RESUMEN
Brazilian artisanal cheeses date from the first Portuguese settlers and evolved via local factors, resulting in unique products that are now part of the patrimony and identity of different Brazilian regions. In this study, we combined several culture-independent approaches, including 16S/ITS metagenetics, assembly- and deep profiling-metagenomics to characterize the originality of the microbiota of five varieties of Brazilian artisanal cheeses from the South and Southeast regions of Brazil. Their core microbiota contained mainly lactic acid bacteria (LAB), of which Lactococcus lactis subsp. lactis was the most frequent, followed by Streptococcus thermophilus in the South region. Moreover, several samples from the Southeast region contained, as dominant LAB, two other food Streptococci belonging to a new species of the salivarius group and S. infantarius. Rinds of samples from the Southeast region were dominated by the halotolerant bacterium Corynebacterium variabile, and the yeasts Diutina catenulata, followed by Debaryomyces hansenii and Kodamaea ohmeri. Rinds from the South region contained mainly LAB due to their short ripening time, and the predominant yeast was D. hansenii. Phylogenomic analysis based on L. lactis metagenome-assembled genomes (MAGs) showed that most Brazilian strains are closely related and form a different clade from those whose genomes are available at this time, indicating that they belong to a specific group. Lastly, functional analysis showed that S. infantarius acquired a â¼ 26 kb DNA fragment from S. thermophilus starter strains that carry the LacSZ system, allowing fast lactose assimilation, an adaptation advantage for growth in milk. Finally, our study identified several areas of concern, such as the presence of somatic cell DNA and high levels of antibiotic resistance genes in several cheese microbiota, suggesting that milk from diseased animals may still be used occasionally. Overall, the data from this study highlight the potential value of the traditional and artisanal cheese production network in Brazil, and provide a metagenomic-based scheme to help manage this resource safely.
Asunto(s)
Queso , Lactobacillales , Lactococcus lactis , Animales , Biodiversidad , Brasil , Queso/análisis , Microbiología de Alimentos , Lactobacillales/genética , Lactococcus lactis/genética , Metagenómica , Streptococcus thermophilus/genética , LevadurasRESUMEN
AIM: To evaluate two folate-producing strains, Streptococcus (Strep.) thermophilus CRL 808 and Strep. thermophilus CRL 415, against chemically induced mucositis in mice. METHODS AND RESULTS: In vitro assays with Caco-2 cells were performed to evaluate the effect of the bacteria in the presence of 5-fluorouracil (5-FU). For in vivo studies, mice were daily injected with 5-FU to induce intestinal mucositis (IM) and orally administered with folate-producing strains during 6 days. Clinical symptoms, histological parameters and cytokine profiles were assessed. The results showed that Strep. thermophilus CRL 808 increased the cytotoxicity of 5-FU against Caco-2 cells. Administration of this strain in mice with chemically induced IM resulted in a reduction in diarrhoea score and restoration of the intestinal architecture. Cytokine analysis showed that the anti-inflammatory effect by the bacterium is not associated with an immune mechanism. Regarding Strep. thermophilus CRL 415, no improvements were observed in any of the parameters evaluated. CONCLUSION: The administration of the folate-producing Strep. thermophilus CRL 808 has the potential to prevent IM induced by 5-FU in mice. SIGNIFICANCE AND IMPACT OF THE STUDY: Folate-producing LAB could be used in chemotherapy patients to reduce the symptoms of IM, improve their nutritional status and increase the effectiveness of 5-FU.
