicrobials were effective in percentages above 70%. Lactobacillus spp. were able to inhibit Staphylococcus spp. in vitro. Thus, our results indicated that the isolated Staphylococcus spp. were sensitive to the most common antimicrobials tested and were inhibited by Lactobacillus spp.Cepas de Staphylococcus spp. molecularmente identificadas foram submetidas à Reação em Cadeia da Polimerase (PCR), utilizando-se iniciadores específicos para a detecção de genes codificadores de enterotoxinas clássicas (SEA, SEB, SEC, SED, SEE) e da Toxina-1 da Síndrome do Choque Tóxico (TSST-1). Foi realizada PCR-Multiplex para detecção dos genes sea, sec, sed e see. Para seb e tst, foram realizadas PCR-Uniplex. Além disso, foi analisado o perfil de susceptibilidade das cepas a antimicrobianos de diferentes classes e foi verificado antagonismo in vitro entre Lactobacillus spp. e as cepas estudadas. Genes codificadores de enteroxinas clássicas, assim como de TSST-1, não foram encontrados. Em relação ao antibiograma, Sulfonamida, Penicilina, Ceftazidima e Oxacilina apresentaram os maiores percentuais de resistência (100, 80, 60 e 40%, respectivamente). Os demais antimicrobianos foram eficientes em percentuais acima de 70%. Lactobacillus spp. foram capazes de inibir o desenvolvimento in vitro de Staphylococcus spp. Conclui-se que as cepas estudadas não possuem genes codificadores da produção de enterotoxinas clássicas e TSST-1, são sensíveis à maioria dos antimicrobianos e são inibidos por bactérias do gênero Lactobacillus

Selection of lactic acid bacteria with probiotic potential isolated from the fermentation process of “Cupuaçu” (Theobroma grandiflorum)

In the present study, nine lactic acid bacteria isolated from the fermentation process of “cupuaçu” (Theobroma grandiflorum) were selected for probiotic use. In vitro (resistance to gastrointestinal environment, in vitro antagonism and co-aggregation with pathogens) and in vivo (intestinal colonization and ex vivo antagonism in germ-free mice, cumulative mortality, translocation to liver and spleen, histopathological examination of liver and ileum and mRNA cytokine gene expression during an experimental infection with S. Typhimurium) assays were used. Among the nine Lactobacillus strains isolated from the “cupuaçu” fermentation, L. plantarum 81 and L. plantarum 90 were selected as potential probiotics based on better results obtained in in vitro evaluations (production of diffusible inhibitory compounds and co-aggregation) as well as in vivo experiments (resistance to gastrointestinal environment, ex vivo antagonism, higher survival after enteropathogen challenge, lower hepatic translocation of enteropathogen, lower histopathological lesions in ileum and liver and anti-inflammatory pattern of immunological response). Concluding, L. plantarum 81 and L. plantarum 90 showed in vitro and in vivo capacities for probiotic use through different mechanisms of protection and its origin would allow an easier adaptation in an alimentary matrix for its administration.Fil: Ornellas, Roberta Maria Santos. Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas; BrasilFil: Santos, Tiza Teles. Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas; BrasilFil: Arcucio, Leonardo Borges. Universidade Federal de Minas Gerais; BrasilFil: Sandes, Sávio Henrique Cicco. Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas; BrasilFil: Oliveira, Mayara Messias. Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas; BrasilFil: Dias, Cristiano Villela. Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas; BrasilFil: de Carvalho Silva, Samuel. Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas; BrasilFil: Uetanabaro, Ana Paula Trovatti. Universidade Estadual de Santa Cruz; BrasilFil: Vinderola, Celso Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Lactología Industrial. Universidad Nacional del Litoral. Facultad de Ingeniería Química. Instituto de Lactología Industrial; ArgentinaFil: Nicoli, Jacques Robert. Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas; Brasi

Probiotic Propionibacterium freudenreichii requires SlpB protein to mitigate mucositis induced by chemotherapy

Propionibacterium freudenreichii CIRM-BIA 129 (P. freudenreichii wild type, WT) is a probiotic bacterium, which exerts immunomodulatory effects. This strain possesses extractable surface proteins, including SlpB, which are involved in antiinflammatory effect and in adhesion to epithelial cells. We decided to investigate the impact of slpB gene mutation on immunomodulation in vitro and in vivo. In an in vitro assay, P. freudenreichii WT reduced expression of IL-8 (p<0.0001) and TNF-α (p<0.0001) cytokines in LPS-stimulated HT-29 cells. P. freudenreichii ΔslpB, lacking the SlpB protein, failed to do so. Subsequently, both strains were investigated in vivo in a 5-FU-induced mucositis mice model. Mucositis is a common side effect of cytotoxic chemotherapy with 5-FU, characterized by mucosal injury, inflammation, diarrhea, and weight loss. The WT strain prevented weight loss, reduced inflammation and consequently histopathological scores. Furthermore, it regulated key markers, including Claudin-1 (cld1, p<0.0005) and IL-17a (Il17a, p<0.0001) genes, as well as IL-12 (p<0.0001) and IL-1β (p<0.0429) cytokines levels. Mutant strain displayed opposite regulatory effect on cld1 expression and on IL-12 levels. This work emphasizes the importance of SlpB in P. freudenreichii ability to reduce mucositis inflammation. It opens perspectives for the development of probiotic products to decrease side effects of chemotherapy using GRAS bacteria with immunomodulatory surface protein properties