A metagenomic study of the preventive effect of Lactobacillus rhamnosus GG on intestinal polyp formation in ApcMin/+ mice.

AIMS: To investigate the in vivo effects of Lactobacillus rhamnosus GG (LGG) on intestinal polyp development and the interaction between this single-organism probiotic and the gut microbiota therein. METHODS AND RESULTS: The ApcMin/+ mouse model was used to study the potential preventive effect of LGG on intestinal polyposis, while shotgun metagenomic sequencing was employed to characterize both taxonomic and functional changes within the gut microbial community. We found that the progression of intestinal polyps in the control group altered the community functional profile remarkably despite small variation in the taxonomic diversity. In comparison, the consumption of LGG helped maintain the overall functional potential and taxonomic profile in the resident microbes, thereby leading to a 25% decrease of total polyp counts. Furthermore, we found that LGG enriched those microbes or microbial activities related to short-chain fatty acid production (e.g. Roseburia and Coprococcus), as well as suppressed the ones that can lead to inflammation (e.g. Bilophila wadsworthia). CONCLUSIONS: Our study using shotgun metagenomics highlights how single probiotic LGG may exert its beneficial effects and decrease polyp formation in mice by maintaining gut microbial functionality. SIGNIFICANCE AND IMPACT OF THE STUDY: This probiotic intervention targeting microbiota may be used in conjugation with other dietary supplements or drugs as part of prevention strategies for early-stage colon cancer, after further clinical validations in human.

Modulation of Intestinal Epithelial Defense Responses by Probiotic Bacteria.

Probiotics are live microorganisms, which when administered in food confer numerous health benefits. In previous studies about beneficial effects of probiotic bacteria to health, particularly in the fields of intestinal mucosa defense responses, specific probiotics, in a strain-dependent manner, show certain degree of potential to reinforce the integrity of intestinal epithelium and/or regulate some immune components. The mechanism of probiotic action is an area of interest. Among all possible routes of modulation by probiotics of intestinal epithelial cell-mediated defense responses, modulations of intestinal barrier function, innate, and adaptive mucosal immune responses, as well as signaling pathways are considered to play important role in the intestinal defense responses against pathogenic bacteria. This review summarizes the beneficial effects of probiotic bacteria to intestinal health together with the mechanisms affected by probiotic bacteria: barrier function, innate, and adaptive defense responses such as secretion of mucins, defensins, trefoil factors, immunoglobulin A (IgA), Toll-like receptors (TLRs), cytokines, gut associated lymphoid tissues, and signaling pathways.

Characterisation of aflatoxin and deoxynivalenol exposure among pregnant Egyptian women.

Mycotoxins such as the aflatoxins and deoxynivalenol (DON) are frequent contaminants of food. Aflatoxin B1 (AFB1) and DON affect the immune system and restrict growth; additionally AFB1 is carcinogenic. To date there are limited descriptive biomarker data concerning maternal exposures during pregnancy, and none on co-exposures to these mycotoxins. This survey was a cross-sectional assessment providing descriptive data on the concentrations of serum aflatoxin-albumin (AF-alb), urinary aflatoxin M1 (AFM1), and urinary DON for 98 pregnant women from Egypt, in relation to diet and socioeconomic status, during the third trimester. AF-alb was detected in 34 of 98 (35%) samples, geometric mean (GM) of positives = 4.9 pg AF-lys mg(-1) albumin (95% confidence interval (CI) = 4.1-5.8 pg mg(-1)), and AFM1 in 44 of 93 (48%) samples, GM of positives = 19.7 pg mg(-1) creatinine (95%CI = 14.8-26.3 pg mg(-1)). AF-alb and AFM1 levels were positively correlated (R = 0.276, p = 0.007). DON was detected in 63 of 93 (68%), GM of positives = 2.8 ng mg(-1) (95%CI = 2.1-3.6 ng mg(-1)). Aflatoxin and DON biomarkers were observed in 41% of the subjects concurrently. The frequency and level of these biomarkers in Egyptian women were modest compared with known high-risk countries. However, this study represents the first biomarker survey to report on the occurrence of DON biomarkers in an African population, in addition to the co-occurrence of these two potent mycotoxins. This combined exposure may be of particular concern during pregnancy given the potential of toxin transfer to the foetus.

