Effects of Loigolactobacillus coryniformis K8 CECT 5711 on the Immune Response of Elderly Subjects to COVID-19 Vaccination: A Randomized Controlled Trial.

Elderly people are particularly vulnerable to COVID-19, with a high risk of developing severe disease and a reduced immune response to the COVID-19 vaccine. A randomized, placebo-controlled, double-blind trial to assess the effect of the consumption of the probiotic Loigolactobacillus coryniformis K8 CECT 5711 on the immune response generated by the COVID-19 vaccine in an elderly population was performed. Two hundred nursing home residents >60 yrs that had not COVID-19 were randomized to receive L. coryniformis K8 or a placebo daily for 3 months. All volunteers received a complete vaccination schedule of a mRNA vaccine, starting the intervention ten days after the first dose. Specific IgG and IgA antibody levels were analyzed 56 days after the end of the immunization process. No differences between the groups were observed in the antibody levels. During the intervention, 19 subjects had COVID-19 (11 receiving K8 vs. 8 receiving placebo, p = 0.457). Subgroup analysis in these patients showed that levels of IgG were significantly higher in those receiving K8 compared to placebo (p = 0.038). Among subjects >85 yrs that did not get COVID-19, administration of K8 tended to increase the IgA levels (p = 0.082). The administration of K8 may enhance the specific immune response against COVID-19 and may improve the COVID-19 vaccine-specific responses in elderly populations.

Effects of a Combination of Extracts from Olive Fruit and Almonds Skin on Oxidative and Inflammation Markers in Hypercholesterolemic Subjects: A Randomized Controlled Trial.

Hydroxytyrosol (HT) from olives and polyphenols from almond skin (ASPs) possess cardioprotective properties. This pilot study evaluates the effect of supplementation with a combination of olive fruit and almond skin extracts on low-density lipoprotein (LDL) cholesterol oxidation, lipid homeostasis, and inflammatory parameters in adults with moderate hypercholesterolemia. A randomized, parallel, double-blind, placebo-controlled pilot study of 8 weeks was performed. The extract group (EG) received the supplement with 7.5 mg HT +210 mg ASPs, and the control group (CG) received a placebo composed of maltodextrin. Oxidized LDL (oxLDL) levels and the oxLDL/LDL ratio were lower in the EG than in the CG after 8 weeks of treatment (18.76 ± 3.91 vs. 10.34 ± 4.22, P < .001 and 0.151 ± 0.025 vs. 0.08 ± 0.023, P < .001, respectively). Interleukin-1ß levels were significantly higher in the CG than in the EG at week 4 (P = .004), IL-6 was significantly higher in the CG than in the EG at week 4 (P = .049), and IL-10 was significantly increased at week 4 in both groups (P = .002 for CG and P = .001 for EG). In conclusion, daily consumption of a combination of an olive fruit extract and an almond skin extract for 8 weeks seems to protect LDL from oxidation and to prevent inflammatory status in moderately hypercholesterolemic subjects.

A Gene Homologous to rRNA Methylase Genes Confers Erythromycin and Clindamycin Resistance in Bifidobacterium breve.

