Human Dendritic Cell DC-SIGN and TLR-2 Mediate Complementary Immune Regulatory Activities in Response to Lactobacillus rhamnosus JB-1

The microbiota is required for optimal host development and ongoing immune homeostasis. Lactobacilli are common inhabitants of the mammalian large intestine and immunoregulatory effects have been described for certain, but not all, strains. The mechanisms underpinning these protective effects are beginning to be elucidated. One such protective organism is Lactobacillus rhamnosus JB-1 (Lb. rhamnosus JB-1). Lb. murinus has no such anti-inflammatory protective effects and was used as a comparator organism. Human monocyte-derived dendritic cells (MDDCs) were co-incubated with bacteria and analysed over time for bacterial adhesion and intracellular processing, costimulatory molecule expression, cytokine secretion and induction of lymphocyte polarization. Neutralising antibodies were utilized to identify the responsible MDDC receptors. Lb. rhamnosus JB-1 adhered to MDDCs, but internalization and intracellular processing was significantly delayed, compared to Lb. murinus which was rapidly internalized and processed. Lb. murinus induced CD80 and CD86 expression, accompanied by high levels of cytokine secretion, while Lb. rhamnosus JB-1 was a poor inducer of costimulatory molecule expression and cytokine secretion. Lb. rhamnosus JB-1 primed MDDCs induced Foxp3 expression in autologous lymphocytes, while Lb. murinus primed MDDCs induced Foxp3, T-bet and Ror-γt expression. DC-SIGN was required for Lb. rhamnosus JB-1 adhesion and influenced IL-12 secretion, while TLR-2 influenced IL-10 and IL-12 secretion. Here we demonstrate that the delayed kinetics of bacterial processing by MDDCs correlates with MDDC activation and stimulation of lymphocytes. Thus, inhibition or delay of intracellular processing may be a novel strategy by which certain commensals may avoid the induction of proinflammatory responses.     

The N-Terminal GYPSY Motif Is Required for Pilin-Specific Sortase SrtC1 Functionality in Lactobacillus rhamnosus Strain GG

Predominantly identified in pathogenic Gram-positive bacteria, sortase-dependent pili are also found in commensal species, such as the probiotic-marketed strain Lactobacillus rhamnosus strain GG. Pili are typically associated with host colonization, immune signalling and biofilm formation. Comparative analysis of the N-terminal domains of pilin-specific sortases from various piliated Gram-positive bacteria identified a conserved motif, called GYPSY, within the signal sequence. We investigated the function and role of the GYPSY residues by directed mutagenesis in homologous (rod-shaped) and heterologous (coccoid-shaped) expression systems for pilus formation. Substitutions of some of the GYPSY residues, and more specifically the proline residue, were found to have a direct impact on the degree of piliation of Lb. rhamnosus GG. The present findings uncover a new signalling element involved in the functionality of pilin-specific sortases controlling the pilus biogenesis of Lb. rhamnosus GG and related piliated Gram-positive species.     

Lactobacillus plantarum CS24.2 prevents Escherichia coli adhesion to HT‐29 cells and also down‐regulates enteropathogen‐induced tumor necrosis factor‐α and interleukin‐8 expression

The aim of the present study was to evaluate the potential of Lactobacillus plantarum CS24.2 to antagonize Escherichia coli adhesion and modulate expression of the responses by HT‐29 cells of inflammatory molecules to E. coli adhesion. Experiments were performed under different adhesion conditions and findings compared with the responses of Lactobacillus rhamnosus GG. Tests of competitive adhesion, adhesion inhibition and displacement assays were performed for lactobacilli (L. rhamnosus GG and L. plantarum CS24.2) and E. coli O26:H11 to HT‐29 cells. Both the lactobacilli significantly reduced E. coli adhesion to HT‐29 cells (P < 0.05). The ability of lactobacilli to modulate tumor necrosis factor‐α and interleukin‐8 expression was analyzed in HT‐29 cells stimulated with E. coli using qRT‐PCR. L. plantarum CS24.2 significantly down regulated expression of both the genes induced by E. coli in HT‐29 cells at 6 hr as well as 24 hr, which was more significant than the corresponding findings for L. rhamnosus GG. The present findings suggest that L. plantarum CS24.2 inhibits pathogen adhesion to a similar extent as does the established probiotic strain L. rhamnosus GG. It may also attenuate tumor necrosis factor‐α and interleukin‐8 expression in HT‐29 cells stimulated with E. coli.     