Transplacental transfer of melamine.

OBJECTIVE: To characterize transplacental transfer of melamine and related mechanisms as well as toxicity using human placental perfusion and cultured cells. METHODS: Transfer and toxicity were analyzed in 4-h perfusions with 10 µM or 1 mM melamine, or 10 µM melamine with 10 nM cyanuric acid (CYA). Efflux transporters were studied in accumulation assay and toxicity in BeWo cells by MTT assay. RESULTS: Of added melamine 34-45% was transferred to fetal circulation and CYA made no difference. Histology, hCG production, and PLAP activity indicated functionality of placental tissue with no grave toxicity. Highest concentration of melamine used (2 mM) with CYA and long treatment time decreased viability of BeWo cells. Inhibitors of ABCB1, ABCG2, ABCC2 did not affect the accumulation of melamine in cells. CONCLUSION: Melamine goes through human term placenta with no contribution of efflux transporters. Toxicity of melamine is low in placental tissue and BeWo cells.

Lactobacillus rhamnosus strain GG restores alkaline phosphatase activity in differentiating Caco-2 cells dosed with the potent mycotoxin deoxynivalenol.

Deoxynivalenol (DON) contamination of cereal crops occurs frequently, and may cause acute exposure at high levels or chronic more moderate exposure. DON has proven toxicity including restriction of enterocyte differentiation, which may play a part in DON induced gastroenteritis. The probiotic bacteria Lactobacillus rhamnosus strain GG (GG) can bind DON, and therefore potentially restrict bioavailability of this toxin. Binding efficacy is not significantly altered by heat treatment, and therefore this in vitro study evaluated whether heat inactivated GG could restore the differentiation process in Caco-2 cells, using alkaline phosphatase (ALP) activity as a marker of differentiation. DON (200ng/mL) caused a significant (p<0.001) 36% reduction in ALP activity (1598+/-137U/mg protein) compared to untreated cells (2502+/-80U/mg). A dose dependant restoration of ALP activity was observed where DON treated cells were co-incubated with heat inactivated GG (1719+/-84; 2007+/-142; 2272+/-160U/mg for GG at 1x10(4) (p>0.9), 1x10(7) (p<0.001), and 1x10(10)CFU/mL (p<0.001), respectively). Co-incubation of the non-binding strain, LC-705 (1x10(10)CFU/mL), with DON did not significantly restore the ALP (1841+/-97U/mg, p<0.077) compared to DON only treated cells. When viable GG were co-incubated with DON a similar restoration of ALP activity was observed as seen for heat inactivated GG. These combined data suggest that the major effect of GG on restoring ALP activity, and therefore Caco-2 cell differentiation, was due to specific binding of DON, with possibly a more minor role of non-specific bacterial interference.

Combining strains of lactic acid bacteria may reduce their toxin and heavy metal removal efficiency from aqueous solution.

AIMS: The primary objective of this study was to compare the removal of cadmium, lead, aflatoxin B1 and microcystin-LR from aqueous solution by Lactobacillus rhamnosus GG, L. rhamnosus LC705, Propionibacterium freudenreichii shermanii JS and Bifidobacterium breve Bbi99/E8, separately and in combination. METHODS AND RESULTS: The removal of toxins and heavy metals was assessed in batch experiments. The removal of all compounds was observed to be strain specific. The removal of lead by a combination of all the strains used was observed to be lower than could be predicted from the removal by single strains (P < 0.05). A similar trend was also observed with the other compounds studied. CONCLUSIONS: The results show that the toxin-removal capacity of a combination of strains of lactic acid bacteria is not the sum of their individual capacities. Therefore, pure single strains should be used when the goal is to remove single compounds. The use of combinations of strains may be beneficial when several compounds are removed together. This needs to be studied in future experiments. SIGNIFICANCE AND IMPACT OF THE STUDY: Lactic acid bacteria have been identified as potent tools for the decontamination of heavy metals, cyanotoxins and mycotoxins. The results of this study should be considered when selecting combinations of bacteria for the simultaneous removal of several toxic compounds.

Lactobacillus rhamnosus strain GG reduces aflatoxin B1 transport, metabolism, and toxicity in Caco-2 Cells.

