Absence of Cyanotoxins in Llayta, Edible Nostocaceae Colonies from the Andes Highlands.

Edible Llayta are cyanobacterial colonies consumed in the Andes highlands. Llayta and four isolated cyanobacteria strains were tested for cyanotoxins (microcystin, nodularin, cylindrospermopsin, saxitoxin and ß-N-methylamino-L-alanine-BMAA) using molecular and chemical methods. All isolates were free of target genes involved in toxin biosynthesis. Only DNA from Llayta amplified the mcyE gene. Presence of microcystin-LR and BMAA in Llayta extracts was discarded by LC/MS analyses. The analysed Llayta colonies have an incomplete microcystin biosynthetic pathway and are a safe food ingredient.

The non-toxigenic strain of Clostridioides difficile Z31 can prevent infection by C. difficile in experimental model piglets.

One of the main challenges associated with Clostridioides difficile infection (CDI) in humans and domestic animals is the lack of an effective preventive strategy. One strategy with promising results is the oral administration of non-toxigenic strains of C. difficile (NTCD). Recently, Z31, a NTCD strain isolated from a healthy dog, showed promising results to prevent CDI in hamsters. Thus, the present study aimed to evaluate the capacity of Z31 to prevent CDI in piglets using an experimental model. Twenty neonatal piglets were randomly distributed in three groups: G1 - 106 spores of Z31 followed by 107 spores of a toxigenic C. difficile strain (n = 7), G2 (positive control) - 107 spores of a toxigenic C. difficile strain (n = 7), and G3 (negative control) - no biological inoculum (n = 6). All animals were kept in individual insulators and observed for 60 h. Data regarding clinical signs, macro and microscopic lesions, toxigenic culture of C. difficile, and detection of A/B toxins in the feces were evaluated. All evaluated parameters were significantly lower in animals that received Z31 compared to the positive control. Thus, oral administration of Z31 was able to prevent CDI in piglets in an experimental model.

[Current Problems Associated with Overseas Health Products].

In recent years, consumer interest in health and health foods (e.g., dietary supplements) has increased, and the types of and market for health foods have also expanded rapidly. The safety of health foods is a significant concern in many countries. Numerous adverse events associated with the consumption of health foods have been reported. There are cases that have resulted in serious liver failure, renal failure, and death in other countries. These products may contain undeclared medical ingredients and toxic chemical compounds that are illegally and intentionally added or contain natural plant toxins. Products containing high levels of heavy metals have also been reported to cause such adverse events. Some products remain on the market, even after regulatory agencies alert consumers and issue warnings to their sellers. Moreover, because people can buy health foods from sources overseas via the Internet, adverse effects associated with the use of such products remain a concern. Two cases of adverse events were reported in Japanese individuals who purchased "OxyElite Pro" products imported privately. They are advertised as weight-loss and muscle-building products and have been associated with many cases of liver failure and hepatitis in the USA. In this paper, regulatory systems and adverse events associated with the use of health foods in other countries are discussed.

A Systematic Literature Review for Evidence of Aphanizomenon flos-aquae Toxigenicity in Recreational Waters and Toxicity of Dietary Supplements: 2000⁻2017.

Previous studies of recreational waters and blue-green algae supplements (BGAS) demonstrated co-occurrence of Aphanizomenon flos-aquae (AFA) and cyanotoxins, presenting exposure risk. The authors conducted a systematic literature review using a GRADE PRISMA-p 27-item checklist to assess the evidence for toxigenicity of AFA in both fresh waters and BGAS. Studies have shown AFA can produce significant levels of cylindrospermopsin and saxitoxin in fresh waters. Toxicity studies evaluating AFA-based BGAS found some products carried the mcyE gene and tested positive for microcystins at levels ≤ 1 µg microcystin (MC)-LR equivalents/g dry weight. Further analysis discovered BGAS samples had cyanotoxins levels exceeding tolerable daily intake values. There is evidence that Aphanizomenon spp. are toxin producers and AFA has toxigenic genes such as mcyE that could lead to the production of MC under the right environmental conditions. Regardless of this ability, AFA commonly co-occur with known MC producers, which may contaminate BGAS. Toxin production by cyanobacteria is a health concern for both recreational water users and BGAS consumers. Recommendations include: limit harvesting of AFA to months when toxicity is lowest, include AFA in cell counts during visible blooms, and properly identify cyanobacteria species using 16S rRNA methods when toxicity levels are higher than advisory levels.

