Evaluation of an in vitro experimental platform of human polarized intestinal epithelial monolayers for the hazard assessment of insecticidal proteins.

Previous work demonstrated the utility of using human-derived intestinal epithelial cell (IEC) lines cultured as polarized monolayers on Transwell® filters to differentiate between hazardous and non-hazardous proteins. The current study seeks to further resolve appropriate concentrations for evaluating proteins of unknown hazard potential using the IEC experimental platform and leverages these parameters for evaluating the potential toxicity of insecticidal proteins characteristic of those expressed in genetically modified (GM) agricultural biotechnology crops. To establish optimal test protein concentrations, effects of several known hazardous (C. perfringens epsilon toxin, Listeriolysin O, Phaseolus vulgaris erythroagglutinin, E. coli Shiga toxin 1, C. difficile Toxin B and wheat germ agglutinin) and non-hazardous (Ara-h2, ß-lactoglobulin, fibronectin and Rubisco) proteins on IEC barrier integrity and cell viability were evaluated at concentration ranges. Two insecticidal proteins (AfIP-1A and AfIP-1B) were evaluated for effects in the IEC assay, a seven-day insecticidal bioassay, and assessed in a high-dose 14-day acute oral toxicity study in mice. The results obtained from the human in vitro IEC assay were consistent with results obtained from an in vivo acute oral toxicity study, both demonstrating that the combination of AfIP-1A and AfIP-1B do not exhibit any identifiable harmful impacts on mammalian cells.

Estimation of production and sedimentation of cyanobacterial toxins (microcystin) based on nutrient budgets in the reservoir of Isahaya Bay, Japan.

The increasing eutrophication of freshwater and brackish habitats globally has led to a corresponding increase in the occurrence of harmful cyanobacterial blooms. Cyanobacteria can produce highly toxic substances such as microcystins (MCs) that affect the health of livestock, wildlife, and humans. The present study broaden the understanding of cyanobacteria ecology and MC dynamics in the field, focusing on the estimation of the production and sedimentation rates of MCs in a natural habitat. The nutrient concentrations of the reservoir water and sediment pore water were monitored at 3-h intervals for 24 h during the summer cyanobacterial bloom. The DIN uptake rate of Microcystis in the Isahaya reservoir was estimated and the large-scale blooms in the reservoir were largely controlled by the interactions between rainfall and nutrient levels in the warm season. By using calculations based on the nitrogen budgets and tracking changes of the MC concentrations in the water column, the total MC production and sedimentation rates were estimated to be 52.2 kg MCs d-1 and 21.5 kg MCs d-1, respectively. Although MCs could be degraded in the environment, the MC sedimentation still comprised 41% of the in-water production.

Identification of epsilon toxin-producing Clostridium perfringens strains in American retail food.

Food samples (n = 216) from New York city were tested for the presence of C. perfringens via PCR for specific toxin genes. Thirty-four (16%) samples were positive for C. perfringens. Of these 34, 31 (91.2%) were type A or E, one (2.9%) was type B, and two (5.9%) were type D.

Potential human health risk assessment of cylindrospermopsin accumulation and depuration in lettuce and arugula.

The cyanobacterial toxin cylindrospermopsin (CYN) has become a globally important secondary metabolite due to the negative effect it has on human and animal health. As a means of evaluating the risk of human exposure to CYN, the bioaccumulation and depuration of the toxin in lettuce (Lactuca sativa L.) and arugula (Eruca sativa Mill.) were investigated, after irrigation with contaminated water. The vegetables were irrigated for 7days with CYN (3, 5 and 10µg/L) contaminated water (bioaccumulation phase), and subsequently, irrigated for 7days with uncontaminated distilled water (depuration phase). In general, the bioaccumulation of CYN in both vegetables decreased with increasing exposure concentration. Bioconcentration factor (BCF) of CYN increased with the progression of the experiment at 3.0µg/L CYN, while the reverse occurred at 5 and 10µg/L CYN. In arugula, BCF increased at all CYN exposure concentrations throughout the study. The depuration of CYN decreased with increasing exposure concentration but was highest in the plants of both species with the highest bioaccumulation of CYN. Specifically, in plants previously irrigated with water contaminated with 3, 5 and 10µg/L CYN, the depuration of the toxin was 60.68, 27.67 and 18.52% for lettuce, and 47, 46.21 and 27.67% for arugula, respectively. Human health risks assessment revealed that the consumption of approximately 10 to 40g of vegetables per meal will expose children and adults to 1.00-6.00ng CYN/kg body mass for lettuce and 2.22-7.70ng CYN/kg body mass for arugula. The irrigation of lettuce and arugula with contaminated water containing low CYN concentrations constitutes a potential human exposure route.