The probiotic Lactobacillus rhamnosus GG is able to bind the potent hepatocarcinogen aflatoxin B1 (AFB1) and thus potentially restrict its rapid absorption from the intestine. In this study we investigated the potential of GG to reduce AFB1 availability in vitro in Caco-2 cells adapted to express cytochrome P-450 (CYP) 3A4, such that both transport and toxicity could be assessed. Caco-2 cells were grown as confluent monolayers on transmembrane filters for 21 days prior to all studies. AFB1 levels in culture medium were measured by high-performance liquid chromatography. In CYP 3A4-induced monolayers, AFB1 transport from the apical to the basolateral chamber was reduced from 11.1%+/-1.9% to 6.4%+/-2.5% (P=0.019) and to 3.3%+/-1.8% (P=0.002) within the first hour in monolayers coincubated with GG (1x10(10) and 5x10(10) CFU/ml, respectively). GG (1x10(10) and 5x10(10) CFU/ml) bound 40.1%+/-8.3% and 61.0%+/-6.0% of added AFB1 after 1 h, respectively. AFB1 caused significant reductions of 30.1% (P=0.01), 49.4% (P=0.004), and 64.4% (P<0.001) in transepithelial resistance after 24, 48, and 72 h, respectively. Coincubation with 1x10(10) CFU/ml GG after 24 h protected against AFB1-induced reductions in transepithelial resistance at both 24 h (P=0.002) and 48 h (P=0.04). DNA fragmentation was apparent in cells treated only with AFB1 cells but not in cells coincubated with either 1x10(10) or 5x10(10) CFU/ml GG. GG reduced AFB1 uptake and protected against both membrane and DNA damage in the Caco-2 model. These data are suggestive of a beneficial role of GG against dietary exposure to aflatoxin.

Lactobacillus rhamnosus strain GG modulates intestinal absorption, fecal excretion, and toxicity of aflatoxin B(1) in rats.

In this study, the modulation of aflatoxin B(1) (AFB(1)) uptake in rats by administration of the probiotic Lactobacillus rhamnosus GG was demonstrated. Fecal AFB(1) excretion in GG-treated rats was increased via bacterial AFB(1) binding. Furthermore, AFB(1)-associated growth faltering and liver injury were alleviated with GG treatment.

Aflatoxin B1 binding by a mixture of Lactobacillus and Propionibacterium: in vitro versus ex vivo.

Aflatoxin B1 (AFB) is a well-known carcinogen and reducing its bioavailability is of great interest for human and animal health. Several probiotic bacteria are able to bind AFB1 in vitro, including Lactobacillus rhamnosus LC-705 and Propionibacterium freudenreichii subsp. shermanii JS. A mixture of these two probiotics is used by the food and feed industry as biopreservative (Bioprofit), making it a promising candidate for future applications. Consequently, this study aims to investigate the in vitro and ex vivo ability of this probiotic mixture to bind AFB1. For in vitro experiments, probiotic mixture was suspended in an AFB1 solution (5 microM), incubated for 1 to 30 min, centrifuged, and AFB1 residues were quantitated in supernatant and pellet. For ex vivo experiments, duodenal loops of chicks were ligated and injected with either AFB1 solution alone or probiotic mixture suspension and AFB1 solution. Lumen content was centrifuged and AFB1 was quantitated in supernatant and pellet. Additionally, AFB1 was extracted from duodenal tissue to calculate tissue uptake. In vitro, 57 to 66% of AFB1 was removed from the solution by the probiotic mixture, but only 38 to 47% could be extracted from the bacterial surface. In ex vivo experiments, only up to 25% of AFB1 was bound by bacteria, and tissue uptake of AFB1 was significantly reduced when probiotic bacteria were present in the duodenal loop. Furthermore, the effect of intestinal mucus on the bacterial binding ability was investigated in vitro and was found to significantly reduce AFB1 binding by the probiotic mixture. However, probiotic mixture could only retard but not prevent AFB1 absorption in duodenal loops. Further work needs to assess the potential of probiotics in different experimental setups.

Intestinal mucus alters the ability of probiotic bacteria to bind aflatoxin B1 in vitro.