Flow Cytometric Testing of Green Fluorescent Protein-Tagged Lactobacillus rhamnosus GG for Response to Defensins

Lactobacillus rhamnosus GG is of general interest as a probiotic. Although L. rhamnosus GG is often used in clinical trials, there are few genetic tools to further determine its mode of action or to develop it as a vehicle for heterologous gene expression in therapy. Therefore, we developed a reproducible, efficient electroporation procedure for L. rhamnosus GG. The best transformation efficiency obtained was 10⁴ transformants per μg of DNA. We validated this protocol by tagging L. rhamnosus GG with green fluorescent protein (GFP) using the nisin-controlled expression (NICE) system. Parameters for overexpression were optimized, which allowed expression of gfp in L. rhamnosus GG upon induction with nisin. The GFP⁺ strain can be used to monitor the survival and behavior of L. rhamnosus GG in vivo. Moreover, implementation of the NICE system as a gene expression switch in L. rhamnosus GG opens up possibilities for improving and expanding the performance of this strain. The GFP-labeled strain was used to demonstrate that L. rhamnosus GG is sensitive to human beta-defensin-2 but not to human beta-defensin-1.     

Lactobacillus rhamnosus BFE 5264 and Lactobacillus plantarum NR74 Promote Cholesterol Excretion Through the Up-Regulation of ABCG5/8 in Caco-2 Cells

The effect of two putative probiotic strains, Lactobacillus rhamnosus BFE5264 and Lactobacillus plantarum NR74, on the control of cholesterol efflux in enterocytes was assessed by focusing on the promotion of ATP-binding cassette sub-family G members 5 and 8 (ABCG5 and ABCG8). Differentiated Caco-2 enterocytes were treated with live bacteria, heat-killed bacteria, a bacterial cell wall fraction, and metabolites and were subjected to cholesterol uptake assay, mRNA analysis, and protein analyses. Following LXR-transfection by incubation with CHO-K1 cells in DNA-lipofectin added media, the luciferase assay was conducted for LXR analysis. Treatment of Caco-2 cells with L. rhamnosus BFE5264 (isolated from traditional fermented Maasai milk) and L. plantarum NR74 (isolated from Korean kimchi) resulted in the up-regulation of LXR, concomitantly with the elevated expression of ABCG5 and ABCG8. This was associated with the promotion of cholesterol efflux at significantly higher levels compared to the positive control strain L. rhamnosus GG (LGG). The experiment with CHO-K1 cells confirmed up-regulation of LXR-beta by the test strains, and treatment with the live L. rhamnosus BFE5264 and L. plantarum NR74 strains significantly increased cholesterol efflux. Heat-killed cells and cell wall fractions of both LAB strains induced the upregulation of ABCG5/8 through LXR activation. By contrast, LAB metabolites did not show any effect on ABCG5/8 and LXR expression. Data from this study suggest that LAB strains, such as L. rhamnosus BFE5264 and L. plantarum NR74, may promote cholesterol efflux in enterocytes, and thus potentially contribute to the prevention of hypercholesterolemia and atherosclerosis.     

A Novel Glutamyl (Aspartyl)-Specific Aminopeptidase A from Lactobacillus delbrueckii with Promising Properties for Application

Lactic acid bacteria (LAB) are auxotrophic for a number of amino acids. Thus, LAB have one of the strongest proteolytic systems to acquit their amino acid requirements. One of the intracellular exopeptidases present in LAB is the glutamyl (aspartyl) specific aminopeptidase (PepA; EC 3.4.11.7). Most of the PepA enzymes characterized yet, belonged to Lactococcus lactis sp., but no PepA from a Lactobacillus sp. has been characterized so far. In this study, we cloned a putative pepA gene from Lb. delbrueckii ssp. lactis DSM 20072 and characterized it after purification. For comparison, we also cloned, purified and characterized PepA from Lc. lactis ssp. lactis DSM 20481. Due to the low homology between both enzymes (30%), differences between the biochemical characteristics were very likely. This was confirmed, for example, by the more acidic optimum pH value of 6.0 for Lb-PepA compared to pH 8.0 for Lc-PepA. In addition, although the optimum temperature is quite similar for both enzymes (Lb-PepA: 60°C; Lc-PepA: 65°C), the temperature stability after three days, 20°C below the optimum temperature, was higher for Lb-PepA (60% residual activity) than for Lc-PepA (2% residual activity). EDTA inhibited both enzymes and the strongest activation was found for CoCl₂, indicating that both enzymes are metallopeptidases. In contrast to Lc-PepA, disulfide bond-reducing agents such as dithiothreitol did not inhibit Lb-PepA. Finally, Lb-PepA was not product-inhibited by L-Glu, whereas Lc-PepA showed an inhibition.     