Asunto(s)
Ácido Fólico/biosíntesis , Lactobacillales/metabolismo , Mucositis/terapia , Probióticos/uso terapéutico , Animales , Células CACO-2 , Citocinas/metabolismo , Diarrea/prevención & control , Fluorouracilo , Humanos , Inflamación/patología , Mucosa Intestinal/patología , Intestinos/patología , Lactobacillales/genética , Ratones , Mucositis/inducido químicamente , Streptococcus thermophilus/genética , Streptococcus thermophilus/metabolismoRESUMEN
Three cos-type virulent Streptococcus thermophilus phages were isolated from failed mozzarella production in Uruguay. Genome analyses showed that these phages are similar to those isolated elsewhere around the world. The CRISPR1 and CRISPR3 arrays of the three S. thermophilus host strains from Uruguay were also characterized and similarities were noted with previously described model strains SMQ-301, LMD-9 and DGCC7710. Spontaneous bacteriophage-insensitive S. thermophilus mutants (BIMs) were obtained after challenging the phage-sensitive wild-type strain Uy02 with the phage 128 and their CRISPR content was analyzed. Analysis of 23 BIMs indicated that all of them had acquired at least one new spacer in their CRISPR1 array. While 14 BIMs had acquired spacer at the 5'-end of the array, 9 other BIMs acquired a spacer within the array. Comparison of the leader sequence in strains Uy02 and DGCC7710 showed a nucleotide deletion at position -1 in Uy02, which may be responsible for the observed ectopic spacer acquisition. Analysis of the spacer sequences upstream the newly acquired ectopic spacer indicated presence of a conserved adenine residue at position -2. This study indicates that natural strains of S. thermophilus can also acquire spacers within a CRISPR array.
Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , ADN Bacteriano/genética , Genoma Viral , Fagos de Streptococcus/genética , Fagos de Streptococcus/patogenicidad , Streptococcus thermophilus/genética , Antibiosis/genética , Secuencia de Bases , Queso/microbiología , Queso/virología , Mapeo Cromosómico , ADN Intergénico/genética , Fermentación , Tecnología de Alimentos/economía , Ontología de Genes , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Anotación de Secuencia Molecular , Mutación , Alineación de Secuencia , Fagos de Streptococcus/ultraestructura , Streptococcus thermophilus/inmunología , Streptococcus thermophilus/virología , Uruguay , VirulenciaRESUMEN
Inflammatory bowel diseases (IBDs) affect the gastrointestinal tract and are characterized by recurrent inflammation that requires lifelong therapies. Probiotics such as lactic acid bacteria (LAB) have been proposed to complement current treatment protocols for these patients; however, their characteristics are strain dependent. In this regard, certain novel characteristics are only possible through the genetic modification of these beneficial micro-organisms. Different delivery systems, such as protein delivery of anti-oxidant enzymes and anti-inflammatory cytokines, have been shown to be effective in preventing and treating IBD in animal models. In this study, the safety of the recombinant LAB (recLAB) Streptococcus thermophilus CRL807 : CAT, S. thermophilus CRL807 : SOD, Lactococcus lactis NCDO2118 pXILCYT : IL-10, L. lactis MG1363 pValac : IL-10 and L. lactis MG1363 pGroESL : IL-10 with proven beneficial effects was compared to their progenitor strains S. thermophilus CRL807, L. lactis NCDO2118 or L. lactis MG1363. The prolonged administration of these genetically modified strains showed that they were just as safe as the native strains from which they derive, as demonstrated by normal animal growth and relative organ weights, absence of microbial translocation from the gastrointestinal tract, normal blood parameters and intestinal histology. The results show the potential use of these recLAB in future therapeutic formulations; however, the use of modern bio-containment systems is required for the future acceptance of these recLAB by the medical community and patients with IBD.
Asunto(s)
Terapia Biológica/métodos , Colitis/prevención & control , Colitis/terapia , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Lactococcus lactis/patogenicidad , Probióticos/efectos adversos , Streptococcus thermophilus/patogenicidad , Experimentación Animal , Animales , Femenino , Lactococcus lactis/genética , Ratones Endogámicos BALB C , Streptococcus thermophilus/genéticaRESUMEN
The aims of this study were to develop strains of lactic acid bacteria (LAB) having both immunomodulatory and antioxidant properties and to evaluate their anti-inflammatory effects both in vitro, in different cellular models, and in vivo, in a mouse model of colitis. Different Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus strains were cocultured with primary cultures of mononuclear cells. Analysis of the pro- and anti-inflammatory cytokines secreted by these cells after coincubation with candidate bacteria revealed that L. delbrueckii subsp. bulgaricus CRL 864 and S. thermophilus CRL 807 display the highest anti-inflammatory profiles in vitro. Moreover, these results were confirmed in vivo by the determination of the cytokine profiles in large intestine samples of mice fed with these strains. S. thermophilus CRL 807 was then transformed with two different plasmids harboring the genes encoding catalase (CAT) or superoxide dismutase (SOD) antioxidant enzymes, and the anti-inflammatory effects of recombinant streptococci were evaluated in a mouse model of colitis induced by trinitrobenzenesulfonic acid (TNBS). Our results showed a decrease in weight loss, lower liver microbial translocation, lower macroscopic and microscopic damage scores, and modulation of the cytokine production in the large intestines of mice treated with either CAT- or SOD-producing streptococci compared to those in mice treated with the wild-type strain or control mice without any treatment. Furthermore, the greatest anti-inflammatory activity was observed in mice receiving a mixture of both CAT- and SOD-producing streptococci. The addition of L. delbrueckii subsp. bulgaricus CRL 864 to this mixture did not improve their beneficial effects. These findings show that genetically engineering a candidate bacterium (e.g., S. thermophilus CRL 807) with intrinsic immunomodulatory properties by introducing a gene expressing an antioxidant enzyme enhances its anti-inflammatory activities.
