Enteropathogenic Escherichia coli modulates the virulence and pathogenicity of Entamoeba dispar.

Amoebiasis is a disease caused by Entamoeba histolytica, affecting the large intestine of humans and occasionally leading to extra-intestinal lesions. Entamoeba dispar is another amoeba species considered commensal, although it has been identified in patients presenting with dysenteric and nondysenteric colitis, as well as amoebic liver abscess. Amoebic virulence factors are essential for the invasion and development of lesions. There is evidence showing that the association of enterobacteria with trophozoites contributes to increased gene expression of amoebic virulence factors. Enteropathogenic Escherichia coli is an important bacterium causing diarrhea, with high incidence rates in the world population, allowing it to interact with Entamoeba sp. in the same host. In this context, this study aims to evaluate the influence of enteropathogenic Escherichia coli on ACFN and ADO Entamoeba dispar strains by quantifying the gene expression of virulence factors, including galactose/N-acetyl-D-galactosamine-binding lectin, cysteine proteinase 2, and amoebapores A and C. Additionally, the study assesses the progression and morphological aspect of amoebic liver abscess and the profile of inflammatory cells. Our results demonstrated that the interaction between EPEC and ACFN Entamoeba dispar strains was able to increase the gene expression of virulence factors, as well as the lesion area and the activity of the inflammatory infiltrate. However, the association with the ADO strain did not influence the gene expression of virulence factors. Together, our findings indicate that the interaction between EPEC, ACFN, and ADO Entamoeba dispar strains resulted in differences in vitro and in vivo gene expression of Gal/GalNAc-binding lectin and CP2, in enzymatic activities of MPO, NAG, and EPO, and consequently, in the ability to cause lesions.

Comparative metatranscriptome analysis of Brazilian milk and water kefir beverages.

The present study compared bacterial and fungal diversity of kefir beverages produced using milk (MK) or sugared water (WK) as propagation matrices and grains from the cities of Curitiba (CU) or Salvador (SA), Brazil, by sequencing the complete set of RNA transcripts produced in four products. In Brazil, milk and sugared water are used as matrices to propagate kefir grains. In all beverages, the bacterial community was composed of Lactobacillaceae and Acetobacteraceae. Saccharomycetaceae was the yeast family more abundant in WK, and Dipodascaceae and Pichiaceae in MK. Regarding KEGG mapping of functional orthologs, the four kefir samples shared 70% of KO entries of yeast genes but only 36% of bacterial genes. Concerning main metabolic processes, the relative abundance of transcripts associated with metabolism (energy metabolism) and environmental information processing (membrane transport) had the highest water/milk kefir ratio observed in Firmicutes. In contrast, transcripts associated with genetic information processing (protein translation, folding, sorting, and degradation) oppositely had the lowest water/milk ratios. Concluding, milk and water kefir have quite different communities of microorganisms. Still, the main mapped functional processes are similar, with only quantitative variation in membrane transport and energy acquisition in the water kefir and protein synthesis and turnover in the milk kefir.

Cell-free supernatant of probiotic bacteria exerted antibiofilm and antibacterial activities against Pseudomonas aeruginosa: A novel biotic therapy.

Aim: This study aims to verify the antibacterial and antibiofilm action of cell-free spent medium (CFSM) from four lactic acid bacteria with potential probiotic characteristics (Lactiplantibacillus plantarum, Lactobacillus acidophilus, Lactobacillus johnsonii, and Lactobacillus delbrueckii) against two Pseudomonas aeruginosa strains. Main methods: The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the CFSM, antibacterial activity by analysing the formation of inhibition zones, and inhibition of planktonic cultures were determined. Whether an increase in the concentration of CFSM influenced the growth of pathogenic strains and the anti-adhesive activity of the CFSM in biofilm formation (crystal violet and MTT assays) were determined, which were all corroborated by using scanning electron microscopy. Key findings: The relationship between the MIC and MBC values showed a bactericidal or bacteriostatic effect for all the cell-free spent media (CFSMs) tested for P. aeruginosa 9027™ and 27853™ strains. The CFSM supplemental doses of 18 or 22%, 20 or 22%, 46 or 48%, and 50 or 54% of L. acidophilus, L. delbrueckii, L. plantarum, and L. johnsonii, respectively, could completely inhibit the growth of both pathogen strains. The antibiofilm activity of the CFSM in three biofilm conditions (pre-coated, co-incubated, and preformed) demonstrated values ranging between 40% and 80% for biofilm inhibition, and similar results were observed for cell viability. Significance: This work provides strong evidence that the postbiotic derived from different Lactobacilli could be practical as an adjuvant therapy for reducing the use of antibiotics, being a good candidate to overcome the growing challenge of hospital infections due to this pathogen.