Screening of Probiotic Activities of Forty-Seven Strains of Lactobacillus spp. by In Vitro Techniques and Evaluation of the Colonization Ability of Five Selected Strains in Humans

The probiotic potential of 47 selected strains of Lactobacillus spp. was investigated. The strains were examined for resistance to pH 2.5 and 0.3% oxgall, adhesion to Caco-2 cells, and antimicrobial activities against enteric pathogenic bacteria in model systems. From the results obtained in vitro, five strains, Lactobacillus rhamnosus 19070-2, L. reuteri DSM 12246, L. rhamnosus LGG, L. delbrueckii subsp. lactis CHCC 2329, and L. casei subsp. alactus CHCC 3137, were selected for in vivo studies. The daily consumption by 12 healthy volunteers of two doses of 10¹⁰ freeze-dried bacteria of the selected strains for 18 days was followed by a washout period of 17 days. Fecal samples were taken at days 0 and 18 and during the washout period at days 5 and 11. Lactobacillus isolates were initially identified by API 50CHL and internal transcribed spacer PCR, and their identities were confirmed by restriction enzyme analysis in combination with pulsed-field gel electrophoresis. Among the tested strains, L. rhamnosus 19070-2, L. reuteri DSM 12246, and L. rhamnosus LGG were identified most frequently in fecal samples; they were found in 10, 8, and 7 of the 12 samples tested during the intervention period, respectively, whereas reisolations were less frequent in the washout period. The bacteria were reisolated in concentrations from 10⁵ to 10⁸ cells/g of feces. Survival and reisolation of the bacteria in vivo appeared to be linked to pH tolerance, adhesion, and antimicrobial properties in vitro.     

Strain-Dependent Augmentation of Tight-Junction Barrier Function in Human Primary Epidermal Keratinocytes by Lactobacillus and Bifidobacterium Lysates

In this study, we investigated whether probiotic lysates can modify the tight-junction function of human primary keratinocytes. The keratinocytes were grown on cell culture inserts and treated with lysates from Bifidobacterium longum, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus fermentum, or Lactobacillus rhamnosus GG. With the exception of L. fermentum (which decreased cell viability), all strains markedly enhanced tight-junction barrier function within 24 h, as assessed by measurements of transepithelial electrical resistance (TEER). However, B. longum and L. rhamnosus GG were the most efficacious, producing dose-dependent increases in resistance that were maintained for 4 days. These increases in TEER correlated with elevated expression of tight-junction protein components. Neutralization of Toll-like receptor 2 abolished both the increase in TEER and expression of tight-junction proteins induced by B. longum, but not L. rhamnosus GG. These data suggest that some bacterial strains increase tight-junction function via modulation of protein components but the different pathways involved may vary depending on the bacterial strain.     

Identification and Application of Plasmids Suitable for Transfer of Foreign DNA to Members of the Genus Gordonia

Gene transfer systems for Gordonia polyisoprenivorans strains VH2 and Y2K based on electroporation and conjugation, respectively, were established. Several parameters were optimized, resulting in transformation efficiencies of >4 × 10⁵ CFU/μg of plasmid DNA. In contrast to most previously described electroporation protocols, the highest efficiencies were obtained by applying a heat shock after the intrinsic electroporation. Under these conditions, transfer and autonomous replication of plasmid pNC9503 was also demonstrated to proceed in G. alkanivorans DSM44187, G. nitida DSM44499^T, G. rubropertincta DSM43197^T, G. rubropertincta DSM46038, and G. terrae DSM43249^T. Conjugational plasmid DNA transfer to G. polyisoprenivorans resulted in transfer frequencies of up to 5 × 10⁻⁶ of the recipient cells. Recombinant strains capable of polyhydroxyalkanoate synthesis from alkanes were constructed.     

