Sonja Nybom

Interaction of probiotics and pathogens--benefits to human health?

The probiotic terminology has matured over the years and currently a unified definition has been formed. Lactic acid bacteria (LAB) and bifidobacteria have been reported to remove heavy metals, cyanotoxins and mycotoxins from aqueous solutions. The binding processes appear to be species and strain specific. The most efficient microbial species and strains in the removal of these compounds vary between components tested. However, it is of interest to note that most strains characterized until now do not bind positive components or nutrients in the diet. This has significant implications to future detoxification biotechnology development. In a similar manner, lactic acid bacteria and bifidobacteria interact directly with viruses and pathogens in food and water as well as toxin producing microbes and some toxins. This review updates information and aims to characterize these interactions in association. The target is to understand probiotic health effects and to relate the mechanisms and actions to future potential of specific probiotic bacteria on decontamination of foods and water, and diets. The same aim is targeted in characterizing the role of probiotics in inactivating pathogens and viruses of health importance to facilitate the establishment of novel means of disease risk reduction related health benefits.

Microbial Degradation of Microcystins

Hepatotoxic microcystins that are produced by freshwater cyanobacteria pose a risk to public health. These compounds may be eliminated by enzymatic degradation. Here, we review the enzymatic pathways for the degradation of these hepatotoxins, some of which are newly discovered processes. The efficiencies of microcystin biodegradation pathways are documented in several papers and are compared here. Additionally, a comprehensive description of the microcystin enzymatic degradation scheme has been supplemented with a proposal for a new biodegradation pathway. Critical comments on less documented hypotheses are also included. The genetic aspects of biodegradation activity are discussed in detail. We also describe some methods that are useful for studying the biological decomposition of microcystins, including screening for microcystin degraders and detecting microcystin degradation products, with an emphasis on mass spectrometric methodology.

Specific strains of probiotic bacteria are efficient in removal of several different cyanobacterial toxins from solution

The ability of specific strains of probiotic bacteria to remove the pure cyanobacterial peptide toxins microcystin-LR, -RR, -LF, and a combination of microcystins from the cyanobacterial extracts Microcystis PCC 7820 and NIES 107, as well as the cyanobacterial cytotoxin cylindrospermopsin, from aqueous solutions was assessed. The probiotic bacterial strains studied were Lactobacillus rhamnosus strains GG and LC-705, Bifidobacterium lactis strains 420 and Bb12 and Bifidobacterium longum 46, all previously shown to be effective in toxin removal. The maximum removal of microcystin-LR, 60.3%, was observed with L. rhamnosus GG, of microcystin-RR, 62.8%, and microcystin-LF, 77.4%, with L. rhamnosus LC-705, and of cylindrospermopsin, 31.6%, with B. longum 46 (toxin concentration 100mugL(-1), 37 degrees C, 24h). Several microcystins could be removed simultaneously as observed by removal of microcystins present in the cyanobacterial extracts. A combination of three probiotic strains enhanced their removal ability as compared to the removal properties of the individual strains. We conclude that specific strains of probiotic bacteria are effective in elimination of different cyanotoxins from solution.

Cyanobacteria and cyanotoxins in fishponds and their effects on fish tissue

Cyanobacteria can produce toxic metabolites known as cyanotoxins. Common and frequently investigated cyanotoxins include microcystins (MCs), nodularin (NOD) and saxitoxins (STXs). During the summer of 2011 extensive cyanobacterial growth was found in several fishponds in Serbia. Sampling of the water and fish (common carp, Cyprinus carpio) was performed. Water samples from 13 fishponds were found to contain saxitoxin, microcystin, and/or nodularin. LC-MS/MS showed that MC-RR was present in samples of fish muscle tissue. Histopathological analyses of fish grown in fishponds with cyanotoxin production showed histopathological damage to liver, kidney, gills, intestines and muscle tissues. This study is among the first so far to report severe hyperplasia of intestinal epithelium and severe degeneration of muscle tissue of fish after cyanobacterial exposure. These findings emphasize the importance of cyanobacterial and cyanotoxin monitoring in fishponds in order to recognize cyanotoxins and their potential effects on fish used for human consumption and, further, on human health.