Does saline enema during the first stage of labour reduce the incidence of Clostridium difficile colonization in neonates? A randomized controlled trial.

BACKGROUND: Maternal rectal enemas may reduce neonatal bacterial exposure during labour, which may reduce the risk of neonatal colonization with Clostridium difficile. The aim of this study was to determine the effectiveness of a saline enema during the first stage of labour in reducing neonatal colonization with C. difficile. METHODS: This study was conducted at Cairo University Hospital, Egypt from January 2016 to July 2016. Asymptomatic mothers with uncomplicated vaginal delivery and their neonates without diarrhoea were included. The study group underwent saline enema, and the control group had no intervention. Stool samples were collected from neonates one week after delivery. The primary outcome was the detection of C. difficile in stool culture and direct detection of C. difficile Toxin A and Toxin B by enzyme-linked immunosorbent assay. FINDINGS: The two groups were comparable (P>0.05) in terms of age, gravidity, parity, body mass index and gestational age. C. difficile was detected in 13.54% and 37.63% of stool cultures from the enema group and the control group, respectively (P<0.001). Direct detection of Toxins A and B was positive in 22.92% of cases in the enema group and 53.76% of cases in the control group (P<0.001). CONCLUSION: This study suggests that a saline enema for the mother during the first stage of labour may be useful in reducing the risk of neonatal gut colonization by C. difficile.

Detection of Cyanotoxins in Algae Dietary Supplements.

Algae dietary supplements are marketed worldwide as natural health products. Although their proprieties have been claimed as beneficial to improve overall health, there have been several previous reports of contamination by cyanotoxins. These products generally contain non-toxic cyanobacteria, but the methods of cultivation in natural waters without appropriate quality controls allow contamination by toxin producer species present in the natural environment. In this study, we investigated the presence of total microcystins, seven individual microcystins (RR, YR, LR, LA, LY, LW, LF), anatoxin-a, dihydroanatoxin-a, epoxyanatoxin-a, cylindrospermopsin, saxitoxin, and ß-methylamino-l-alanine in 18 different commercially available products containing Spirulina or Aphanizomenon flos-aquae. Total microcystins analysis was accomplished using a Lemieux oxidation and a chemical derivatization using dansyl chloride was needed for the simultaneous analysis of cylindrospermopsin, saxitoxin, and ß-methylamino-l-alanine. Moreover, the use of laser diode thermal desorption (LDTD) and ultra-high performance liquid chromatography (UHPLC) both coupled to high resolution mass spectrometry (HRMS) enabled high performance detection and quantitation. Out of the 18 products analyzed, 8 contained some cyanotoxins at levels exceeding the tolerable daily intake values. The presence of cyanotoxins in these algal dietary supplements reinforces the need for a better quality control as well as consumer's awareness on the potential risks associated with the consumption of these supplements.

Variable Cyanobacterial Toxin and Metabolite Profiles across Six Eutrophic Lakes of Differing Physiochemical Characteristics.

Future sustainability of freshwater resources is seriously threatened due to the presence  of  harmful  cyanobacterial  blooms,  and  yet,  the  number,  extent,  and  distribution  of  most  cyanobacterial toxins-including "emerging" toxins and other bioactive compounds-are poorly  understood.  We  measured  15  cyanobacterial  compounds-including  four  microcystins  (MC),  saxitoxin (SXT), cylindrospermopsin (CYL), anatoxin-a (ATX) and homo-anatoxin-a (hATX), two  anabaenopeptins (Apt), three cyanopeptolins (Cpt), microginin (Mgn), and nodularin (NOD)-in  six freshwater lakes that regularly experience noxious cHABs. MC, a human liver toxin, was present  in all six lakes and was detected in 80% of all samples. Similarly, Apt, Cpt, and Mgn were detected  in all lakes in roughly 86%, 50%, and 35% of all samples, respectively. Despite being a notable  brackish  water  toxin,  NOD  was  detected  in  the  two  shallowest  lakes-Wingra  (4.3  m)  and  Koshkonong (2.1 m). All compounds were highly variable temporally, and spatially. Metabolite  profiles were significantly different between lakes suggesting lake characteristics influenced the  cyanobacterial community and/or metabolite production. Understanding how cyanobacterial toxins  are  distributed  across  eutrophic  lakes  may  shed  light  onto  the  ecological  function  of  these  metabolites, provide valuable information for their remediation and removal, and aid in the  protection of public health.