A laboratory assessment of the potential effect of Cry1Ab/Cry2Aj-containing Bt maize pollen on Folsomia candida by toxicological and biochemical analyses.

The common soil arthropod Folsomia candida can survive well when fed only maize pollen and thus may be exposed to insecticidal proteins by ingesting insect-resistant genetically engineered maize pollen containing Bacillus thuringiensis (Bt) proteins when being released into the soil. Laboratory experiments were conducted to assess the potential effects of Cry1Ab/Cry2Aj-producing transgenic Bt maize (Shuangkang 12-5) pollen on F. candida fitness. Survival, development, and the reproduction were not significantly reduced when F. candida fed on Bt maize pollen rather than on non-Bt maize pollen, but these parameters were significantly reduced when F. candida fed on non-Bt maize pollen containing the protease inhibitor E-64 at 75 µg/g pollen. The intrinsic rate of increase (rm) was not significantly reduced when F. candida fed on Bt maize pollen but was significantly reduced when F. candida fed on non-Bt maize pollen containing E-64. The activities of antioxidant-related enzymes in F. candida were not significantly affected when F. candida fed on Bt maize pollen but were significantly increased when F. candida fed on non-Bt pollen containing E-64. The results demonstrate that consumption of Bt maize pollen containing Cry1Ab/Cry2Aj has no lethal or sublethal effects on F. candida.

Identification of LukPQ, a novel, equid-adapted leukocidin of Staphylococcus aureus.

Bicomponent pore-forming leukocidins are a family of potent toxins secreted by Staphylococcus aureus, which target white blood cells preferentially and consist of an S- and an F-component. The S-component recognizes a receptor on the host cell, enabling high-affinity binding to the cell surface, after which the toxins form a pore that penetrates the cell lipid bilayer. Until now, six different leukocidins have been described, some of which are host and cell specific. Here, we identify and characterise a novel S. aureus leukocidin; LukPQ. LukPQ is encoded on a 45 kb prophage (ΦSaeq1) found in six different clonal lineages, almost exclusively in strains cultured from equids. We show that LukPQ is a potent and specific killer of equine neutrophils and identify equine-CXCRA and CXCR2 as its target receptors. Although the S-component (LukP) is highly similar to the S-component of LukED, the species specificity of LukPQ and LukED differs. By forming non-canonical toxin pairs, we identify that the F-component contributes to the observed host tropism of LukPQ, thereby challenging the current paradigm that leukocidin specificity is driven solely by the S-component.

Desperate times call for desperate measures: benefits and costs of toxin-antitoxin systems.

Toxin-antitoxin (TA) loci were first described as killing systems for plasmid maintenance. The surprisingly abundant presence of TA loci in bacterial chromosomes has stimulated an extensive research in the recent decade aimed to understand the biological importance of these potentially deadly systems. Accumulating evidence suggests that the evolutionary success of genomic TA systems could be explained by their ability to increase bacterial fitness under stress conditions. While TA systems remain quiescent under favorable growth conditions, the toxins can be activated in response to stress resulting in growth suppression and development of stress-tolerant dormant state. Yet, several studies suggest that the TA-mediated stress protection is costly and traded off against decreased fitness under normal growth conditions. Here, we give an overview of the fitness benefits of the chromosomal TA systems, and discuss the costs of TA-mediated stress protection.

Dracorhodin Perochlorate attenuates Staphylococcus aureus USA300 virulence by decreasing α-toxin expression.