Bifidobacteria are mutualistic intestinal bacteria, and their presence in the human gut has been associated with health-promoting activities. The presence of antibiotic resistance genes in this genus is controversial, since, although bifidobacteria are nonpathogenic microorganisms, they could serve as reservoirs of resistance determinants for intestinal pathogens. However, until now, few antibiotic resistance determinants have been functionally characterized in this genus. In this work, we show that Bifidobacterium breve CECT7263 displays atypical resistance to erythromycin and clindamycin. In order to delimit the genomic region responsible for the observed resistance phenotype, a library of genomic DNA was constructed and a fragment of 5.8 kb containing a gene homologous to rRNA methylase genes was able to confer erythromycin resistance in Escherichia coli This genomic region seems to be very uncommon, and homologs of the gene have been detected in only one strain of Bifidobacterium longum and two other strains of B. breve In this context, analysis of shotgun metagenomics data sets revealed that the gene is also uncommon in the microbiomes of adults and infants. The structural gene and its upstream region were cloned into a B. breve-sensitive strain, which became resistant after acquiring the genetic material. In vitro conjugation experiments did not allow us to detect gene transfer to other recipients. Nevertheless, prediction of genes potentially acquired through horizontal gene transfer events revealed that the gene is located in a putative genomic island.IMPORTANCEBifidobacterium breve is a very common human intestinal bacterium. Often described as a pioneer microorganism in the establishment of early-life intestinal microbiota, its presence has been associated with several beneficial effects for the host, including immune stimulation and protection against infections. Therefore, some strains of this species are considered probiotics. In relation to this, because probiotic bacteria are used for human and animal consumption, one of the safety concerns over these bacteria is the presence of antibiotic resistance genes, since the human gut is a densely populated habitat that could favor the transfer of genetic material to potential pathogens. In this study, we analyzed the genetic basis responsible for the erythromycin and clindamycin resistance phenotype of B. breve CECT7263. We were able to identify and characterize a novel gene homologous to rRNA methylase genes which confers erythromycin and clindamycin resistance. This gene seems to be very uncommon in other bifidobacteria and in the gut microbiomes of both adults and infants. Even though conjugation experiments showed the absence of transferability under in vitro conditions, it has been predicted to be located in a putative genomic island recently acquired by specific bifidobacterial strains.

In vitro and in vivo anti-microbial activity evaluation of inactivated cells of Lactobacillus salivarius CECT 5713 against Streptococcus mutans.

OBJECTIVE: Defining the etiology of dental caries is a complex problem. The microbiological approach has included Streptococcus mutans as one of the bacterial species involved in this disease. This research investigates the inhibitory effects of heat-inactivated Lactobacillus salivarius CECT 5713 against S. mutans using in vitro and in vivo assays. DESIGN: On the one hand, the effect of non-viable L. salivarius CECT 5713 on the in vitro adhesion of S. mutans to hydroxyapatite discs was evaluated. On the other hand, levels of Streptococcus mutans, amount of salivary flow and salivary pH before and after taking the rinse with the non-viable L. salivarius CECT 5713 in healthy volunteers were assessed (self-controlled open-label pilot study). RESULTS: The levels of S. mutans seemed to decrease in the in vitro and in vivo assays (p<0.05). The in vitro effect of non-viable L. salivarius was maintained until 36 months of storage. In addition, the reduction of S. mutans salivary concentration in the volunteers was statistically significant from the third day until two weeks of treatment. CONCLUSIONS: Heat-inactivated L. salivarius CECT 5713 prevents S. mutans adhesion to hydroxyapatite and could be used as a strategy to reduce the salivary concentration of this oral pathogen.

Draft Genome Sequence of Lactobacillus reuteri CECT8605.

Lactobacillus reuteri CECT8605 has shown potential probiotic properties in both in vitro and in vivo assays. Besides its beneficial characteristics, general aspects concerning genetic stability and safety for human consumption have been studied. Its genome sequence has been a useful tool to support preliminary conclusions based on empirical observations.

Resequencing the Genome of Bifidobacterium breve Strain CECT7263.

The probiotic properties of Bifidobacterium breve CECT7263, as well as its safety, have been the focus of in several studies since 2008, including the sequencing of its genome in 2012. This study aims to complete the available genomic data to deepen the knowledge of some phenotypic characteristics of this strain.

Lactobacillus fermentum CECT 5716 Reduces Staphylococcus Load in the Breastmilk of Lactating Mothers Suffering Breast Pain: A Randomized Controlled Trial.

