Effect of High Pressure and Heat on Bacterial Toxins

Even though the inactivation of microorganisms by high pressure treatment is a subject of intense investigations, the effect of high pressure on bacterial toxins has not been studied so far. In this study, the influence of combined pressure/temperature treatment (0.1 to 800 MPa and 5 to 121 °C) on bacterial enterotoxins was determined. Therefore, heat-stable enterotoxin (STa) of cholera toxin (CT) from Vibrio cholerae, staphylococcal enterotoxins A-E, haemolysin BL (HBL) from Bacillus cereus, and Escherichia coli (STa) were subjected to different treatment schemes. Structural alterations were monitored in enzyme immunoassays (EIAs). Cytotoxicity of the pressure treated supernatant of toxigenic B. cereus DSM 4384 was investigated with Vero cells. High pressure of 200 to 800 MPa at 5 °C leads to a slight increase of the reactivity of the STa of E. coli. However, reactivity decreased at 800 MPa and 80 °C to (66±21) % after 30 min and to (44±0.3) % after 128 min. At ambient pressure no decrease in EIA reactivity could be observed after 128 min. Pressurization (0.1 to 800 MPa) of heat stable monomeric staphylococcal toxins at 5 and 20 °C showed no effect. A combined heat (80 °C) and pressure (0.1 to 800 MPa) treatment lead to a decrease in the immuno-reactivity to 20 % of its maximum. For cholera toxin a significant loss in latex agglutination was observable only at 80 °C and 800 MPa for holding times higher than 20 min. Interestingly, the immuno-reactivity of B. cereus HBL toxin increased with the increase of pressure (182 % at 800 MPa, 30 °C), and high pressure showed only minor effects on cytotoxicity to Vero cells. Our results indicate that pressurization can increase inactivation observed by heat treatment, and combined treatments may be effective at lower temperatures and/or shorter incubation time

Effect of High Pressure and Heat on Bacterial Toxins

Even though the inactivation of microorganisms by high pressure treatment is a subject of intense investigations, the effect of high pressure on bacterial toxins has not been studied so far. In this study, the influence of combined pressure/temperature treatment (0.1 to 800 MPa and 5 to 121 °C) on bacterial enterotoxins was determined. Therefore, heat-stable enterotoxin (STa) of cholera toxin (CT) from Vibrio cholerae, staphylococcal enterotoxins A-E, haemolysin BL (HBL) from Bacillus cereus, and Escherichia coli (STa) were subjected to different treatment schemes. Structural alterations were monitored in enzyme immunoassays (EIAs). Cytotoxicity of the pressure treated supernatant of toxigenic B. cereus DSM 4384 was investigated with Vero cells. High pressure of 200 to 800 MPa at 5 °C leads to a slight increase of the reactivity of the STa of E. coli. However, reactivity decreased at 800 MPa and 80 °C to (66±21) % after 30 min and to (44±0.3) % after 128 min. At ambient pressure no decrease in EIA reactivity could be observed after 128 min. Pressurization (0.1 to 800 MPa) of heat stable monomeric staphylococcal toxins at 5 and 20 °C showed no effect. A combined heat (80 °C) and pressure (0.1 to 800 MPa) treatment lead to a decrease in the immuno-reactivity to 20 % of its maximum. For cholera toxin a significant loss in latex agglutination was observable only at 80 °C and 800 MPa for holding times higher than 20 min. Interestingly, the immuno-reactivity of B. cereus HBL toxin increased with the increase of pressure (182 % at 800 MPa, 30 °C), and high pressure showed only minor effects on cytotoxicity to Vero cells. Our results indicate that pressurization can increase inactivation observed by heat treatment, and combined treatments may be effective at lower temperatures and/or shorter incubation time

Ecological importance of cross-feeding of the intermediate metabolite 1,2-propanediol between bacterial gut symbionts