α-Toxin, a pore-forming toxin secreted by most Staphylococcus aureus, plays critical role in the pathogenesis associated with various infectious diseases. The USA300 which is a major international epidemic methicilin-resisrant S. aureus has spread rapidly to multiple countries and become an emerging public health concern. In this study, the in vitro efficacy of Dracorhodin Perochlorate (DP) against USA300 virulence was evaluated. Using susceptibility testing, immunoblots, rabbit blood haemolytic assay and real-time RT-PCR, we observed that the α-toxin production was decreased when USA300 was co-cultured with different sub-inhibitory concentration of DP. Further, the protective effect of DP against USA300-mediated injury of human alveolar epithelial cells (A549) and MH-S cells was evaluated by cytotoxicity assays, and the result revealed that DP, at final concentration of 16 µg/ml, is a potent antagonist for USA300-mediated cell damage. Importantly, those beneficial effects might partially correlate with hla and RNAIII suppression by DP, leading to the inhibition of α-toxin production in culture supernatant. Overall, these results suggest that DP could attenuate the virulence of USA300 by decreasing α-toxin production without inhibiting bacterial growth, and this compound may represent an ideal candidate for the development of anti-virulence agent combating S. aureus infection.

Assessment of uptake and phytotoxicity of cyanobacterial extracts containing microcystins or cylindrospermopsin on parsley (Petroselinum crispum L.) and coriander (Coriandrum sativum L).

Blooms of harmful cyanobacteria that synthesize cyanotoxins are increasing worldwide. Agronomic plants can uptake these cyanotoxins and given that plants are ultimately ingested by humans, this represents a public health problem. In this research, parsley and coriander grown in soil and watered through 7 days with crude extracts containing microcystins (MCs) or cylindrospermopsin (CYN) in 0.1-1 µg mL-1 concentration range were evaluated concerning their biomass, biochemical parameters and uptake of cyanotoxins. Although biomass, chlorophylls (a and b), carotenoids and glutathione-S-transferase of parsley and coriander exposed to the crude extracts containing MC or CYN had shown variations, these values were not statistically significantly different. Protein synthesis is not inhibited in coriander exposed to MC or CYN and in parsley exposed to MC. Also, glutathione reductase (GR) and glutathione peroxidase (GPx) in parsley and coriander was not affected by exposure to MC, and in coriander, the CYN did not induce statistically significant differences in these two antioxidative enzymes. Only parsley showed statistically significant increase in protein content exposed to 0.5 µg CYN mL-1 (3.981 ± 0.099 mg g-1 FW) compared to control (2.484 ± 0.145 mg g-1 FW), statistically significant decrease in GR exposed to 0.1 µg CYN mL-1 (0.684 ± 0.117 nmol min-1 mg-1 protein) compared to control (1.30 ± 0.06 nmol min-1 mg-1 protein) and statistically significant increase in GPx exposed to 1 µg CYN mL-1 (0.054 ± 0.026 nmol min-1 mg-1 protein) compared to 0.5 µg CYN mL-1 (0.003 ± 0.001 nmol min-1 mg-1 protein). These changes may be due to the induction of defensive mechanisms by plants by the presence of toxic compounds in the soil or probably to a low generation of reactive oxygen species. Furthermore, the parsley and coriander leaves and stems after 10 days of exposure did not accumulate microcystins or cylindrospermopsin.

The impact of long-lasting microbial larvicides in reducing malaria transmission and clinical malaria incidence: study protocol for a cluster randomized controlled trial.