Clostridium difficile toxin testing by National Health Service (NHS) acute Trusts in England: 2008-2013.

In October 2007, a governmental 3-year target to reduce Clostridium difficile infection (CDI) by 30%, with financial penalties levied for failure, was introduced in England. This target was met within just 1 year, leading to speculation of 'gaming', with hospitals empirically treating possible CDI in the absence of a microbiological diagnosis, to avoid having to report confirmed cases. An analysis of aggregate mandatory data on levels of testing for C. difficile toxin showed little evidence of a fall in testing during the steepest infection rate reductions, suggesting that this was not a major factor in the decline in CDI.

Berberine blocks the relapse of Clostridium difficile infection in C57BL/6 mice after standard vancomycin treatment.

Vancomycin is a preferred antibiotic for treating Clostridium difficile infection (CDI) and has been associated with a rate of recurrence of CDI of as high as 20% in treated patients. Recent studies have suggested that berberine, an alternative medical therapy for gastroenteritis and diarrhea, exhibits several beneficial effects, including induction of anti-inflammatory responses and restoration of the intestinal barrier function. This study investigated the therapeutic effects of berberine on preventing CDI relapse and restoring the gut microbiota in a mouse model. Berberine was administered through gavage to C57BL/6 mice with established CDI-induced intestinal injury and colitis. The disease activity index (DAI), mean relative weight, histopathology scores, and levels of toxins A and B in fecal samples were measured. An Illumina sequencing-based analysis of 16S rRNA genes was used to determine the overall structural change in the microbiota in the mouse ileocecum. Berberine administration significantly promoted the restoration of the intestinal microbiota by inhibiting the expansion of members of the family Enterobacteriaceae and counteracting the side effects of vancomycin treatment. Therapy consisting of vancomycin and berberine combined prevented weight loss, improved the DAI and the histopathology scores, and effectively decreased the mortality rate. Berberine prevented CDIs from relapsing and significantly improved survival in the mouse model of CDI. Our data indicate that a combination of berberine and vancomycin is more effective than vancomycin alone for treating CDI. One of the possible mechanisms by which berberine prevents a CDI relapse is through modulation of the gut microbiota. Although this conclusion was generated in the case of the mouse model, use of the combination of vancomycin and berberine and represent a novel therapeutic approach targeting CDI.

Eutrophic urban ponds suffer from cyanobacterial blooms: Dutch examples.

Ponds play an important role in urban areas. However, cyanobacterial blooms counteract the societal need for a good water quality and pose serious health risks for citizens and pets. To provide insight into the extent and possible causes of cyanobacterial problems in urban ponds, we conducted a survey on cyanobacterial blooms and studied three ponds in detail. Among 3,500 urban ponds in the urbanized Dutch province of North Brabant, 125 showed cyanobacterial blooms in the period 2009-2012. This covered 79% of all locations registered for cyanobacterial blooms, despite the fact that urban ponds comprise only 11% of the area of surface water in North Brabant. Dominant bloom-forming genera in urban ponds were Microcystis, Anabaena and Planktothrix. In the three ponds selected for further study, the microcystin concentration of the water peaked at 77 µg l(-1) and in scums at 64,000 µg l(-1), which is considered highly toxic. Microcystin-RR and microcystin-LR were the most prevalent variants in these waters and in scums. Cyanobacterial chlorophyll-a peaked in August with concentrations up to 962 µg l(-1) outside of scums. The ponds were highly eutrophic with mean total phosphorus concentrations between 0.16 and 0.44 mg l(-1), and the sediments were rich in potential releasable phosphorus. High fish stocks dominated by carp lead to bioturbation, which also favours blooms. As urban ponds in North Brabant, and likely in other regions, regularly suffer from cyanobacterial blooms and citizens may easily have contact with the water and may ingest cyanobacterial material during recreational activities, particularly swimming, control of health risk is of importance. Monitoring of cyanobacteria and cyanobacterial toxins in urban ponds is a first step to control health risks. Mitigation strategies should focus on external sources of eutrophication and consider the effect of sediment P release and bioturbation by fish.