A Selected Lactobacillus rhamnosus Strain Promotes EGFR-Independent Akt Activation in an Enterotoxigenic Escherichia coli K88-Infected IPEC-J2 Cell Model

Enterotoxigenic Escherichia coli (ETEC) are important intestinal pathogens that cause diarrhea in humans and animals. Although probiotic bacteria may protect against ETEC-induced enteric infections, the underlying mechanisms are unknown. In this study, porcine intestinal epithelial J2 cells (IPEC-J2) were pre-incubated with and without Lactobacillus rhamnosus ATCC 7469 and then exposed to F4⁺ ETEC. Increases in TLR4 and NOD2 mRNA expression were observed at 3 h after F4⁺ ETEC challenge, but these increases were attenuated by L. rhamnosus treatment. Expression of TLR2 and NOD1 mRNA was up-regulated in cells pre-treated with L. rhamnosus. Pre-treatment with L. rhamnosus counteracted F4⁺ ETEC-induced increases in TNF-α concentration. Increased PGE₂. concentrations were observed in cells infected with F4⁺ ETEC and in cells treated with L. rhamnosus only. A decrease in phosphorylated epidermal growth factor receptor (EGFR) was observed at 3 h after F4⁺ ETEC challenge in cells treated with L. rhamnosus. Pre-treatment with L. rhamnosus enhanced Akt phosphorylation and increased ZO-1 and occludin protein expression. Our findings suggest that L. rhamnosus protects intestinal epithelial cells from F4⁺ ETEC-induced damage, partly through the anti-inflammatory response involving synergism between TLR2 and NOD1. In addition, L. rhamnosus promotes EGFR-independent Akt activation, which may activate intestinal epithelial cells in response to bacterial infection, in turn increasing tight junction integrity and thus enhancing the barrier function and restricting pathogen invasion. Pre-incubation with L. rhamnosus was superior to co-incubation in reducing the adhesion of F4⁺ ETEC to IPEC-J2 cells and subsequently attenuating F4⁺ ETEC-induced mucin layer destruction and suppressing apoptosis. Our data indicate that a selected L. rhamnosus strain interacts with porcine intestinal epithelial cells to maintain the epithelial barrier and promote intestinal epithelial cell activation in response to bacterial infection, thus protecting cells from the deleterious effects of F4⁺ ETEC.     

Survival of Lactobacillus reuteri DSM 17938 and Lactobacillus rhamnosus GG in the Human Gastrointestinal Tract with Daily Consumption of a Low-Fat Probiotic Spread