BACKGROUND: The massive scale-up of insecticide-treated nets (ITNs) and indoor residual spraying (IRS) has led to a substantial increase in malaria vector insecticide resistance as well as in increased outdoor transmission, both of which hamper the effectiveness and efficiency of ITN and IRS. Long-lasting microbial larvicide can be a cost-effective new supplemental intervention tool for malaria control. METHODS/DESIGN: We will implement the long-lasting microbial larvicide intervention in 28 clusters in two counties in western Kenya. We will test FourStar controlled release larvicide (6 % by weight Bacillus thuringiensis israelensis and 1 % Bacillus sphaerius) by applying FourStar controlled release granule formulation, 90-day briquettes, and 180-day briquettes in different habitat types. The primary endpoint is clinical malaria incidence rate and the secondary endpoint is malaria vector abundance and transmission intensity. The intervention will be conducted as a two-step approach. First, we will conduct a four-cluster trial (two clusters per county, with one of the two clusters randomly assigned to the intervention arm) to optimize the larvicide application scheme. Second, we will conduct an open-label, cluster-randomized trial to evaluate the effectiveness and cost-effectiveness of the larvicide. Fourteen clusters in each county will be assigned to intervention (treatment) or no intervention (control) by a block randomization on the basis of clinical malaria incidence, vector density, and human population size per site. We will treat each treatment cluster with larvicide for three rounds at 4-month intervals, followed by no treatment for the following 8 months. Next, we will switch the control and treatment sites. The former control sites will receive three rounds of larvicide treatment at appropriate time intervals, and former treatment sites will receive no larvicide. We will monitor indoor and outdoor vector abundance using CO2-baited CDC light traps equipped with collection bottle rotators. Clinical malaria data will be aggregated from government-run malaria treatment centers. DISCUSSION: Since current first-line vector intervention methods do not target outdoor transmission and will select for higher insecticide resistance, new methods beyond bed nets and IRS should be considered. Long-lasting microbial larviciding represents a promising new tool that can target both indoor and outdoor transmission and alleviate the problem of pyrethroid resistance. It also has the potential to diminish costs by reducing larvicide reapplications. If successful, it could revolutionize malaria vector control in Africa, just as long-lasting bed nets have done. TRIAL REGISTRATION: U.S. National Institute of Health, study ID NCT02392832 . Registered on 3 February 2015.

Microcystin-LR bioaccumulation and depuration kinetics in lettuce and arugula: Human health risk assessment.

Microcystin-LR (MC-LR) is one of the most toxic and common microcystins (MCs) variant found in aquatic ecosystems. Little is known about the possibility of recovering microcystins contaminated agricultural crops. The objectives of this study were to determine the bioaccumulation and depuration kinetics of MC-LR in leaf tissues of lettuce and arugula, and estimate the total daily intake (ToDI) of MC-LR via contaminated vegetables by humans. Arugula and lettuce were irrigated with contaminated water having 5 and 10µgL(-1) of MC-LR for 7days (bioaccumulation), and subsequently, with uncontaminated water for 7days (depuration). Quantification of MC-LR was performed by LC-MS/MS. The one-compartment biokinetic model was employed for MC-LR bioaccumulation and depuration data analysis. MC-LR was only accumulated in lettuce. After 7days of irrigation with uncontaminated water, over 25% of accumulated MC-LR was still retained in leaf tissues of plants treated with 10µgL(-1) MC-LR. Total daily toxin intake by adult consumers (60kg-bw) exceeded the 0.04µgMC-LRkg(-1) limit recommended by WHO. Bioaccumulation was found to be linearly proportional to the exposure concentration of the toxin, increasing over time; and estimated to become saturated after 30days of uninterrupted exposure. On the other hand, MC-LR depuration was less efficient at higher exposure concentrations. This is because biokinetic half-life calculations gave 2.9 and 3.7days for 5 and 10µgL(-1) MC-LR treatments, which means 29-37days are required to eliminate the toxin. For the first time, our results demonstrated the possibility of MC-LR decontamination of lettuce plants.

Assessment of the mutagenic and genotoxic activity of cyanobacterial toxin beta-N-methyl-amino-L-alanine in Salmonella typhimurium.