Changes in bovine milk bacterial microbiome from healthy and subclinical mastitis affected animals of the Girolando, Gyr, Guzera, and Holstein breeds / Cambios en el microbioma bacteriano de la leche bovina de animales sanos y afectados por mastitis subclínica de las razas Girolando, Gyr, Guzera y Holstein

Raw milk samples were collected from 200 dairy cows belonging to Girolando 1/2, Gyr, Guzera, and Holstein breeds, and the bacterial diversity was explored using 16S rRNA amplicon sequencing. SCC analysis showed that 69 animals were classified as affected with subclinical mastitis. The milk bacterial microbiome was dominated by Firmicutes, Proteobacteria, and Actinobacteria, with an increase of Firmicutes in animals with subclinical mastitis and Proteobacteria in healthy animals. At the family and genus level, the milk bacterial microbiome was dominated by Staphylococcus, Acinetobacter, Pseudomonas, members of the family Enterobacteriaceae, Lactococcus, Aerococcus, members of the family Rhizobiaceae, Anaerobacillus, Streptococcus, members of the family Intrasporangiaceae, members of the family Planococcaceae, Corynebacterium, Nocardioides, and Chryseobacterium. Significant differences in alpha and beta diversity analysis suggest an effect of udder health status and breed on the composition of raw bovine milk microbiota. LEfSe analysis showed 45 and 51 discriminative taxonomic biomarkers associated with udder health status and with one of the four breeds respectively, suggesting an effect of subclinical mastitis and breed on the microbiota of milk in cattle.(AU)

Changes in bovine milk bacterial microbiome from healthy and subclinical mastitis affected animals of the Girolando, Gyr, Guzera, and Holstein breeds.

Raw milk samples were collected from 200 dairy cows belonging to Girolando 1/2, Gyr, Guzera, and Holstein breeds, and the bacterial diversity was explored using 16S rRNA amplicon sequencing. SCC analysis showed that 69 animals were classified as affected with subclinical mastitis. The milk bacterial microbiome was dominated by Firmicutes, Proteobacteria, and Actinobacteria, with an increase of Firmicutes in animals with subclinical mastitis and Proteobacteria in healthy animals. At the family and genus level, the milk bacterial microbiome was dominated by Staphylococcus, Acinetobacter, Pseudomonas, members of the family Enterobacteriaceae, Lactococcus, Aerococcus, members of the family Rhizobiaceae, Anaerobacillus, Streptococcus, members of the family Intrasporangiaceae, members of the family Planococcaceae, Corynebacterium, Nocardioides, and Chryseobacterium. Significant differences in alpha and beta diversity analysis suggest an effect of udder health status and breed on the composition of raw bovine milk microbiota. LEfSe analysis showed 45 and 51 discriminative taxonomic biomarkers associated with udder health status and with one of the four breeds respectively, suggesting an effect of subclinical mastitis and breed on the microbiota of milk in cattle.

Prospecting of potentially probiotic lactic acid bacteria from bovine mammary ecosystem: imminent partners from bacteriotherapy against bovine mastitis / Prospección de bacterias ácido lácticas potencialmente probióticas del ecosistema mamario bovino: socios inminentes de la bacterioterapia contra la mastitis bovina

Mastitis is one of the most important causes of loss of cattle production, burdening producers due to the increased cost of milk production and decreased herd productivity. The development of alternative methods for the treatment and prevention of mastitis other than traditional chemical antibiotic therapy needs to be implemented to meet international pressures to reduce the use of these drugs and promote the elimination of multiresistant microbial strains from the environment. Treatment with probiotic bacteria or yeast strains offers a possible strategy for the control of mastitis. The objective of this work was to isolate, identify, and characterize lactic bacteria from milk and the intramammary duct of Gyr, Guzerat, Girolando 1/2, and Holstein cattle breeds from Brazil. Samples of 115 cows were taken, a total of 192 bacteria isolates belonging to 30 species were obtained, and 81 were selected to evaluate their probiotic potential in in vitro characterization tests. In general, bacteria isolated from the mammary gland have low autoaggregation, cell surface hydrophobicity, and co-aggregation with mastitis etiological bacteria Staphylococcus aureus and Escherichia coli. Also, they have biofilm assembly capacity, inability to produce exopolysaccharides, high production of H2O2, and strong antagonism against mastitis pathogens. Ten lactic bacteria isolates were used in co-culture with human MDA-MB-231 breast epithelial cells to assess their adhesion capacity and impairment of the S. aureus invasion. Our results, therefore, contribute to the future production of new prevention and treatment tools for bovine mastitis.(AU)

Prospecting of potentially probiotic lactic acid bacteria from bovine mammary ecosystem: imminent partners from bacteriotherapy against bovine mastitis.