Probiotics are live microorganisms which, when administered in adequate amounts, confer a health benefit on the host. Therefore, probiotic strains should be able to survive passage through the human gastrointestinal tract. Human gastrointestinal tract survival of probiotics in a low-fat spread matrix has, however, never been tested. The objective of this randomized, double-blind, placebo-controlled human intervention study was to test the human gastrointestinal tract survival of Lactobacillus reuteri DSM 17938 and Lactobacillus rhamnosus GG after daily consumption of a low-fat probiotic spread by using traditional culturing, as well as molecular methods. Forty-two healthy human volunteers were randomly assigned to one of three treatment groups provided with 20 g of placebo spread (n = 13), 20 g of spread with a target dose of 1 × 10⁹ CFU of L. reuteri DSM 17938 (n = 13), or 20 g of spread with a target dose of 5 × 10⁹ CFU of L. rhamnosus GG (n = 16) daily for 3 weeks. Fecal samples were obtained before and after the intervention period. A significant increase, compared to the baseline, in the recovery of viable probiotic lactobacilli in fecal samples was demonstrated after 3 weeks of daily consumption of the spread containing either L. reuteri DSM 17938 or L. rhamnosus GG by selective enumeration. In the placebo group, no increase was detected. The results of selective enumeration were supported by quantitative PCR, detecting a significant increase in DNA resulting from the probiotics after intervention. Overall, our results indicate for the first time that low-fat spread is a suitable carrier for these probiotic strains.The human intestinal microflora or microbiota constitutes a metabolically active microbial environment. This community is relatively stable in the guts of healthy individuals (20). Some of the microbial groups harbor species that are potentially harmful, whereas others, such as the bifidobacteria and lactobacilli, are regarded as beneficial (8). Specific members of the genera Lactobacillus and Bifidobacterium are being applied in functional foods as probiotics (25). Probiotics are live microorganisms which, when administered in adequate amounts, confer a health benefit on the host (9). The current scientific consensus is that probiotics should be alive to exert their beneficial effect in the human gastrointestinal (GI) tract (6). Consequently, probiotics should remain alive in the product, such that the daily effective dose per serving is still present at the end of the shelf life (14). Food matrices, production processes, or product usages that involve heating can affect the viability of probiotics (24).Typically, those members selected for probiotic application are chosen for their resistance to passage through the upper GI tract and thus are able to transiently colonize the gut (25). Human GI tract survival of probiotics should lead to shedding of live cells in fecal samples. GI tract survival is, however, dependent on both the strain and the food matrix involved (27). Fecal recovery of several probiotic strains has been demonstrated in different food matrices, including fermented milk and yoghurt (10, 26, 29), fruit drinks (21), a cereal bar (22), supplements (13, 17, 27), and infant formula (29).For this study, we have selected two well-established probiotic strains to test the suitability of a low-fat spread as a probiotic carrier, namely, Lactobacillus reuteri DSM 17938 (BioGaia, Sweden) and Lactobacillus rhamnosus GG (ATCC 53103; Valio, Finland). L. reuteri DSM 17938 was derived from L. reuteri ATCC 55730 by curing of two plasmids harboring antibiotic resistance genes (23). A series of in vitro experiments confirmed the retention of the functional properties of the daughter strain, as no differences in colony morphology, fermentation patterns, production of reuterin, generation time, mucus-binding ability, or tolerance to bovine bile were found between L. reuteri ATCC 55730 and DSM 17938. The daughter strain is somewhat more resistant to low pH and grows to a higher density in vitro (23). Several studies have been published which provide data on the survival of L. reuteri ATCC 55730 in the human GI tract at doses of 4 × 10⁸ to 1 × 10¹⁰ CFU/day in freeze-dried matrices and chewable tablets (32-34). Furthermore, L. reuteri DSM 17938 was demonstrated to survive human GI tract passage in the same way as L. reuteri ATCC 55730 (23).L. rhamnosus GG has been isolated from a healthy human intestinal flora by Goldin et al. (10). L. rhamnosus GG is relatively resistant to acid and bile, adheres in vitro to epithelial cells, and can produce an antimicrobial substance (10, 15). A wide range of studies have been published which provide data on the survival of L. rhamnosus GG in the human GI tract (3, 4, 10, 18, 19, 27-30), as well as transient colonization of the intestinal microbiota in healthy adults in various formats, including freeze-dried powder, capsules, and tablets or via fermented milk drinks, yoghurt, or fruit juice. Saxelin et al. (28) evaluated the dose-response effect of orally administered L. rhamnosus GG in powder form on fecal colonization in healthy adults, which indicated that consumption of approximately 10¹⁰ to 10¹¹ CFU/day was required to reach detectable levels in fecal samples from volunteers. This was also the case when L. rhamnosus GG was administered in gelatin capsules (29). Additionally, Saxelin et al. (27) observed that milk, but possibly also other protective compounds in food, can improve the survival of L. rhamnosus GG. Fecal recovery of L. rhamnosus GG in milk-based products was shown at dose levels of around 2 × 10⁹ CFU/day.It is, however, not known whether probiotics can survive passage through the human GI tract after the consumption of a low-fat spread. The objective of this randomized, double-blind, placebo-controlled human intervention study was therefore to test the human GI tract survival of L. reuteri DSM 17938 and L. rhamnosus GG after daily consumption of a low-fat probiotic spread by using traditional culturing, as well as molecular methods. The primary outcome parameter of this study was a significant change from the baseline in the number of probiotic bacteria of the respective strains in fecal samples.     