Several probiotics are known to bind aflatoxin B(1) (AFB(1)) to their surfaces and to adhere to intestinal mucus. In this study, preincubation of two probiotic preparations with either AFB(1) or mucus reduced the subsequent surface binding of mucus and AFB(1), respectively, in a strain-dependent manner.

Kinetics of adsorption and desorption of aflatoxin B1 by viable and nonviable bacteria.

The reactions involved in the binding (adsorption) and release (desorption) of aflatoxin B1 (AFB1) to and from the surface of bacteria were investigated. Viable and heat-killed Lactobacillus rhamnosus GG, L. rhamnosus LC-705, and Propionibacterium freudenreichii subsp. shermanii JS were incubated in phosphate-buffered saline containing variable concentrations (0.0017 to 13.3 microg/ml) of AFB1. The relationship between the bacterial surface hydrophobicity and the AFB1 adsorption affinity was also investigated. A linear relationship was observed between the specific rate of AFB1 adsorption and the AFB1 concentration for all bacteria. The nature of desorption of adsorbed AFB1 was investigated by repetitive aqueous washes. A linear relationship was observed between the natural log value of the concentration of AFB1 adsorbed and the number of washes for all bacteria studied. The desorption constants were strain-dependent and were lower for heat-killed bacteria than for viable bacteria. Heat treatment appears to alter the surface properties of the bacteria rather than expose new adsorption sites. No correlation was found between the hydrophobicity and the AFB1 adsorption affinity.

Removal of common Fusarium toxins in vitro by strains of Lactobacillus and Propionibacterium.

This study was conducted to examine the ability of selected strains of Lactobacillus and Propionibacterium to remove common Fusarium toxins, trichothecenes, from liquid media. The trichothecenes studied were deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-AcDON), nivalenol (NIV), fusarenon (FX), diacetoxyscirpenol (DAS), T-2 toxin (T-2) and HT-2 toxin (HT-2). The Lactobacillus rhamnosus strain GG (LGG), Lactobacillus rhamnosus strain LC-705 (LC-705) and Propionibacterium freudenreichii ssp. shermanii JS (PJS) were incubated in PBS buffer containing 20 microg toxin ml(-1) for 1h at 37 degrees C, and after centrifugation the concentration of the toxins was measured in the supernatant fraction. Both viable and heat-killed forms of LGG and PJS were more efficient than LC-705 in removing the toxins from the liquid media. LGG and PJS removed four of the seven tested toxins (the removal varying from 18 to 93%) and LC-705 two toxins (10-64%). Of the toxins, 3-AcDON was not removed by any of the bacteria; HT-2 was removed by the non-viable LGG and also slightly by non-viable LC-705; DAS was removed by all three bacteria tested. Binding is postulated as the possible mechanism of the removal, since no difference was observed between the ability of viable and heat-killed bacteria in removing the trichothecenes, and no degradation products of the toxins were detected by gas chromatography (GC)-mass spectrometry (MS) analysis. It is concluded that significant differences exist in the ability of the bacteria to bind trichothecenes in vitro.

Aflatoxin B1 binding by dairy strains of lactic acid bacteria and bifidobacteria.

Various food commodities including dairy products may be contaminated with aflatoxins, which, even in small quantities, have detrimental effects on human and animal health. Several microorganisms have been reported to bind or degrade aflatoxins in foods and feeds. This study assessed the binding of aflatoxin B1 (AFB1) from contaminated solution by 20 strains of lactic acid bacteria and bifidobacteria. The selected strains are used in the food industry and comprised 12 Lactobacillus, five Bifidobacterium, and three Lactococcus strains. Bacteria and AFB1 were incubated (24 h, +37 degrees C) and the amount of unbound AFB1 was quantitated by HPLC. Between 5.6 and 59.7% AFB1 was bound from solution by these strains. Two Lactobacillus amylovorus strains and one Lactobacillus rhamnosus strain removed more than 50% AFB1 and were selected for further study. Bacterial binding of AFB1 by these strains was rapid, and more than 50% AFB1 was bound throughout a 72-h incubation period. Binding was reversible, and AFB1 was released by repeated aqueous washes. These findings further support the ability of specific strains of lactic acid bacteria to bind selected dietary contaminants.

Surface binding of aflatoxin B(1) by lactic acid bacteria.