Mastitis is one of the most important causes of loss of cattle production, burdening producers due to the increased cost of milk production and decreased herd productivity. The development of alternative methods for the treatment and prevention of mastitis other than traditional chemical antibiotic therapy needs to be implemented to meet international pressures to reduce the use of these drugs and promote the elimination of multiresistant microbial strains from the environment. Treatment with probiotic bacteria or yeast strains offers a possible strategy for the control of mastitis. The objective of this work was to isolate, identify, and characterize lactic bacteria from milk and the intramammary duct of Gyr, Guzerat, Girolando 1/2, and Holstein cattle breeds from Brazil. Samples of 115 cows were taken, a total of 192 bacteria isolates belonging to 30 species were obtained, and 81 were selected to evaluate their probiotic potential in in vitro characterization tests. In general, bacteria isolated from the mammary gland have low autoaggregation, cell surface hydrophobicity, and co-aggregation with mastitis etiological bacteria Staphylococcus aureus and Escherichia coli. Also, they have biofilm assembly capacity, inability to produce exopolysaccharides, high production of H2O2, and strong antagonism against mastitis pathogens. Ten lactic bacteria isolates were used in co-culture with human MDA-MB-231 breast epithelial cells to assess their adhesion capacity and impairment of the S. aureus invasion. Our results, therefore, contribute to the future production of new prevention and treatment tools for bovine mastitis.

Co-infection by Salmonella enterica subsp. Enterica serovar typhimurium and Entamoeba dispar pathogenic strains enhances colitis and the expression of amoebic virulence factors.

Amebiasis is the most severe protozoan infection affecting the human intestine, and the second leading cause of death among parasitic diseases. The mechanisms of amoebic virulence factors acquisition are poorly understood, and there are few studies showing the interaction between Entamoeba dispar and bacteria. Salmonella enterica subsp. enterica serovar typhimurium is also a common cause of gastroenteritis in humans. Considering the high rates of amebiasis and salmonellosis, it is possible that these diseases may co-exist in the human intestine, leading to co-infection. Due to the scarcity of studies showing the influence of enteropathogenic bacteria on amoebic virulence, our research group proposed to evaluate the impact of S. typhimurium on E. dispar trophozoites. We assessed whether co-infection of S. typhimurium and E. dispar can change the progression of amoebic colitis, and the inflammatory response profile in the caecum mucosa, using a co-infection experimental model in rats. In vitro assays was used to investigate whether S. typhimurium induces changes in amoebic virulence phenotype. In the present work, we found that S. typhimurium co-infection exacerbates amoebic colitis and intestinal inflammation. The in vitro association of S. typhimurium and E. dispar trophozoites contributed to increase the expression of amoebic virulence factors. Also, we demonstrated, for the first time, the cysteine proteinase 5 expression in E. dispar MCR, VEJ and ADO strains, isolated in Brazil. Together, our results show that S. typhimurium and E. dispar co-infection worsens amoebic colitis, possibly by increasing the expression of amoebic virulence factors.

Weissella paramesenteroides WpK4 plays an immunobiotic role in gut-brain axis, reducing gut permeability, anxiety-like and depressive-like behaviors in murine models of colitis and chronic stress.

The relationship between inflammatory bowel disease (IBD) and mood disorders is complex and involves overlapping metabolic pathways, which may determine comorbidity. Several studies have been shown that this comorbidity could worsen IBD clinical course. The treatment of ulcerative colitis is complex, and involves traditional therapy to promote the function of epithelial barrier, reducing exacerbated inflammatory responses. Recently, it has been shown that some probiotic strains could modulate gut-brain axis, reducing depressive and anxiety scores in humans, including IBD patients. Accordingly, this study aimed to evaluate the role of Weissella paramesenteroides WpK4 in murine models of ulcerative colitis and chronic stress. It was observed that bacterium ingestion improved health of colitis mice, reducing intestinal permeability, besides improving colon histopathological appearance. In stressed mice, bacterial consumption was associated with a reduced anxiety-like and depressive-like behaviors. In both assays, the beneficial role of W. paramesenteroides WpK4 was related to its immunomodulatory feature. It is possible to state that W. paramesenteroides WpK4 exerted their beneficial roles in gut-brain axis through their immunomodulatory effects with consequences in several metabolic pathways related to intestinal permeability and hippocampal physiology.