Glutathione Reductase from Lactobacillus sanfranciscensis DSM20451T: Contribution to Oxygen Tolerance and Thiol Exchange Reactions in Wheat Sourdoughs

The effect of the glutathione reductase (GshR) activity of Lactobacillus sanfranciscensis DSM20451^T on the thiol levels in fermented sourdoughs was determined, and the oxygen tolerance of the strain was also determined. The gshR gene coding for a putative GshR was sequenced and inactivated by single-crossover integration to yield strain L. sanfranciscensis DSM20451^TΔgshR. The gene disruption was verified by sequencing the truncated gshR and surrounding regions on the chromosome. The gshR activity of L. sanfranciscensis DSM20451^TΔgshR was strongly reduced compared to that of the wild-type strain, demonstrating that gshR indeed encodes an active GshR enzyme. The thiol levels in wheat doughs fermented with L. sanfranciscensis DSM20451 increased from 9 μM to 10.5 μM sulfhydryl/g of dough during a 24-h sourdough fermentation, but in sourdoughs fermented with L. sanfranciscensis DSM20451^TΔgshR and in chemically acidified doughs, the thiol levels decreased to 6.5 to 6.8 μM sulfhydryl/g of dough. Remarkably, the GshR-negative strains Lactobacillus pontis LTH2587 and Lactobacillus reuteri BR11 exerted effects on thiol levels in dough comparable to those of L. sanfranciscensis. In addition to the effect on thiol levels in sourdough, the loss of GshR activity in L. sanfranciscensis DSM20451^TΔgshR resulted in a loss of oxygen tolerance. The gshR mutant strain exhibited a strongly decreased aerobic growth rate on modified MRS medium compared to either the growth rate under anaerobic conditions or that of the wild-type strain, and aerobic growth was restored by the addition of cysteine. Moreover, the gshR mutant strain was more sensitive to the superoxide-generating agent paraquat.     

Lactobacillus rhamnosus GG Inhibits the Toxic Effects of Staphylococcus aureus on Epidermal Keratinocytes

Few studies have evaluated the potential benefits of the topical application of probiotic bacteria or material derived from them. We have investigated whether a probiotic bacterium, Lactobacillus rhamnosus GG, can inhibit Staphylococcus aureus infection of human primary keratinocytes in culture. When primary human keratinocytes were exposed to S. aureus, only 25% of the keratinocytes remained viable following 24 h of incubation. However, in the presence of 10⁸ CFU/ml of live L. rhamnosus GG, the viability of the infected keratinocytes increased to 57% (P = 0.01). L. rhamnosus GG lysates and spent culture fluid also provided significant protection to keratinocytes, with 65% (P = 0.006) and 57% (P = 0.01) of cells, respectively, being viable following 24 h of incubation. Keratinocyte survival was significantly enhanced regardless of whether the probiotic was applied in the viable form or as cell lysates 2 h before or simultaneously with (P = 0.005) or 12 h after (P = 0.01) S. aureus infection. However, spent culture fluid was protective only if added before or simultaneously with S. aureus. With respect to mechanism, both L. rhamnosus GG lysate and spent culture fluid apparently inhibited adherence of S. aureus to keratinocytes by competitive exclusion, but only viable bacteria or the lysate could displace S. aureus (P = 0.04 and 0.01, respectively). Furthermore, growth of S. aureus was inhibited by either live bacteria or lysate but not spent culture fluid. Together, these data suggest at least two separate activities involved in the protective effects of L. rhamnosus GG against S. aureus, growth inhibition and reduction of bacterial adhesion.     

Survival of Probiotic Lactobacilli in Acidic Environments Is Enhanced in the Presence of Metabolizable Sugars

Lactobacillus rhamnosus GG is an industrially significant probiotic strain with proven health benefits. In this study, the effect of glucose on L. rhamnosus GG survival was analyzed in simulated gastric juice at pH 2.0. It was found that the presence of 19.4 mM glucose resulted in up to 6-log₁₀-enhanced survival following 90 min of exposure. Further work with dilute HCl confirmed that glucose was the sole component responsible. Comparative analysis with other Lactobacillus strains revealed that enhanced survival was apparent in all strains, but at different pH values. The presence of glucose at concentrations from 1 to 19.4 mM enhanced L. rhamnosus GG survival from 6.4 to 8 log₁₀ CFU ml⁻¹ in simulated gastric juice. The mechanisms behind the protective effect of glucose were investigated. Addition of N′,N′-dicyclohexylcarbodiimide to simulated gastric juice caused survival to collapse, which was indicative of a prominent role in inhibition of F₀F₁-ATPase. Further work with neomycin-resistant mutants that exhibited 38% to 48% of the F₀F₁-ATPase activity of the parent confirmed this, as the survival in the presence of glucose of these mutants decreased 3 × 10⁶-fold compared with the survival of the wild type (which had a viability of 8.02 log₁₀ CFU ml⁻¹). L. rhamnosus GG survival in acidic conditions occurred only in the presence of sugars that it could metabolize efficiently. To confirm the involvement of glycolysis in the glucose effect, iodoacetic acid was used to inhibit glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity. The reduction in GAPDH activity caused survival to decrease by 8.30 log₁₀ CFU ml⁻¹ in the presence of glucose. The data indicate that glucose provides ATP to F₀F₁-ATPase via glycolysis, enabling proton exclusion and thereby enhancing survival during gastric transit.     