Toxin content and cytotoxicity of algal dietary supplements.

Blue-green algae (Spirulina sp., Aphanizomenon flos-aquae) and Chlorella sp. are commercially distributed as organic algae dietary supplements. Cyanobacterial dietary products in particular have raised serious concerns, as they appeared to be contaminated with toxins e.g. microcystins (MCs) and consumers repeatedly reported adverse health effects following consumption of these products. The aim of this study was to determine the toxin contamination and the in vitro cytotoxicity of algae dietary supplement products marketed in Germany. In thirteen products consisting of Aph. flos-aquae, Spirulina and Chlorella or mixtures thereof, MCs, nodularins, saxitoxins, anatoxin-a and cylindrospermopsin were analyzed. Five products tested in an earlier market study were re-analyzed for comparison. Product samples were extracted and analyzed for cytotoxicity in A549 cells as well as for toxin levels by (1) phosphatase inhibition assay (PPIA), (2) Adda-ELISA and (3) LC-MS/MS. In addition, all samples were analyzed by PCR for the presence of the mcyE gene, a part of the microcystin and nodularin synthetase gene cluster. Only Aph. flos-aquae products were tested positive for MCs as well as the presence of mcyE. The contamination levels of the MC-positive samples were ≤ 1 µg MC-LR equivalents g(-1) dw. None of the other toxins were found in any of the products. However, extracts from all products were cytotoxic. In light of the findings, the distribution and commercial sale of Aph. flos-aquae products, whether pure or mixed formulations, for human consumption appear highly questionable.

Spatiotemporal patterns and ecophysiology of toxigenic microcystis blooms in Lake Taihu, China: implications for water quality management.

Whole lake monitoring of hypertrophic Lake Taihu, China, was conducted during the summers of 2009-2010, with the intent of identifying environmental factors influencing Microcystis bloom formation and promoting the growth of toxigenic strains (mcyE possessing). Low N:P ratios (replete N & P)appeared to select for toxigenic populations of Microcystis spp., whereas nontoxic Microcystis spp. strains were dominant in more nutrient limited regions of the lake. Chlorophyll a (Adj. R(2) = 0.83, p < 0.0001) was equally predicative of microcystin variance across the lake as fluorescence based real-time quantitative PCR (qPCR) measurements of microcystin synthetase E (mcyE) gene equivalents (Adj. R(2) = 0.85, p < 0.0001). Interestingly, chlorophyll a was identified as a more robust and useful metric for predicting microcystin concentrations than qPCR measurements enumerating the total Microcystis population based on c-phycocyanin (α subunit; cpcA) gene equivalents (Adj. R(2) = 0.61, p < 0.0001). Overall, the lakewide composition of Microcystis spp. was highly variable over time and space, and on average the population consisted of 36 ± 12% potentially toxic cells. On the basis of this study's findings, a framework for the design and implementation of a water safety plan for Taihu water quality managers and public health officials is proposed.

Expression of the cholera toxin B subunit (CT-B) in maize seeds and a combined mucosal treatment against cholera and traveler's diarrhea.