A neurotoxin ß-N-methylamino-L-alanine (L-BMAA) is a non-protein amino acid produced by most cyanobacteria ubiquitously present in aquatic and terrestrial environments. Due to its global presence in surface waters, a widespread human exposure is possible and therefore this toxin represents a health risk for humans and animals. L-BMAA has been linked to the development of a variety of neurodegenerative diseases. Its neurotoxic activity has been extensively studied, while nothing is known on its genotoxic properties. In the present study we evaluated for the first time L-BMAA mutagenic potential using Ames assay on several Salmonella typhimurium strains (TA97a, TA98, TA100, TA102 and TA1535). The results showed that the toxin (up to 0.9 mg/plate) did not induce mutations without or with S9 metabolic activation. Its genotoxic activity was further studied with the SOS/umuC assay on S. typhimurium TA1535/pSK1002 and the results showed that it was not cytotoxic nor genotoxic for bacteria. The present study represents the first evidence that L-BMAA is not mutagenic nor genotoxic for bacteria even at concentrations much higher than those typically found in the environment. However, as most of the cyanobacterial toxins are not bacterial mutagens it is very important to further elucidate its genotoxic activity in eukaryotic cells.

Pocket analysis of the full-length cholix toxin. An assessment of the structure-dynamics of the apo catalytic domain.

Cholix toxin from Vibrio cholerae is the third member of the diphtheria toxin (DT) group of mono-ADP-ribosyltransferase (mART) bacterial toxins. It shares structural and functional properties with Pseudomonas aeruginosa exotoxin A and Corynebacterium diphtheriae DT. Cholix toxin is an important model for the development of antivirulence approaches and therapeutics against these toxins from pathogenic bacteria. Herein, we have used the high-resolution X-ray structure of full-length cholix complexed with NAD(+) to describe the properties of the NAD(+)-binding pocket at the residue level, including the role of crystallographic water molecules in the NAD(+) substrate interaction. The full-length apo cholix structure is used to describe the putative NAD(+)-binding site(s) and to correlate biochemical with crystallographic data to study the stoichiometry and orientation of bound NAD(+) molecules. We quantitatively describe the NAD(+) substrate interactions on a residue basis for the main 22 pocket residues in cholixf, a glycerol and 5 contact water molecules as part of the recognition surface by the substrate according to the conditions of crystallization. In addition, the dynamic properties of an in silico version of the catalytic domain were investigated in order to understand the lack of electronic density for one of the main flexible loops (R-loop) in the pocket of X-ray complexes. Implications for a rational drug design approach for mART toxins are derived.

Dietary mechanism behind the costs associated with resistance to Bacillus thuringiensis in the cabbage looper, Trichoplusia ni.

Beneficial alleles that spread rapidly as an adaptation to a new environment are often associated with costs that reduce the fitness of the population in the original environment. Several species of insect pests have evolved resistance to Bacillus thuringiensis (Bt) toxins in the field, jeopardizing its future use. This has most commonly occurred through the alteration of insect midgut binding sites specific for Bt toxins. While fitness costs related to Bt resistance alleles have often been recorded, the mechanisms behind them have remained obscure. We asked whether evolved resistance to Bt alters dietary nutrient intake, and if reduced efficiency of converting ingested nutrients to body growth are associated with fitness costs and variation in susceptibility to Bt. We fed the cabbage looper Trichoplusia ni artificial diets differing in levels of dietary imbalance in two major macronutrients, protein and digestible carbohydrate. By comparing a Bt-resistant T. ni strain with a susceptible strain we found that the mechanism behind reduced pupal weights and growth rates associated with Bt-resistance in T. ni was reduced consumption rather than impaired conversion of ingested nutrients to growth. In fact, Bt-resistant T. ni showed more efficient conversion of nutrients than the susceptible strain under certain dietary conditions. Although increasing levels of dietary protein prior to Bt challenge had a positive effect on larval survival, the LC50 of the resistant strain decreased when fed high levels of excess protein, whereas the LC50 of the susceptible strain continued to rise. Our study demonstrates that examining the nutritional basis of fitness costs may help elucidate the mechanisms underpinning them.

Evaluation of the diagnostic performance of the xpert Clostridium difficile assay and its comparison with the toxin A/B enzyme-linked fluorescent assay and in-house real-time PCR assay used for the detection of toxigenic C. difficile.