Paenibacillus taohuashanense sp. nov., a nitrogen-fixing species isolated from rhizosphere soil of the root of Caragana kansuensis Pojark

A nitrogen-fixing bacterium, designated strain gs65^T, was isolated from a rhizosphere soil sample of Caragana kansuensis Pojark. Phylogenetic analysis based on a fragment of the nifH gene and the full-length 16S rRNA gene sequence revealed that strain gs65^T is a member of the genus Paenibacillus. High levels of 16S rRNA gene similarity were found between strain gs65^T and Paenibacillus borealis DSM 13188^T (97.5 %), Paenibacillus odorifer ATCC BAA-93^T (97.3 %), Paenibacillus durus DSM 1735^T (97.0 %) and Paenibacillus sophorae DSM23020^T (96.9 %). Levels of 16S rRNA gene sequence similarity between strain gs65^T and the type strains of other recognized members of the genus Paenibacillus were below 97.0 %. Levels of DNA–DNA relatedness between strain gs65^T and P. borealis DSM 13188^T, P. odorifer ATCC BAA-93^T (97.3 %), P. durus DSM 1735^T and P. sophorae DSM23020^T were 35.9, 38.0, 34.2 and 35.5 % respectively. The DNA G+C content of strain gs65^T was determined to be 51.6 mol%. The major fatty acids were found to be iso-C_14:0, anteiso-C_15:0 and iso-C_16:0. On the basis of its phenotypic characteristics and levels of DNA–DNA hybridization, strain gs65^T is considered to represent a novel species of the genus Paenibacillus, for which the name Paenibacillus taohuashanense sp. nov. is proposed. The type strain is gs65^T (=CGMCC 1.12175^T = DSM 25809^T).     

Binding Rather Than Metabolism May Explain the Interaction of Two Food-Grade Lactobacillus Strains with Zearalenone and Its Derivative ά-Zearalenol

The interaction between two Fusarium mycotoxins, zearalenone (ZEN) and its derivative ¯α-zearalenol (¯α-ZOL), with two food-grade strains of Lactobacillus was investigated. The mycotoxins (2 μg ml⁻¹) were incubated with either Lactobacillus rhamnosus strain GG or L. rhamnosus strain LC705. A considerable proportion (38 to 46%) of both toxins was recovered from the bacterial pellet, and no degradation products of ZEN and ¯α-ZOL were detected in the high-performance liquid chromatograms of the supernatant of the culturing media and the methanol extract of the pellet. Both heat-treated and acid-treated bacteria were capable of removing the toxins, indicating that binding, not metabolism, is the mechanism by which the toxins are removed from the media. Binding of ZEN or ¯α-ZOL by lyophilized L. rhamnosus GG and L. rhamnosus LC705 was a rapid reaction: approximately 55% of the toxins were bound instantly after mixing with the bacteria. Binding was dependent on the bacterial concentration, and coincubation of ZEN with ¯α-ZOL significantly affected the percentage of the toxin bound, indicating that these toxins may share the same binding site on the bacterial surface. These results can be exploited in developing a new approach for detoxification of mycotoxins from foods and feeds.     