INTRODUCTION: Recent results indicate that human mastitis and painful breastfeeding may be characterized by a mammary bacterial dysbiosis, a process in which the population of potential pathogens increases at the expense of the normal mammary microbiota. The objective of the present study is the evaluation of three different doses of Lactobacillus fermentum CECT5716 to reduce the load of Staphylococcus in the breastmilk of women suffering from painful breastfeeding. MATERIALS AND METHODS: A randomized double-blinded controlled study with four study groups was performed. Three groups received the probiotic strain for 3 weeks at doses of 3×10(9) colony-forming units (CFU)/day, 6×10(9) CFU/day, or 9×10(9) CFU/day. The fourth group received a placebo of maltodextrin. The main outcome of the study was Staphylococcus counts in breastmilk. The secondary outcomes were Streptococcus, Lactobacillus, and total bacteria counts in breastmilk, immunoglobulin A and interleukin 8 concentrations in breastmilk, and breast pain scores. RESULTS: At the end of the study, a significant decrease in the Staphylococcus load was observed in the probiotic groups compared with the baseline loads (p=0.045), whereas the control group maintained similar levels over time. A significant difference in the pain score was observed among the groups receiving the three probiotic doses compared with the control group (p=0.035, p=0.000, and p=0.028, respectively). A dose-response effect could not be observed because the three doses tested induced similar effects, and no significant differences were detected. CONCLUSIONS: We conclude that L. fermentum CECT5716 is an efficient treatment for breast pain during lactation associated with a high level of Staphylococcus in breastmilk.

Prevention of diet-induced obesity by apple polyphenols in Wistar rats through regulation of adipocyte gene expression and DNA methylation patterns.

This study was conducted to determine the mechanisms implicated in the beneficial effects of apple polyphenols (APs) against diet-induced obesity in Wistar rats, described in a previous study from our group. Supplementation of high-fat sucrose diet with AP prevented adiposity increase by inhibition of adipocyte hypertrophy. Rats supplemented with AP exhibited improved glucose tolerance while adipocytes isolated from these rats showed an enhanced lipolytic response to isoproterenol. AP intake led to reduced Lep, Plin, and sterol regulatory element binding transcription factor 1 (Srebf1) mRNA levels and increased aquaporin 7 (Aqp7), adipocyte enhancer binding protein 1 (Aebp1), and peroxisome proliferator-activated receptor gamma co-activator 1 alpha (Ppargc1a) mRNA levels in epididymal adipocytes. In addition, we found different methylation patterns of Aqp7, Lep, Ppargc1a, and Srebf1 promoters in adipocytes from apple-supplemented rats compared to high-fat sucrose fed rats. The administration of AP protects against body weight gain and fat deposition and improves glucose tolerance in rats. We propose that AP exerts the antiobesity effects through the regulation of genes involved in adipogenesis, lipolysis, and fatty acid oxidation, in a process that could be mediated in part by epigenetic mechanisms.

Intestinal anti-inflammatory activity of hydroalcoholic extracts of Phlomis purpurea L. and Phlomis lychnitis L. in the trinitrobenzenesulphonic acid model of rat colitis.

ETHNOPHARMACOLOGICAL RELEVANCE: Different species from genus Phlomis, frequently native from the the eastern Mediterranean zone, have been used in traditional medicine as an anti-inflammatory remedy. Among other constituents, they contain polyphenols that show antioxidant properties, which are interesting for the treatment of inflammatory pathologies associated with oxidative stress in humans, such as inflammatory bowel disease (IBD). The aim of this study was to evaluate the intestinal anti-inflammatoy effect of hydroalcoholic extracts of Phlomis lychnitis and P. purpurea in the trinitrobenzenesulphonic acid (TNBS) model of rat colitis, a well characterized experimental model with some resemblance to human IBD. MATERIALS AND METHODS: Hydroalcoholic extracts of both plants were characterized by determining their polyphenolic content and then assayed in the TNBS model of rat colitis. For this purpose, female Wistar rats were assigned to seven groups (n=10): healthy control, untreated TNBS-colitis and five TNBS- colitis groups treated with Phlomis lychnitis (10 and 20mg/kg), P. purpurea (10 and 25mg/kg) and sulphasalazine (200mg/kg), as a positive control. Treatments started the same day of TNBS colitis induction, and rats were sacrificed one week later. Colonic inflammation was evaluated both histologically and biochemically. RESULTS: The histological (macroscopic and microscopic) analysis of colonic samples revealed that both extracts showed an anti-inflammatory effect, which was confirmed biochemically by a decreased colonic MPO activity, a maker of neutrophil infiltration, an increased colonic glutathione content, which counteracts the oxidative status associated with the inflammatory process, and a down-regulated iNOS expression. However, only the extract of P. purpurea reduced the expression of the proinflammatory cytokines IL-1ß and IL-17, the chemokines CINC-1 and MCP-1, as well as the adhesion molecule ICAM-1, ameliorating the altered immune response associated with the colonic inflammation. Furthermore, both P. lychnitis and P. purpurea extracts were able to significantly increase the expression of markers of epithelial integrity such as MUC-2, MUC-3 and villin, thus revealing an improvement in the altered colonic permeability that characterizes colonic inflammation. CONCLUSIONS: Both extracts showed intestinal anti-inflammatory activity in the TNBS model of rat colitis, thus confirming their traditional use in digestive inflammatory complaints. In addition to their antioxidant properties, other mechanisms can contribute to this beneficial effect, like an improvement in the intestine epithelial barrier and a downregulation of the immune response.