Quantitative PCR detection and improved sample preparation of microcystin-producing Anabaena, Microcystis and Planktothrix.

Blooms of toxic cyanobacteria, associated with illness and mortality in humans and animals, are becoming increasingly common worldwide. The safe use of surface waters for drinking water production and recreation necessitates assessment of toxigenic cyanobacteria. We have developed simple and reliable sample preparation and qPCR methods to detect microcystin-producing strains of three major bloom-forming genera, Anabaena, Microcystis and Planktothrix. The mcyB second thiolation motif, previously not recognized as a potential target for qPCR, was used as a basis for primer and genus-specific probe design. Assay specificity and sensitivity was confirmed with cultured cyanobacterial strains and the effect of different sample preparation methods on quantification was investigated. Sample filtration and cell lysis reduced assay time and resulted in more efficient amplification compared to DNA extraction. Positive correlation (p<0.005) between mcyB copy numbers and microcystin concentrations was observed in environmental samples. The results encourage the use of qPCR in water risk management.

Effect of glucose in removal of microcystin-LR by viable commercial probiotic strains and strains isolated from dadih fermented milk.

The removal of the cyanobacterial peptide toxin microcystin-LR at 4 and 37 degrees C by six commercial probiotic strains and Lactobacillus plantarum strains IS-10506 and IS-20506 isolated from dadih, Indonesian traditional fermented milk, was assessed in this study. The aim was to evaluate the main factors influencing the viability and metabolic activity of the probiotic strains, as well as their capacity to remove microcystin-LR. Both L. plantarum strains isolated from dadih, as well as Bifidobacterium lactis strains Bb12 and 420, were shown to be more resistant, and >85% remained viable in phosphate-buffered saline (PBS) solution for 48 h of incubation at either temperature, while the viability of the other four commercial bacteria decreased markedly over time. The effect of glucose on viability and removal of toxin was shown to be a strain-specific and strain-dependent property, but in general, the efficiency of microcystin-LR removal increased when glucose was added to the solution. A maximum removal of 95% was observed for L. plantarum strain IS-20506 (37 degrees C, 10 (11) colony-forming units mL(-1)) with 1-2% glucose supplementation and 75% in PBS alone.

Characterization of microcystin-LR removal process in the presence of probiotic bacteria.

Toxic cyanobacteria have been reported in lakes and reservoirs in several countries. The presence of toxins in drinking water creates a potential risk of toxin transference for water consumers. Besides chemical and physical methods of cyanotoxin removal from water, biodegradation methods would be useful. The aim of the current study was to identify bacterial removal mechanisms of the hepatotoxin microcystin-LR. This was studied by testing the hypothesis of enzymatic degradation of microcystin-LR in the presence of probiotic lactic acid bacterial and bifidobacterial strains and the participation of the proteolytic system of the bacteria in this process. The results suggest that extracellularly located cell-envelope proteinases are involved in the decomposition of microcystin-LR. In particular, a correlation between proteolytic activity and microcystin removal was found and both these parameters were dependent on glucose as an energy source. In addition, EDTA, which was indicated as a main inhibitor of proteinases of the investigated strain, was shown to limit the rate of microcystin removal. The removal of microcystins was shown to be different from the known microcystin-degradation pathway of Sphingomonas. (14)C-labeled microcystin was not found inside the cells and bacterial cell extracts were not able to remove the toxin, which supports the involvement of extracellularly located proteinases. The results confirm the hypothesis of enzymatic degradation of microcystins in the presence of probiotic bacteria.

