Toxin immunosensors and sensor arrays for food quality control

The global efforts to improve consumer protection and public health lead to an increasing number of analytical approaches applicable to food analysis and process control. Biosensor systems are efficient analytical tools to monitor production processes or storage of nutrition and to control contamination outbreaks as they are easy-to-use, fast, and with minimal effort on sample preparation. Relevant targets of immunosensors implemented to food safety are prevalent bacterial toxins (staphylococcal enterotoxins and clostridial toxins), plant toxins (Ricin), mycotoxins (aflatoxins and ochratoxin A), marine toxins, and other pathogenic bacterial contaminations (Listeria, Salmonella, Staphylococcus aureus, or Escherichia coli). These cause acute intoxication and also chronic diseases in humans consuming contaminated food. Promising approaches for the determination of different types of toxins in food matrices will be outlined. The corresponding sensor systems use immunological receptor units such as antibodies or antigens and include optical (fluorescence and surface plasmon resonance), electrochemical, or acoustical readout methods. This review is focused on recent developments of sensor formats devoted to food safety control and is structured according to the type of toxin or contaminant that is recognized. It is intended to give an overview on emerging sensor technologies and their potential applications for the rapid analysis of the most important food poisoning agents.     

Coupling Immunomagnetic Separation on Magnetic Beads with Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Detection of Staphylococcal Enterotoxin B

The growing importance of mass spectrometry for the identification and characterization of bacterial protein toxins is a consequence of the improved sensitivity and specificity of mass spectrometry-based techniques, especially when these techniques are combined with affinity methods. Here we describe a novel method based on the use of immunoaffinity capture and matrix-assisted laser desorption ionization-time of flight mass spectrometry for selective purification and detection of staphylococcal enterotoxin B (SEB). SEB is a potent bacterial protein toxin responsible for food poisoning, as well as a potential biological warfare agent. Unambiguous detection of SEB at low-nanogram levels in complex matrices is thus an important objective. In this work, an affinity molecular probe was prepared by immobilizing anti-SEB antibody on the surface of para-toluene-sulfonyl-functionalized monodisperse magnetic particles and used to selectively isolate SEB. Immobilization and affinity capture procedures were optimized to maximize the density of anti-SEB immunoglobulin G and the amount of captured SEB, respectively, on the surface of magnetic beads. SEB could be detected directly “on beads” by placing the molecular probe on the matrix-assisted laser desorption ionization target plate or, alternatively, “off beads” after its acidic elution. Application of this method to complex biological matrices was demonstrated by selective detection of SEB present in different matrices, such as cultivation media of Staphylococcus aureus strains and raw milk samples.     

Bacterial toxin and effector glycosyltransferases

Clostridial glucosylating cytotoxins, including Clostridium difficile toxins A and B, Clostridium novyi α-toxin, and Clostridium sordellii lethal toxin, are major virulence factors and causative agents of human diseases. These toxins mono-O-glucosylate (or mono-O-GlcNAcylate) a specific threonine residue of Rho/Ras-proteins, which is essential for the function of the molecular switches. Recently, a related group of glucosyltransferases from Legionella pneumophila has been identified. These Legionella glucosyltransferases modify the large GTPase elongation factor eEF1A at a serine residue by mono-O-glucosylation, thereby inhibiting protein synthesis of target cells. Recent results on structures, functions and biological roles of both groups of bacterial toxin glucosyltransferases will be discussed.     

L-Phase variants ofAgromyces ramosus

Agromyces ramosus, which is a numerically prevalent bacterium in soil, was easily induced into the L-phase by growing it on agar media containing low levels of penicillin or glycine. The L-forms were stable after initial contact with the inducing agent and could not be reverted to the bacterial form by any of the procedures tried. These results are discussed in relation to a possible natural occurrence of L-forms of this bacterium in soil.     