Screening of polyphenolic plant extracts for anti-obesity properties in Wistar rats.

BACKGROUND: Polyphenols have been reported to prevent chronic diseases such as cardiovascular diseases, cancers, diabetes and neurodegenerative diseases. The objective of the study was to conduct a screening for potential anti-obesity polyphenolic plant extracts using a diet-induced animal model. Rats were fed a high-fat-sucrose (HFS) diet with or without supplementation of different polyphenolic plant extracts (almond, apple, cinnamon, orange blossom, hamamelis, lime blossom, grape vine, and birch) for 56-64 days. RESULTS: Body weight gain was lower in rats supplemented with apple, cinnamon, hamamelis and birch extracts as compared to HFS non-supplemented group. Moreover, apple and cinnamon extracts prevented the increase in fat mass promoted by the HFS diet. Insulin resistance, estimated by the homostatic model assessment-insulin resistance (HOMA-IR) index, was reduced in rats fed apple, cinnamon, hamamelis and birch extracts. Apple extract also prevented the HFS-induced hyperglycaemia and hyperleptinaemia. CONCLUSION: Only apple and cinnamon extracts were finally considered as potentially important anti-obesogenic extracts, due to their body fat-lowering effects, while the improvement of obesity-related metabolic complications by apple polyphenols highlights this extract as a promising functional food ingredient for the management of obesity and its metabolic complications.

Metabolism of prebiotic products containing beta(2-1) fructan mixtures by two Lactobacillus strains.

The capacity of two probiotic strains, isolated from human breast milk, to use several beta(2-1) fructan mixtures as carbon and energy source in in vitro cultures has been tested. Results showed that both strains, Lactobacillus gasseri CECT5714 and Lactobacillus fermentum CECT5716, reached higher growth levels on culture media containing fructooligosaccharide mixtures produced by enzymatic synthesis, compared to those obtained by inulin hydrolysis. Furthermore, the shortest beta(2-1) fructan, kestose, was the only prebiotic compound in the mixtures significantly metabolized in all growth media tested. Analysis of short-chain fatty acid production showed no correlation between the fatty acid profile produced and the carbon source used in each experiment. These data could serve to select appropriate beta(2-1) fructans to be used as prebiotics for L. gasseri CECT5714 and L. fermentum CECT5716 and to design suitable symbiotic food products containing the mentioned lactobacilli.

Expression of an Aspergillus niger glucose oxidase in saccharomyces cerevisiae and its use to optimize fructo-oligosaccharides synthesis.

Fructo-oligosaccharides (FOS) represent the most abundantly supplied and utilized group of nondigestible oligosaccharides as food ingredients. These prebiotics can be produced from sucrose using the transglycosylating activity of beta-fructofuranosidases (EC 3.2.1.26) at high concentrations of the starting material. The main problem during FOS synthesis is that the activity of the enzyme is inhibited by the glucose generated during the reaction, and therefore the maximum FOS content in commercial products reaches up to 60% on a dry substance basis. The glucose oxidase (gox) gene from Aspergillus niger BT18 was cloned and integrated, as part of an expression cassette, into the ribosomal DNA of a Saccharomyces cerevisiae host strain. One of the recombinant strains with a high copy number of the gox gene and showing a high GOX specific activity was used to produce the enzyme. Addition of the extracellular glucose oxidase to the FOS synthesis reaction helped to remove the glucose generated, avoiding the inhibition of the fungal beta-fructofuranosidase. As a result, a final syrup containing up to 90% of FOS was obtained.