The non-toxic B subunit (CT-B) of cholera toxin from Vibrio cholerae is a strong immunogen and amplifies the immune reaction to conjugated antigens. In this work, a synthetic gene encoding for CT-B was expressed under control of a γ-zein promoter in maize seeds. Levels of CT-B in maize plants were determined via ganglioside dependent ELISA. The highest expression level recorded in T(1) generation seeds was 0.0014% of total aqueous soluble protein (TASP). Expression level of the same event in the T(2) generation was significantly increased to 0.0197% of TASP. Immunogenicity of maize derived CT-B was evaluated in mice with an oral immunization trial. Anti-CTB IgG and anti-CTB IgA were detected in the sera and fecal samples of the orally immunized mice, respectively. The mice were protected against holotoxin challenge with CT. An additional group of mice was administrated with an equal amount (5 µg per dose each) of mixed maize-derived CT-B and LT-B (B subunit of E. coli heat labile toxin). In the sera and fecal samples obtained from this group, the specific antibody levels were enhanced compared to either the same or a higher amount of CT-B alone. These results suggest that a synergistic action may be achieved using a CT-B and LT-B mixture that can lead to a more efficacious combined vaccine to target diarrhea induced by both cholera and enterotoxigenic strains of Escherichia coli.

Detection of anthrax lef with DNA-based photonic crystal sensors.

Bacillus anthracis has posed a threat of becoming biological weapons of mass destruction due to its virulence factors encoded by the plasmid-borne genes, such as lef for lethal factor. We report the development of a fast and sensitive anthrax DNA biosensor based on a photonic crystal structure used in a total-internal-reflection configuration. For the detection of the lef gene, a single-stranded DNA lef probe was biotinylated and immobilized onto the sensor via biotin-streptavidin interactions. A positive control, lef-com, was the complementary strand of the probe, while a negative control was an unrelated single-stranded DNA fragment from the 16S rRNA gene of Acinetobacter baumannii. After addition of the biotinylated lef probe onto the sensor, significant changes in the resonance wavelength of the sensor were observed, resulting from binding of the probe to streptavidin on the sensor. The addition of lef-com led to another significant increase as a result of hybridization between the two DNA strands. The detection sensitivity for the target DNA reached as low as 0.1 nM. In contrast, adding the unrelated DNAs did not cause an obvious shift in the resonant wavelength. These results demonstrate that detection of the anthrax lef by the photonic crystal structure in a total-internal-reflection sensor is highly specific and sensitive.

Cyanobacteria-/cyanotoxin-contaminations and eutrophication status before Wuxi drinking water crisis in Lake Taihu, China.

After the appalling "Wuxi Drinking Water Crisis", increasing investigations concerning the contaminations of cyanobacterial blooms and their toxins in Lake Taihu have been performed and reported in the last two years. However, information regarding these issues before the crisis in 2007 remained insufficient. To provide some background data for further comparisons, the present study reported our investigations conducted in 2004, associated with the cyanotoxin contaminations as well as the eutrophication status in Lake Taihu. Results from the one-year-study near a drinking water resource for Wuxi City indicated that, unlike the status in recent two years, cyanobacteria and chlorophyta are the co-dominance species throughout the year. The highest toxin concentration (34.2 ng/mL) in water columns occurred in August. In bloom biomass, the peak value of intracellular toxin (0.59 microg/mg DW) was determined in October, which was lag behind that in water column. In addition, MC-RR was the major toxin variant throughout the year. During the study period, nutrients levels of total nitrogen and phosphorus were also recorded monthly. Results from the present study will lead to a better understanding of the eutrophication status and the potential risks before "Wuxi Drinking Water Crisis".

Determination of the cyanobacterial toxin cylindrospermopsin in algal food supplements.

For the analysis of blue-green algal food supplements for cylindrospermopsin (CYN), a C18 solid-phase extraction column and a polygraphitized carbon solid-phase extraction column in series was an effective procedure for the clean-up of extracts. Determination of CYN was by liquid chromatography with ultraviolet light detection. At extract spiking levels of CYN equivalent to 25-500 µg g(-1), blue-green algal supplement recoveries were in the range 70-90%. CYN was not detected in ten samples of food supplements and one chocolate product, all containing blue-green algae. The limit of detection for the method was 16 µg g(-1), and the limit of quantification was 52 µg g(-1).

In vitro and in vivo safety assessment of edible blue-green algae, Nostoc commune var. sphaeroides Kützing and Spirulina plantensis.