"Physicochemical, immunomodulatory and safety aspects of milks fermented with Lactobacillus paracasei isolated from kefir".

The use of Lactobacillus paracasei strains isolated from kefir grains as starters for the development of functional dairy products was evaluated. The physicochemical and immunomodulatory properties of milks fermented with L. paracasei CIDCA8339, CIDCA83123 and CIDCA83124 were analyzed. The three strains produced bioactive metabolites during fermentation, since the fermented milk supernatants were able to downregulate >75% of the induced innate immune response in vitro. Although all strains presented absence of hemolytic activity and susceptibility to antibiotics, L. paracasei CIDCA8339 presented more attractive probiotic and technological properties. Mice consuming the fermented milk with L. paracasei CIDCA 8339 did not present significant modifications in sIgA levels or TNF-α, TGF-ß and IL-10 mRNA expression in ileum. Additionally, a decrease of INF-γ level in ileum and no microbiological translocation to liver and spleen was observed. These results demonstrate that L. paracasei CIDCA8339 represents a safe promising potential probiotic strain for the development of functional foods.

Low-intensity shock wave therapy for erectile dysfunction and the influence of disease duration.

OBJECTIVE: Low-intensity shock-wave treatment (LiSWT) is a therapy for erectile dysfunction (ED) with good results reported in the literature. The aim of this study was to evaluate the results of LiSWT on patients treated for ED and the influence of ED duration in treatment outcomes. MATERIAL AND METHODS: We performed an open-label single-arm prospective study of patients treated with LiSWT for ED. Patients were assessed with the IIEF-5 at baseline and at six weeks and three months after LiSWT, and with penile dynamic Doppler ultrasound before treatment and six weeks after. Patients were divided into two groups accordingly to ED evolution time: ≤ 24 months and > 24 months. RESULTS: Twenty-five patients were enrolled, 13 had ED ≤ 24 months and 12 > 24 months. Median baseline IIEF-5 was 14, at 6 weeks post LiSWT was 16 (p < 0.001) and at 3 months post LiSWT was 18 (p < 0.001). Mean baseline peak systolic velocity (PSV) was 29.3 ± 13.0 cm/s, after LiSWT was 35.9 ± 15.2 cm/s (p 0.001). Mean baseline end-diastolic velocity (EDV) was 2.6 ± 4.8 cm/s and after LiSWT was 1.3 ± 4.3 cm/s (p 0.015). No statistical significative difference was identified between the two groups. CONCLUSIONS: LiSWT is a safe, harmless and repeatable treatment tool for ED with good outcomes reported. Our results suggest that length of disease duration doesn´t negatively influences treatment results.

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 anti-inflammatory 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-1ß (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.

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-ß 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.

Selection of starter cultures for the production of sour cassava starch in a pilot-scale fermentation process

ABSTRACT Sour cassava starch (Polvilho azedo) is obtained from a spontaneous fermentation conducted by microorganisms from raw materials and fermentation tanks. This product is traditionally used in the baking industry for the manufacture of biscuits and Brazilian cheese breads. However, the end of fermentation is evaluated empirically, and the process occurs without standardization, which results in products of inconsistent quality. Predominant microbiota from a cassava flour manufacturer was isolated in order to select starter cultures for the production of sour cassava starch in a pilot-scale fermentation process. Lactic acid bacteria and yeasts were isolated, enumerated and grouped by Restriction Fragment Length Polymorphism, and PCR fingerprinting, respectively. One isolate of each molecular profile was identified by sequencing of the rRNA gene. LAB were prevalent throughout the entire process. Lactobacillus brevis (21.5%), which produced the highest values of acidity, and Lactobacillus plantarum (13.9%) were among the most frequent species. Pichia scutulata (52.2%) was the prevalent yeast and showed amylolytic activity. The aforementioned species were tested as single and mixed starter cultures in a pilot-scale fermentation process for 28 days. L. plantarum exhibited better performance as a starter culture, which suggests its potential for the production of sour cassava starch.

Microencapsulation of Lactic Acid Bacteria Improves the Gastrointestinal Delivery and in situ Expression of Recombinant Fluorescent Protein.