Inactivation of the lys7 gene, encoding saccharopine reductase in Penicillium chrysogenum, leads to accumulation of the secondary metabolite precursors piperideine-6-carboxylic acid and pipecolic acid from alpha-aminoadipic acid.

Pipecolic acid serves as a precursor of the biosynthesis of the alkaloids slaframine and swainsonine (an antitumor agent) in some fungi. It is not known whether other fungi are able to synthesize pipecolic acid. Penicillium chrysogenum has a very active alpha-aminoadipic acid pathway that is used for the synthesis of this precursor of penicillin. The lys7 gene, encoding saccharopine reductase in P. chrysogenum, was target inactivated by the double-recombination method. Analysis of a disrupted strain (named P. chrysogenum SR1-) showed the presence of a mutant lys7 gene lacking about 1,000 bp in the 3'-end region. P. chrysogenum SR1- lacked saccharopine reductase activity, which was recovered after transformation of this mutant with the intact lys7 gene in an autonomously replicating plasmid. P. chrysogenum SR1- was a lysine auxotroph and accumulated piperideine-6-carboxylic acid. When mutant P. chrysogenum SR1- was grown with L-lysine as the sole nitrogen source and supplemented with DL-alpha-aminoadipic acid, a high level of pipecolic acid accumulated intracellularly. A comparison of strain SR1- with a lys2-defective mutant provided evidence showing that P. chrysogenum synthesizes pipecolic acid from alpha-aminoadipic acid and not from L-lysine catabolism.

Co-transformation with autonomous replicating and integrative plasmids in Penicillium chrysogenum is highly efficient and leads in some cases to rescue of the intact integrative plasmid.

The efficiency of co-transformation in Penicillium chrysogenum Wisconsin 54-1255 pyrG(-) and the fate of the transforming DNA were studied using an integrative (pEF43) and an autonomous replicating plasmid (pAM9L). The results showed a co-transformation frequency of nearly 70% of all transformants tested. The total efficiency of transformation was shown to be dependent on the plasmid marker used as transformant selection (i.e., markers in the integrative or autonomous replicating vector). Analysis of the plasmids re-isolated from several co-transformants showed that different populations of plasmids co-exist in the fungal host. Interestingly, in all co-transformants studied, the integrative plasmid was found to be replicating autonomously without integrating into the host genome. In some cases, co-integrates were formed by recombination between autonomous replicating (pAM9L) and integrative (pEF43) plasmids. However, unexpectedly in some cases, the non-reorganised pEF43 integrative plasmid used in the co-transformation assays was rescued from some co-transformants.

A novel epimerization system in fungal secondary metabolism involved in the conversion of isopenicillin N into penicillin N in Acremonium chrysogenum.

The epimerization step that converts isopenicillin N into penicillin N during cephalosporin biosynthesis has remained uncharacterized despite its industrial relevance. A transcriptional analysis of a 9-kb region located downstream of the pcbC gene revealed the presence of two transcripts that correspond to the genes named cefD1 and cefD2 encoding proteins with high similarity to long chain acyl-CoA synthetases and acyl-CoA racemases from Mus musculus, Homo sapiens, and Rattus norvegicus. Both genes are expressed in opposite orientations from a bidirectional promoter region. Targeted inactivation of cefD1 and cefD2 was achieved by the two-marker gene replacement procedure. Disrupted strains lacked isopenicillin N epimerase activity, were blocked in cephalosporin C production, and accumulated isopenicillin N. Complementation in trans of the disrupted nonproducer mutant with both genes restored epimerase activity and cephalosporin biosynthesis. However, when cefD1 or cefD2 were introduced separately into the double-disrupted mutant, no epimerase activity was detected, indicating that the concerted action of both proteins encoded by cefD1 and cefD2 is required for epimerization of isopenicillin N into penicillin N. This epimerization system occurs in eukaryotic cells and is entirely different from the known epimerization systems involved in the biosynthesis of bacterial beta-lactam antibiotics.