Blue-green algae (BGA) have been consumed as food and herbal medicine for centuries. However, safety for their consumption has not been well investigated. This study was undertaken to evaluate in vitro and in vivo toxicity of cultivated Nostoc commune var. sphaeroides Kützing (NO) and Spirulina platensis (SP). Neither NO nor SP contained detectable levels of microcystin (MC)-LA, MC-RR, MC-LW and MC-LR by LC/MS/MS. Cell viability remained ∼70-80% when HepG2 cells were incubated with 0-500 µg/ml of hexane, chloroform, methanol and water-extractable fractions of NO and SP. Four-week-old male and female C57BL/6J mice were fed an AIN-93G/M diet supplemented with 0%, 2.5% or 5% of NO and SP (wt/wt) for 6 months. For both genders, BGA-rich diets did not induce noticeable abnormality in weight gain and plasma alanine aminotransferase (ALT) and aspartate aminotransferase concentrations except a significant increase in plasma ALT levels by 2.5% NO supplementation in male mice at 6 month. Histopathological analysis of livers, however, indicated that BGA did not cause significant liver damage compared with controls. In conclusion, our results suggest that NO and SP are free of MC and the long-term dietary supplementation of up to 5% of the BGA may be consumed without evident toxic side-effects.

Control of important clostridial diseases of sheep.

Clostridia cause many different diseases, all characterized by sudden death, most occurring worldwide. Diseases caused by clostridia can be divided into 4 groups: those affecting the alimentary system (the enterotoxemias), those affecting the parenchymatous organs, those causing myonecrosis and toxemia, and those causing neurologic disorders. Their mode of action is to produce one or more potent toxins when multiplying under favorable conditions. Considerable variation exists between different strains of the same organism. Specific trigger factors are required to induce toxin production. Excellent control is obtained by the use of toxoid vaccines. Protection is passed to the lamb via the colostrum.

Citizen monitoring: Testing hypotheses about the interactive influences of eutrophication and mussel invasion on a cyanobacterial toxin in lakes.

An existing volunteer monitoring network in the state of Michigan was exploited to conduct a statewide survey of the cyanobacterial toxin, microcystin, and to test hypotheses about the interactive influences of eutrophication and dreissenid mussel invasion. A total of 77 lakes were sampled by citizen volunteers for microcystin, total phosphorus (TP) and chlorophyll a. Microcystin was measured in depth-integrated samples collected from the euphotic zone as well as in surface-water samples collected along the shoreline. Average microcystin in samples collected by volunteers was not different from samples collected side-by-side by professionals. Euphotic-zone microcystin was positively related to TP in lakes without dreissenids (uninvaded) but not in lakes with dreissenids (invaded). Regression-tree analysis indicated that euphotic-zone microcystin was eight times higher in the presence of dreissenids for lakes with TP between 5 and 10microgL(-1). In contrast, euphotic-zone microcystin was almost identical in invaded and uninvaded lakes with TP between 10 and 26microgL(-1). Across all lakes, microcystin concentrations at the surface were on average more than double, and in some cases an order-of-magnitude greater than, concentrations in the euphotic-zone. Given these results, it seems prudent to include dreissenid invasion status in forecasting models for microcystin, and to include shoreline sampling in monitoring programs aimed at assessing recreational exposure to cyanobacterial toxins.

[Clostridium-difficile-associated diarrhea]. / Diarrea asociada a Clostridium difficile.

Clostridium difficile is the most frequent cause of nosocomial diarrhea and is a significant cause of morbidity among hospitalized patients. The inflammation is produced as a result of a non-specific response to toxins. In the last few years, a hypervirulent strain, NAP1/BI/027, has been reported. Symptoms usually consist of abdominal pain and diarrhea. The diagnosis should be suspected in any patient who develops diarrhea during antibiotic therapy or 6-8 weeks after treatment. Diagnosis should be confirmed by the detection of CD toxin in stool and by colonoscopy in special situations. The treatment of choice is metronidazole or vancomycin. In some patients who do not respond to this therapy or who have complications, subtotal colectomy may be required. Relapse is frequent and must be distinguished from reinfection. Prevention and control in healthcare settings requires careful attention.