Microcystin accumulation and potential effects on antioxidant capacity of leaves and fruits of Capsicum annuum

ABSTRACT Surface water, often used for irrigation purposes, may sometimes be contaminated with blooming cyanobacteria and thereby may contain their potent and harmful toxins. Cyanotoxins adversely affect many terrestrial plants, and accumulate in plant tissues that are subsequently ingested by humans. Studies were undertaken to (1) examine the bioaccumulation of microcystins (MCs) in leaves and fruits of pepper Capsicum annuum and (2) examine the potential effects of MCs on antioxidant capacity of these organs. Plants were irrigated with water containing MCs for a period of 3 mo. Data showed that MCs did not accumulate in leaves; however, in fruits the presence of the MC-LR (0.118 ng/mg dry weight) and dmMC-LR (0.077 ng/mg dry weight) was detected. The concentrations of MC-LR in fruit approached the acceptable guideline values and tolerable daily intake for this toxin. Lipid peroxidation levels and flavonoids content were significantly enhanced in both organs of treated plants, while total phenolic concentrations were not markedly variable between control and treated plants. Significant decrease in 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging capacity was noted for both organs. The levels of superoxide anion in fruits and hydroxyl radical in leaves were markedly reduced. Data suggest that exposure to MCs significantly reduced antioxidant capacity of experimental plants, indicating that MCs affected antioxidant systems in C. annuum.

Removal of Cholera Toxin from Aqueous Solution by Probiotic Bacteria

Cholera remains a serious health problem, especially in developing countries where basic hygiene standards are not met. The symptoms of cholera are caused by cholera toxin, an enterotoxin, which is produced by the bacterium Vibrio cholerae. We have recently shown that human probiotic bacteria are capable of removing cyanobacterial toxins from aqueous solutions. In the present study we investigate the ability of the human probiotic bacteria, Lactobacillus rhamnosus strain GG (ATCC 53103) and Bifidobacteriumlongum 46 (DSM 14583), to remove cholera toxin from solution in vitro. Lactobacillus rhamnosus strain GG and Bifidobacteriumlongum 46 were able to remove 68% and 59% of cholera toxin from aqueous solutions during 18 h of incubation at 37 °C, respectively. The effect was dependent on bacterial concentration and L. rhamnosus GG was more effective at lower bacterial concentrations. No significant effect on cholera toxin concentration was observed when nonviable bacteria or bacterial supernatant was used.

Cyanobacterial effects in Lake Ludoš, Serbia - Is preservation of a degraded aquatic ecosystem justified?

Cyanobacteria are present in many aquatic ecosystems in Serbia. Lake Ludoš, a wetland area of international significance and an important habitat for waterbirds, has become the subject of intense research interest because of practically continuous blooming of cyanobacteria. Analyses of water samples indicated a deterioration of ecological condition and water quality, and the presence of toxin-producing cyanobacteria (the most abundant Limnothrix redekei, Pseudanabaena limnetica, Planktothrix agardhii and Microcystis spp.). Furthermore, microcystins were detected in plants and animals from the lake: in macrophyte rhizomes (Phragmites communis, Typha latifolia and Nymphaea elegans), and in the muscle, intestines, kidneys, gonads and gills of fish (Carassius gibelio). Moreover, histopathological deleterious effects (liver, kidney, gills and intestines) and DNA damage (liver and gills) were observed in fish. A potential treatment for the reduction of cyanobacterial populations employing hydrogen peroxide was tested during this study. The treatment was not effective in laboratory tests although further in-lake trials are needed to make final conclusions about the applicability of the method. Based on our observations of the cyanobacterial populations and cyanotoxins in the water, as well as other aquatic organisms and, a survey of historical data on Lake Ludoš, it can be concluded that the lake is continuously in a poor ecological state. Conservation of the lake in order to protect the waterbirds (without urgent control of eutrophication) actually endangers them and the rest of the biota in this wetland habitat, and possibly other ecosystems. Thus, urgent measures for restoration are required, so that the preservation of this Ramsar site would be meaningful.