Cross-feeding based on the metabolite 1,2-propanediol has been proposed to have an important role in the establishment of trophic interactions among gut symbionts, but its ecological importance has not been empirically established. Here, we show that in vitro growth of Lactobacillus reuteri ATCC PTA 6475 is enhanced through 1,2-propanediol produced by Bifidobacterium breve UCC2003 and Escherichia coli MG1655 from the metabolization of fucose and rhamnose, respectively. Work with isogenic mutants showed that the tropic interaction is dependent on the pduCDE operon in L. reuteri, which encodes for the ability to use 1,2-propanediol, and the L-fucose permease (fucP) gene in B. breve, which is required for 1,2-propanediol formation from fucose. Experiments in gnotobiotic mice revealed that, although the pduCDE operon bestows a fitness burden on L. reuteri ATCC PTA 6475 in the mouse digestive tract, the ecological performance of the strain was enhanced in the presence of B. breve UCC2003 and the mucus-degrading species Bifidobacterium bifidum The use of the respective pduCDE and fucP mutants of L. reuteri and B. breve in the mouse experiments indicated that the trophic interaction was specifically based on 1,2-propanediol. Overall, our work established the ecological importance of cross-feeding relationships based on 1,2-propanediol for the fitness of a bacterial symbiont in the vertebrate gut.Importance Through experiments in gnotobiotic mice that employed isogenic mutants of bacterial strains that produce (Bifidobacterium breve) and utilize (Lactobacillus reuteri) 1,2-propanediol, this study provides mechanistic insight into the ecological ramifications of a trophic interaction between gut symbionts. The findings improve our understanding on how cross-feeding influences the competitive fitness of L. reuteri in the vertebrate gut and revealed a putative selective force that shaped the evolution of the species. The findings are relevant as they provide a basis to design rational microbial-based strategies to modulate gut ecosystems, which could employ mixtures of bacterial strains that establish trophic interactions or a personalized approach based on the ability of a resident microbiota to provide resources for the incoming microbe

Conversion of (poly)phenolic compounds in food fermentations by lactic acid bacteria: Novel insights into metabolic pathways and functional metabolites

Lactobacillaceae are among the major fermentation organisms in most food fermentations but the metabolic pathways for conversion of (poly)phenolic compounds by lactobacilli have been elucidated only in the past two decades. Hydroxycinnamic and hydroxybenzoic acids are metabolized by separate enzymes which include multiple esterases, decarboxylases and hydroxycinnamic acid reductases. Glycosides of phenolic compounds including flavonoids are metabolized by glycosidases, some of which are dedicated to glycosides of plant phytochemicals rather than oligosaccharides. Metabolism of phenolic compounds in food fermentations often differs from metabolism in vitro, likely reflecting the diversity of phenolic compounds and the unknown stimuli that induce expression of metabolic genes. Current knowledge will facilitate fermentation strategies to achieve improved food quality by targeted conversion of phenolic compounds

Studies on the Mode of Action of Reutericyclin

The mode of action of reutericyclin was determined with fluorescent dyes that probed the permeability of the cytoplasmic membrane by large molecules, protons, and potassium. A comparison of reutericyclin activity with those of nisin, nigericin, and valinomycin demonstrated that reutericyclin does not form pores but selectively dissipates the transmembrane proton potential

Cruciferin improves stress resistance and simulated gastrointestinal survival of probiotic Limosilactobacillus reuteri in the model encapsulation system

Encapsulation is a viable strategy to improve the stability and survival of probiotics during processing, storage, and consumption. Cruciferin, a major canola protein with high denaturation temperature and resistance to gastric degradation, has potential for encapsulation and protection of probiotics against harsh conditions in processing and gastrointestinal tract. Cruciferin/alginate capsules were fabricated to encapsulate probiotics, and were characterized using confocal and scanning electron microscopy (SEM). The bacterial viability was studied during storage, processing, and gastro-intestinal transit. Limosilactobacillus reuteri TMW 1.656 was encapsulated in spherical cruciferin/alginate capsules (2.2 ± 0.1 mm) prepared using an extrusion method. SEM images of the capsules showed that the bacteria were entrapped within the porous structure which was formed by the complexation of cruciferin and alginate. The confocal microscopy images confirmed that cruciferin and alginate were homogeneously distributed throughout the capsules. The shelf life of the bacteria in the presence of cruciferin and alginate increased up to 8 weeks at 4 °C, while unencapsulated (free) bacteria lost their viability after 2 weeks storage. The heat resistance of encapsulated bacteria exposed to 65 °C and 70 °C was improved by up to ∼ 4 and 2 log cycles, respectively, compared to unencapsulated bacteria. Encapsulation also protected L. reuteri against gastric low pH and enzymes; the viability was 3 logs higher when compared to unencapsulated bacteria. The capsules were degraded in simulated intestinal fluid, leading to the release of the encapsulated bacteria, whereas the wall materials increased the resistance of released bacteria to bile salts. Comparison between the viability of unencapsulated bacteria in presence of cruciferin/alginate mixtures and bacteria encapsulated in the capsules revealed that capsule formation provided physical barriers to the harsh conditions and played a key role in the protection of bacteria. This study showed that cruciferin/alginate capsules are capable to improve stability and shelf life of Limosilactobacillus reuteri