BACKGROUND: Clostridium difficile genes or toxin can be detected using several laboratory techniques. In this study, we compared the performance of the Xpert C. difficile assay with that of a toxin A/B enzyme-linked fluorescent immunoassay (ELFA) and an in-house real-time PCR assay for the tcdB gene. METHODS: From April 2011 through January 2012, 138 soft or liquid stool samples from 138 adult patients at Paik Hospital were tested using the toxin A/B ELFA, in-house real-time PCR assay, and Xpert C. difficile assay to detect toxigenic C. difficile. Specimens were considered true positives if results were positive in both the in-house real-time PCR for tcdB gene and Xpert C. difficile assays. RESULTS: Sensitivity of the toxin A/B ELFA, in-house tcdB gene real-time PCR, and Xpert C. difficile assay were 67.6%, 97.3%, and 100.0%, respectively. The specificity of the in-house tcdB gene real-time PCR assay was 100%, while the specificity was 98.0% for the other two methods. The turnaround time (TAT) was 50 min for the Xpert C. difficile assay, 75 min for the toxin A/B ELFA, and 160 min for the in-house real-time PCR assay. CONCLUSION: The Xpert C. difficile assay and the in-house real-time PCR assay had higher sensitivity than the toxin A/B ELFA; however, the specificities of the three assays were similar. Considering its rapid TAT and high sensitivity, use of the Xpert C. difficile assay is highly recommended for rapid and accurate diagnosis of C. difficile infection.

Growth feedback as a basis for persister bistability.

A small fraction of cells in many bacterial populations, called persisters, are much less sensitive to antibiotic treatment than the majority. Persisters are in a dormant metabolic state, even while remaining genetically identical to the actively growing cells. Toxin and antitoxin modules in bacteria are believed to be one possible cause of persistence. A two-gene operon, HipBA, is one of many chromosomally encoded toxin and antitoxin modules in Escherichia coli and the HipA7 allelic variant was the first validated high-persistence mutant. Here, we present a stochastic model that can generate bistability of the HipBA system, via the reciprocal coupling of free HipA to the cellular growth rate. The actively growing state and the dormant state each correspond to a stable state of this model. Fluctuations enable transitions from one to the other. This model is fully in agreement with experimental data obtained with synthetic promoter constructs.

Bayesian decision tree for the classification of the mode of motion in single-molecule trajectories.

Membrane proteins move in heterogeneous environments with spatially (sometimes temporally) varying friction and with biochemical interactions with various partners. It is important to reliably distinguish different modes of motion to improve our knowledge of the membrane architecture and to understand the nature of interactions between membrane proteins and their environments. Here, we present an analysis technique for single molecule tracking (SMT) trajectories that can determine the preferred model of motion that best matches observed trajectories. The method is based on Bayesian inference to calculate the posteriori probability of an observed trajectory according to a certain model. Information theory criteria, such as the Bayesian information criterion (BIC), the Akaike information criterion (AIC), and modified AIC (AICc), are used to select the preferred model. The considered group of models includes free Brownian motion, and confined motion in 2nd or 4th order potentials. We determine the best information criteria for classifying trajectories. We tested its limits through simulations matching large sets of experimental conditions and we built a decision tree. This decision tree first uses the BIC to distinguish between free Brownian motion and confined motion. In a second step, it classifies the confining potential further using the AIC. We apply the method to experimental Clostridium Perfingens [Formula: see text]-toxin (CP[Formula: see text]T) receptor trajectories to show that these receptors are confined by a spring-like potential. An adaptation of this technique was applied on a sliding window in the temporal dimension along the trajectory. We applied this adaptation to experimental CP[Formula: see text]T trajectories that lose confinement due to disaggregation of confining domains. This new technique adds another dimension to the discussion of SMT data. The mode of motion of a receptor might hold more biologically relevant information than the diffusion coefficient or domain size and may be a better tool to classify and compare different SMT experiments.

A quantitative microbiological exposure assessment model for Bacillus cereus in REPFEDs.