Vagococcus teuberi sp. nov., isolated from the Malian artisanal sour milk fènè

Ten bacterial isolates belonging to the genus Vagococcus were obtained from Malian sour milk fènè produced from spontaneously fermented cow milk. However, these isolates could not be assigned to a species upon initial comparative 16S rRNA gene sequence analysis and were therefore further characterized. Rep-PCR fingerprinting of the isolates yielded four strain clusters represented by strains CG-21^T (=DSM 21459^T), 24CA, CM21 and 9H. Sequence identity of the 16S rRNA gene of DSM 21459^T to its closest relative species Vagococcus penaei was 97.9%. Among the four rep strain clusters, DSM 21459^T and 24CA shared highest 16S rRNA gene sequence identity of 99.6% while CM21 and 9H shared 98.6–98.8% with DSM 21459^T and V. penaei CD276^T. DSM 21459^T and 24CA were thus subjected to a polyphasic typing approach. The genome of DSM 21459^T featured a G + C content of 34.1 mol% for a 2.17-bp chromosome and a 15-kbp plasmid. Average nucleotide identity (ANI) of DSM 21459^T to Vagococcus fluvialis bH819, V. penaei CD276^T were 72.88%, 72.63%, respectively. DNA–DNA hybridization (DDH) similarities of strain DSM 21459^T to other Vagococcus species were <42.0%. ANI and DDH findings strongly supported the 16S rRNA gene phylogenetic tree delineations. The fatty acid patterns of DSM 21459^T was palmitic acid (C _16:0, 24.5%), oleic acid (C _18:1-ω9c, 32.8%), stearic acid (C _18:0, 18.9%). General physiological characterization of DSM 21459^T and 24CA were consistent with those of the genus Vagococcus. Strain DSM 21459^T and further strains are therefore considered to belong to a novel species, for which the nomenclature Vagococcus teuberi sp. nov. is proposed. The type strain is named CG-21^T (=DSM 21459^T and LMG 24695^T).     

Surface Binding of Aflatoxin B1 by Lactic Acid Bacteria

Specific lactic acid bacterial strains remove toxins from liquid media by physical binding. The stability of the aflatoxin B₁ complexes formed with 12 bacterial strains in both viable and nonviable (heat- or acid-treated) forms was assessed by repetitive aqueous extraction. By the fifth extraction, up to 71% of the total aflatoxin B₁ remained bound. Nonviable bacteria retained the highest amount of aflatoxin B₁. Lactobacillus rhamnosus strain GG (ATCC 53103) and L. rhamnosus strain LC-705 (DSM 7061) removed aflatoxin B₁ from solution most efficiently and were selected for further study. The accessibility of bound aflatoxin B₁ to an antibody in an indirect competitive inhibition enzyme-linked immunosorbent assay suggests that surface components of these bacteria are involved in binding. Further evidence is the recovery of around 90% of the bound aflatoxin from the bacteria by solvent extraction. Autoclaving and sonication did not release any detectable aflatoxin B₁. Variation in temperature (4 to 37°C) and pH (2 to 10) did not have any significant effect on the amount of aflatoxin B₁ released. Binding of aflatoxin B₁ appears to be predominantly extracellular for viable and heat-treated bacteria. Acid treatment may permit intracellular binding. In all cases, binding is of a reversible nature, but the stability of the complexes formed depends on strain, treatment, and environmental conditions.     

Adhesion of the probiotic strains <Emphasis Type="Italic">Enterococcus mundtii</Emphasis> ST4SA and <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> 423 to Caco-2 cells under conditions simulating the intestinal tract,and in the presence of antibiotics and anti-inflammatory medicaments

Adhesion of Enterococcus mundtii ST4SA and Lactobacillus plantarum 423 to Caco-2 (human carcinoma epithelial) cells was visualized by fluorescent staining. Both strains showed good adhesion compared to L. casei MB1, L. casei Shirota, L. johnsonii La1 and L. rhamnosus GG. No correlation was found between hydrophobicity, aggregation and adhesion to Caco-2 cells. Presence of antibiotics and anti-inflammatory medicaments reduced adhesion of bacterial strains to Caco-2 cells. Proteins sensitive to pepsin, trypsin and pronase are involved in the adhesion of E. mundtii ST4SA and L. plantarum 423 to Caco-2 cells. Adhesion of Listeria monocytogenes ScottA to Caco-2 cells was not prevented by E. mundtii ST4SA and L. plantarum 423. Cell-free culture supernatants of strains ST4SA and 423, containing the antimicrobial peptides plantaricin 423 and peptide ST4SA, prevented the invasion of L. monocytogenes ScottA into Caco-2 cells.