Specific lactic acid bacterial strains remove toxins from liquid media by physical binding. The stability of the aflatoxin B(1) 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 B(1) remained bound. Nonviable bacteria retained the highest amount of aflatoxin B(1). Lactobacillus rhamnosus strain GG (ATCC 53103) and L. rhamnosus strain LC-705 (DSM 7061) removed aflatoxin B(1) from solution most efficiently and were selected for further study. The accessibility of bound aflatoxin B(1) 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 B(1). Variation in temperature (4 to 37 degrees C) and pH (2 to 10) did not have any significant effect on the amount of aflatoxin B(1) released. Binding of aflatoxin B(1) 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.

Ability of dairy strains of lactic acid bacteria to bind aflatoxin M1 in a food model.

Aflatoxin M1 (AFM1) is a highly toxic compound found in milk. Its occurrence poses a threat to the health of consumers, especially young children, and leads to economic losses due to contaminated milk. The problem is global but more severe in developing countries. Consequently, there is a great demand for novel strategies to prevent the contamination and adverse effects of AFM1. To develop a safe and practical decontamination method, a preliminary study was carried out with specific lactic acid bacteria strains that were tested for their ability to remove AFM1 from liquid media. All strains, whether viable or heat-killed, could reduce the AFM1 content of a liquid medium. Two most effective strains were also tested using contaminated skim and full cream milk. The results indicate that specific lactic acid bacteria used in dairy products can offer novel means of decontaminating aflatoxin M1 from milk.

Ability of Lactobacillus and Propionibacterium strains to remove aflatoxin B, from the chicken duodenum.

The ability of Lactobacillus rhamnosus strains GG and LC-705 to remove AFB1 from the intestinal luminal liquid medium has been tested in vivo using a chicken intestinal loop technique. In this study, the GG strain of L. rhamnosus decreased AFB1 concentration by 54% in the soluble fraction of the luminal fluid within 1 min. This strain was more efficient in binding AFB1 compared with L. rhamnosus strain LC-705 (P < 0.05) that removed 44% of AFBl under similar conditions. Accumulation of AFB1 into the intestinal tissue was also determined. There was a 74% reduction in the uptake of AFB1 by the intestinal tissue, in the presence of L. rhamnosus strain GG compared with 63% and 37% in the case of Propionibacterium freudenreichii ssp. shermanii JS and L. rhamnosus strain LC-705, respectively. The complexes formed in vitro between either L. rhamnosus strain GG or L. rhamnosus strain LC-705 and AFB1 were stable under the luminal conditions for a period of 1 h.

Binding of aflatoxin B1 alters the adhesion properties of Lactobacillus rhamnosus strain GG in a Caco-2 model.

Lactic acid bacteria have been previously reported to possess antimycotoxigenic activities both in vitro and in vivo. The objective of this study was to investigate the effect of aflatoxin B1 on adhesion capability of Lactobacillus rhamnosus strain GG using a Caco-2 adhesion model. Removal of aflatoxin B1 by L. rhamnosus strain GG reduced the adhesion capability of this strain from 30% to 5%. It is therefore concluded that aflatoxins may influence the adhesion properties of probiotics able to sequester them, and subsequently these bacteria may reduce the accumulation of aflatoxins in the intestine via increased excretion of an aflatoxin-bacteria complex.

The effect of orally administered viable probiotic and dairy lactobacilli on mouse lymphocyte proliferation.

Four common Lactobacillus strains were screened for their effects on proliferation of mouse splenic lymphocytes. Mice received perorally 10(9) viable bacteria kg(-1) body weight for 7 days. Lactobacillus acidophilus treatment enhanced ex vivo basal proliferation (by 43%) and B-cell response at suboptimal and optimal concentrations of lipopolysaccharide (LPS) (by 27-28%). Conversely, Lactobacillus casei, Lactobacillus gasseri and Lactobacillus rhamnosus inhibited both basal proliferation (by 14-51%) and mitogen-stimulated lymphoproliferation, particularly at supra-optimal concentrations of concanavalin A (by 43-68%) and LPS (by 23-62%). Therefore, these Lactobacillus strains demonstrate strain-specific effects on B- and T-cells and may also alter the splenocyte sensitivity to the cytotoxic effects of mitogens.