Identification and quantification of virulence factors of enterotoxigenic Escherichia coli by high-resolution melting curve quantitative PCR

Abstract Background Diagnosis of enterotoxigenic E. coli (ETEC) associated diarrhea is complicated by the diversity of E.coli virulence factors. This study developed a multiplex quantitative PCR assay based on high-resolution melting curves analysis (HRM-qPCR) to identify and quantify genes encoding five ETEC fimbriae related to diarrhea in swine, i.e. K99, F41, F18, F6 and K88. Methods Five fimbriae expressed by ETEC were amplified in multiple HRM-qPCR reactions to allow simultaneous identification and quantification of five target genes. The assay was calibrated to allow quantification of the most abundant target gene, and validated by analysis of 30 samples obtained from piglets with diarrhea and healthy controls, and comparison to standard qPCR detection. Results The five amplicons with melting temperatures (Tm) ranging from 74.7 ± 0.06 to 80.5 ± 0.15 °C were well-separated by HRM-qPCR. The area of amplicons under the melting peak correlated linearly to the proportion of the template in the calibration mixture if the proportion exceeded 4.8% (K88) or <1% (all other amplicons). The suitability of the method was evaluated using 30 samples from weaned pigs aged 6–7 weeks; 14 of these animals suffered from diarrhea in consequence of poor sanitary conditions. Genes encoding fimbriae and enterotoxins were quantified by HRM-qPCR and/or qPCR. The multiplex HRM-qPCR allowed accurate analysis when the total gene copy number of targets was more than 1 × 105 / g wet feces and the HRM curves were able to simultaneously distinguish fimbriae genes in the fecal samples. The relative quantification of the most abundant F18 based on melting peak area was highly correlated (P < 0.001; r2 = 0.956) with that of individual qPCR result but the correlation for less abundant fimbriae was much lower. Conclusions The multiplex HRM assay identifies ETEC virulence factors specifically and efficiently. It correctly indicated the predominant fimbriae type and additionally provides information of presence/ absence of other fimbriae types and it could find broad applications for pathogen diagnosis

Contribution of glutamate decarboxylase in <it>Lactobacillus reuteri</it> to acid resistance and persistence in sourdough fermentation

Abstract Background Acid stress impacts the persistence of lactobacilli in industrial sourdough fermentations, and in intestinal ecosystems. However, the contribution of glutamate to acid resistance in lactobacilli has not been demonstrated experimentally, and evidence for the contribution of acid resistance to the competitiveness of lactobacilli in sourdough is lacking. It was therefore the aim of this study to investigate the ecological role of glutamate decarboxylase in L. reuteri. Results A gene coding for a putative glutamate decarboxylase, gadB, was identified in the genome of L. reuteri 100-23. Different from the organization of genetic loci coding for glutamate decarboxylase in other lactic acid bacteria, gadB was located adjacent to a putative glutaminase gene, gls3. An isogenic deletion mutant, L. reuteri ∆gadB, was generated by a double crossover method. L. reuteri 100-23 but not L. reuteri ∆gadB converted glutamate to γ-aminobutyrate (GABA) in phosphate butter (pH 2.5). In sourdough, both strains converted glutamine to glutamate but only L. reuteri 100-23 accumulated GABA. Glutamate addition to phosphate buffer, pH 2.5, improved survival of L. reuteri 100-23 100-fold. However, survival of L. reuteri ∆gadB remained essentially unchanged. The disruption of gadB did not affect growth of L. reuteri in mMRS or in sourdough. However, the wild type strain L. reuteri 100-23 displaced L. reuteri ∆gadB after 5 cycles of fermentation in back-slopped sourdough fermentations. Conclusions The conversion of glutamate to GABA by L. reuteri 100-23 contributes to acid resistance and to competitiveness in industrial sourdough fermentations. The organization of the gene cluster for glutamate conversion, and the availability of amino acids in cereals imply that glutamine rather than glutamate functions as the substrate for GABA formation. The exceptional coupling of glutamine deamidation to glutamate decarboxylation in L. reuteri likely reflects adaptation to cereal substrates.</p