One of the pathogens of concern in refrigerated and processed foods of extended durability (REPFED) is psychrotrophic Bacillus cereus, because of its ability to survive pasteurisation and grow at low temperatures. In this study a quantitative microbiological exposure assessment (QMEA) of psychrotrophic B. cereus in REPFEDs is presented. The goal is to quantify (i) the prevalence and concentration of B. cereus during production and shelf life, (ii) the number of packages with potential emetic toxin formation and (iii) the impact of different processing steps and consumer behaviour on the exposure to B. cereus from REPFEDs. The QMEA comprises the entire production and distribution process, from raw materials over pasteurisation and up to the moment it is consumed or discarded. To model this process the modular process risk model (MPRM) was used (Nauta, 2002). The product life was divided into nine modules, each module corresponding to a basic process: (1) raw material contamination, (2) cross contamination during handling, (3) inactivation during preparation, (4) growth during intermediate storage, (5) partitioning of batches in portions, (6) mixing portions to create the product, (7) recontamination during assembly and packaging, (8) inactivation during pasteurisation and (9) growth during shelf life. Each of the modules was modelled and built using a combination of newly gathered and literature data, predictive models and expert opinions. Units (batch/portion/package) with a B. cereus concentration of 10(5)CFU/g or more were considered 'risky' units. Results show that the main drivers of variability and uncertainty are consumer behaviour, strain variability and modelling error. The prevalence of B. cereus in the final products is estimated at 48.6% (±0.01%) and the number of packs with too high B. cereus counts at the moment of consumption is estimated at 4750 packs per million (0.48%). Cold storage at retail and consumer level is vital in limiting the exposure. Four key points were identified (i) raw material contamination, (ii) recontamination during packaging, (iii) reduction during pasteurisation and cooking and (iv) cold storage at retail and consumer level.

A review of current knowledge on toxic benthic freshwater cyanobacteria--ecology, toxin production and risk management.

Benthic cyanobacteria are found globally in plethora of environments. Although they have received less attention than their planktonic freshwater counterparts, it is now well established that they produce toxins and reports of their involvement in animal poisonings have increased markedly during the last decade. Most of the known cyanotoxins have been identified from benthic cyanobacteria including: the hepatotoxic microcystins, nodularins and cylindrospermopsins, the neurotoxic saxitoxins, anatoxin-a and homoanatoxin-a and dermatotoxins, such as lyngbyatoxin. In most countries, observations of toxic benthic cyanobacteria are fragmented, descriptive and in response to animal toxicosis events. Only a limited number of long-term studies have aimed to understand why benthic proliferations occur, and/or how toxin production is regulated. These studies have shown that benthic cyanobacterial blooms are commonly a mixture of toxic and non-toxic genotypes and that toxin concentrations can be highly variable spatially and temporally. Physiochemical parameters responsible for benthic proliferation vary among habitat type with physical disturbance (e.g., flow regimes, wave action) and nutrients commonly identified as important. As climatic conditions change and anthropogenic pressures on waterways increase, it seems likely that the prevalence of blooms of benthic cyanobacteria will increase. In this article we review current knowledge on benthic cyanobacteria: ecology, toxin-producing species, variables that regulate toxin production and bloom formation, their impact on aquatic and terrestrial organisms and current monitoring and management strategies. We suggest research needs that will assist in filling knowledge gaps and ultimately allow more robust monitoring and management protocols to be developed.

TAC from Mycobacterium tuberculosis: a paradigm for stress-responsive toxin-antitoxin systems controlled by SecB-like chaperones.

Bacterial type II toxin-antitoxins (TAs) are two-component systems that modulate growth in response to specific stress conditions, thus promoting adaptation and persistence. The major human pathogen Mycobacterium tuberculosis potentially encodes 75 TAs and it has been proposed that persistence induced by active toxins might be relevant for its pathogenesis. In this work, we focus on the newly discovered toxin-antitoxin-chaperone (TAC) system of M. tuberculosis, an atypical stress-responsive TA system tightly controlled by a molecular chaperone that shows similarity to the canonical SecB chaperone involved in Sec-dependent protein export in Gram-negative bacteria. We performed a large-scale genome screening to reconstruct the evolutionary history of TAC systems and found that TAC is not restricted to mycobacteria and seems to have disseminated in diverse taxonomic groups by horizontal gene transfer. Our results suggest that TAC chaperones are evolutionary related to the solitary chaperone SecB and have diverged to become specialized toward their cognate antitoxins.