The microencapsulation process of bacteria has been used for many years, mainly in the food industry and, among the different matrixes used, sodium alginate stands out. This matrix forms a protective wall around the encapsulated bacterial culture, increasing its viability and protecting against environmental adversities, such as low pH, for example. The aim of the present study was to evaluate both in vitro and in vivo, the capacity of the encapsulation process to maintain viable lactic acid bacteria (LAB) strains for a longer period of time and to verify if they are able to reach further regions of mouse intestine. For this purpose, a recombinant strain of LAB (L. lactis ssp. cremoris MG1363) carrying the pExu vector encoding the fluorescence protein mCherry [L. lactis MG1363 (pExu:mCherry)] was constructed. The pExu was designed by our group and acts as a vector for DNA vaccines, enabling the host cell to produce the protein of interest. The functionality of the pExu:mCherry vector, was demonstrated in vitro by fluorescence microscopy and flow cytometry after transfection of eukaryotic cells. After this confirmation, the recombinant strain was submitted to encapsulation protocol with sodium alginate (1%). Non-encapsulated, as well as encapsulated strains were orally administered to C57BL/6 mice and the expression of mCherry protein was evaluated at different times (0-168 h) in different bowel portions. Confocal microscopy showed that the expression of mCherry was higher in animals who received the encapsulated strain in all portions of intestine analyzed. These results were confirmed by qRT-PCR assay. Therefore, this is the first study comparing encapsulated and non-encapsulated L. lactis bacteria for mucosal DNA delivery applications. Our results showed that the microencapsulation process is an effective method to improve DNA delivery, ensuring a greater number of viable bacteria are able to reach different sections of the bowel.

Coagulase-Negative Staphylococci Isolated from Human Bloodstream Infections Showed Multidrug Resistance Profile.

Coagulase-negative staphylococci (CNS) are important pathogens causing nosocomial infections worldwide with increasing resistance to antimicrobials. The aim of this study was to characterize resistance aspects of CNS isolated from patients with bloodstream infections acquired in hospitals in Belo Horizonte, MG, Brazil. Staphylococcus strains were characterized using repetitive sequence-based polymerase chain reaction (rep-PCR) fingerprinting with (GTG)5 primer. Phenotypic resistance was analyzed using AST-P5085 card (bioMérieuxVitek®). PCR was used to detect mecA, vanA, blaZ, ermA/B/C, aac-aphD, and SCC-mec. For statistical analyses, we used hierarchical cluster, chi-square test (χ2), and correspondence. Several clusters were formed within the same species using (GTG)5 primer, and strains showed resistance to the following antimicrobials: benzylpenicillin (100%); oxacillin (93.1%); gentamicin (36.3%); ciprofloxacin (63.7%); moxifloxacin (32.7%); norfloxacin (81.0%); erythromycin (86.2%); clindamycin (75.8%); linezolid, teicoplanin and vancomycin (1.7%); tigecycline (0%); fusidic acid (10.35%); rifampicin (13.7%); and trimethoprim/sulfamethoxazole (46.5%). Regarding genotypic analyses, 40%, 0%, 78%, 42%, 100%, 24%, and 30% were positive for mecA, vanA, blaZ, ermA, ermB, ermC, and aac-aphD, respectively. Regarding staphylococcal cassette mec (SCCmec) type, 3.4% presented type I; 5.0% type II; 27.1% type III; 20.3% type IIIA; and 32.2% type IIIB. Six clusters were formed and frequency distributions of resistant strains to oxacillin, gentamicin, ciprofloxacin, moxifloxacin, norfloxacin, erythromycin, clindamycin, linezolid, teicoplanin, vancomycin, fusidic acid, rifampicin, and trimethoprim/sulfamethoxazole, and mecA, blaZ, ermC, aac-aphD, and SCCmec type differed (p < 0.001). In conclusion, the strains investigated in this study were multidrug resistant and carried multiple antibiotic resistance genes.

Porcine stomachs with and without gastric ulcer differ in Lactobacillus load and strain characteristics.