Asunto(s)
Antioxidantes/metabolismo , Factores Inmunológicos/metabolismo , Inflamación/inmunología , Ingeniería Metabólica , Streptococcus thermophilus/inmunología , Animales , Catalasa/genética , Catalasa/metabolismo , Células Cultivadas , Colitis/inducido químicamente , Colitis/inmunología , Colitis/microbiología , Colitis/patología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/microbiología , Ratones , Streptococcus thermophilus/genética , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Ácido Trinitrobencenosulfónico/administración & dosificación , Ácido Trinitrobencenosulfónico/toxicidadRESUMEN
The effects of the expression of a small heat shock protein (shsp) gene from Streptococcus thermophilus on stress resistance in Lactococcus lactis under different environmental stresses were investigated in this study. pMG36e-shsp, an expression vector, was first constructed by inserting a shsp open reading frame (ORF) cloned from S. thermophilus strain St-QC into pMG36e. Then, a food-grade expression vector, pMG-shsp, was generated by deleting the erythromycin resistance gene from pMG36e-shsp. The transformation rate of pMG-shsp was comparable to that of pMG36e-shsp when each of these two vectors was introduced into L. lactis. These results demonstrated that the shsp ORF could successfully used as a food-grade selection marker in both pMG-shsp and pMG36e-shsp. Furthermore, the growth characteristics were almost the same between L. lactis ML23 transformants harboring pMG36e or pMG-shsp. The survival rate of L. lactis ML23 expressing the shsp ORF were increased to 0.032%, 0.006%, 0.0027%, 0.03%, and 0.16% under the following environmental stresses: heat, acid, ethanol, bile salt and H2O2, respectively. These results indicated that the expression of the shsp gene in the food-grade vector pMG-shsp conferred resistance to environmental stresses without affecting the growth characteristics of L. lactis ML23.
Asunto(s)
Humanos , Resistencia a Medicamentos , Eritromicina/farmacología , Microbiología de Alimentos , Expresión Génica , Lactobacillus , Streptococcus thermophilus/genética , Métodos , VirulenciaRESUMEN
AIMS: To determine whether the presence and type of exopolysaccharides (EPS), slime-EPS or capsular, and the structural characteristics of the polymers produced by Streptococcus thermophilus strains could interfere with or be involved in phage adsorption. METHODS AND RESULTS: Phage-host interactions between eight EPS-producing Strep. thermophilus strains (CRL419, 638, 804, 810, 815, 817, 821, 1190) and five streptococcus specific phages (phiYsca, phi3, phi5, phi6, phi8) isolated from Argentinean faulty fermentation failed yoghurts were evaluated. No relationship was found between the EPS chemical composition and the phage sensitivity/resistance phenotype. In general, the capsular-producing strains were more sensitive to phage attacks than the noncapsular-producing strains. Streptococcus thermophilus CRL1190 (capsular-producing) was the only strain sensitive to all bacteriophages and showed the highest efficiency of plating. Phage adsorption to a capsular-negative, EPS low-producing mutant of strain CRL1190 was reduced, especially for phiYcsa and phi8. CONCLUSIONS: The presence of capsular polysaccharide surrounding the cells of Strep. thermophilus strains could play a role in the adsorption of specific phages to the cells. SIGNIFICANCE AND IMPACT OF THE STUDY: Capsular-producing Strep. thermophilus strains should be evaluated for their bacteriophage sensitivity if they are included in starter cultures for the fermented food industry.