Although Lactobacillus species are recognized as normal inhabitants of porcine gastric mucosa, the association of these bacteria with health status or gastric ulcer disease has never been considered. We investigated the bacterial load of Lactobacillus isolated from the antrum, corpus, and pars esophagea of stomachs with (n = 13) and without (n = 10) ulcer of the pars esophagea of slaughtered pigs. We also evaluated in vitro antagonistic properties against typical pathogens of strains isolated from stomachs without ulcer. To quantify Lactobacillus, gastric mucosa samples obtained with 5 mm biopsy punches were smeared on MRS agar and colonies were counted after 48 h of incubation under anaerobic conditions. The score of Lactobacillus was significantly greater in the antrum and corpus of stomachs without ulcer (P < 0.001 for both) when compared with stomachs with ulcer. Fingerprint profiles, obtained by repetitive sequence-based PCR using (GTG)5 primers, showed that the isolates were highly diverse. The reduction of Lactobacillus load in porcine stomachs may be a contributing factor for gastric ulcer. Strains isolated from healthy stomachs, which showed a wide spectrum of antagonistic activity against pathogens, may be viewed as an untapped source of bacteria with potential beneficial properties that deserve to be further investigated.

Selection of starter cultures for the production of sour cassava starch in a pilot-scale fermentation process.

Sour cassava starch (Polvilho azedo) is obtained from a spontaneous fermentation conducted by microorganisms from raw materials and fermentation tanks. This product is traditionally used in the baking industry for the manufacture of biscuits and Brazilian cheese breads. However, the end of fermentation is evaluated empirically, and the process occurs without standardization, which results in products of inconsistent quality. Predominant microbiota from a cassava flour manufacturer was isolated in order to select starter cultures for the production of sour cassava starch in a pilot-scale fermentation process. Lactic acid bacteria and yeasts were isolated, enumerated and grouped by Restriction Fragment Length Polymorphism, and PCR fingerprinting, respectively. One isolate of each molecular profile was identified by sequencing of the rRNA gene. LAB were prevalent throughout the entire process. Lactobacillus brevis (21.5%), which produced the highest values of acidity, and Lactobacillus plantarum (13.9%) were among the most frequent species. Pichia scutulata (52.2%) was the prevalent yeast and showed amylolytic activity. The aforementioned species were tested as single and mixed starter cultures in a pilot-scale fermentation process for 28 days. L. plantarum exhibited better performance as a starter culture, which suggests its potential for the production of sour cassava starch.

Biological activity of the non-microbial fraction of kefir: antagonism against intestinal pathogens.

Kefir is a fermented milk obtained by the activity of kefir grains which are composed of lactic and acetic acid bacteria, and yeasts. Many beneficial health effects have been associated with kefir consumption such as stimulation of the immune system and inhibition of pathogenic microorganisms. The biological activity of kefir may be attributed to the presence of a complex microbiota as well as the microbial metabolites that are released during fermentation. The aim of this work was to characterise the non-microbial fraction of kefir and to study its antagonism against Escherichia coli, Salmonella spp. and Bacillus cereus. During milk fermentation there was a production of organic acids, mainly lactic and acetic acid, with a consequent decrease in pH and lactose content. The non-microbial fraction of kefir added to nutrient broth at concentrations above 75% v/v induced a complete inhibition of pathogenic growth that could be ascribed to the presence of un-dissociated lactic acid. In vitro assays using an intestinal epithelial cell model indicated that pre-incubation of cells with the non-microbial fraction of kefir did not modify the association/invasion of Salmonella whereas pre-incubation of Salmonella with this fraction under conditions that did not affect their viability significantly decreased the pathogen's ability to invade epithelial cells. Lactate exerted a protective effect against Salmonella in a mouse model, demonstrating the relevance of metabolites present in the non-microbial fraction of kefir produced during milk fermentation.

Selection of new lactic acid bacteria strains bearing probiotic features from mucosal microbiota of healthy calves: Looking for immunobiotics through in vitro and in vivo approaches for immunoprophylaxis applications.

From the birth, since their mucosal microbiota and immune system are not fully developed, newborn calves are susceptible to several mucosal pathogenic microorganisms. Operating through humoral and non-humoral mechanisms in the host, several lactic acid bacteria strains bearing probiotic features are often employed in livestock as food supplement, improving animal production performance, promoting health and reducing the severity of mucosal infections. Accordingly, we isolated, species-level identified and screened for their probiotic potentials seventy lactic acid bacteria strains from upper airway, vaginal and intestinal mucosa of healthy calves. Based on in vitro approaches, we selected three strains: Lactobacillus fermentum V3B-08 isolated from upper airway mucosa, Weissella hellenica V1V-30 isolated from vaginal mucosa and Lactobacillus farciminis B4F-06 isolated from intestinal mucosa were used to mono-colonize germ-free mice in the same site in which these strains were isolated, aiming to characterize their immunomodulatory features. These strains were able to colonize germ-free mice mucosa and trigger sIgA synthesis at a local level, in addition to stimulating, in different ways, adaptive immune responses at a systemic level.