Investigation of Reported Aflatoxin Production by Fungi Outside the Aspergillus flavus Group

A screening study of 121 fungus isolates, representing 29 species, for aflatoxin synthesis demonstrated this property only in Aspergillus flavus and A. parasiticus. Eight of the organisms found negative were isolates reported by other investigators to produce aflatoxin. Since similar negative reports have come from several other workers, it is concluded that only the A. flavus group of Aspergillus can presently be certified as sources of these toxins. Reasons for possible false-positive findings are discussed along with precautionary measures and differential analytical procedures useful in aflatoxin screening studies.     

Genome mapping ofClostridium perfringens strains with I-CeuI shows many virulence genes to be plasmid-borne

The intron-encoded endonuclease I-CeuI fromChlamydomonas eugametos was shown to cleave the circular chromosomes of allClostridium perfringens strains examined at single sites in the rRNA operons, thereby generating ten fragments suitable for the rapid mapping of virulence genes by pulsed-field gel electrophoresis (PFGE). This method easily distinguishes between plasmid and chromosomal localisations, as I-CeuI only cuts chromosomal DNA. Using this approach, the genes for three of the four typing toxins,β, ε, and?, in addition to the enterotoxin andλ-toxin genes, were shown to be plasmid-borne. In a minority of strains, associated with food poisoning, where the enterotoxin toxin gene was located on the chromosome, genes for two of the minor toxins,? andμ, were missing.     

Comparison of ELISA and SPR biosensor technology for the detection of paralytic shellfish poisoning toxins

An enzyme labeled immunosorbent assay (ELISA) and surface plasmon resonance (SPR) biosensor assay for the detection of paralytic shellfish poisoning (PSP) toxins were developed and a comparative evaluation was performed. A polyclonal antibody (BC67) used in both assay formats was raised to saxitoxin–jeffamine–BSA in New Zealand white rabbits. Each assay format was designed as an inhibition assay. Shellfish samples (n = 54) were evaluated by each method using two simple rapid extraction procedures and compared to the AOAC high performance liquid chromatography (HPLC) and the mouse bioassay (MBA). The results of each assay format were comparable with the HPLC and MBA methods and demonstrate that an antibody with high sensitivity and broad specificity to PSP toxins can be applied to different immunological techniques. The method of choice will depend on the end-users needs. The reduced manual labor and simplicity of operation of the SPR biosensor compared to ELISA, ease of sample extraction and superior real time semi-quantitative analysis are key features that could make this technology applicable in a high-throughput monitoring unit.     

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

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

Three Dimensional Structure of the MqsR:MqsA Complex: A Novel TA Pair Comprised of a Toxin Homologous to RelE and an Antitoxin with Unique Properties

One mechanism by which bacteria survive environmental stress is through the formation of bacterial persisters, a sub-population of genetically identical quiescent cells that exhibit multidrug tolerance and are highly enriched in bacterial toxins. Recently, the Escherichia coli gene mqsR (b3022) was identified as the gene most highly upregulated in persisters. Here, we report multiple individual and complex three-dimensional structures of MqsR and its antitoxin MqsA (B3021), which reveal that MqsR:MqsA form a novel toxin:antitoxin (TA) pair. MqsR adopts an α/β fold that is homologous with the RelE/YoeB family of bacterial ribonuclease toxins. MqsA is an elongated dimer that neutralizes MqsR toxicity. As expected for a TA pair, MqsA binds its own promoter. Unexpectedly, it also binds the promoters of genes important for E. coli physiology (e.g., mcbR, spy). Unlike canonical antitoxins, MqsA is also structured throughout its entire sequence, binds zinc and coordinates DNA via its C- and not N-terminal domain. These studies reveal that TA systems, especially the antitoxins, are significantly more diverse than previously recognized and provide new insights into the role of toxins in maintaining the persister state.     

Production and excretion of okadaic acid,pectenotoxin-2 and a novel dinophysistoxin from the DSP-causing marine dinoflagellate Dinophysis acuta – Effects of light,food availability and growth phase

Diarrhetic shellfish poisoning (DSP) toxins constitute a severe economic threat to shellfish industries and a major food safety issue for shellfish consumers. The prime producers of the DSP toxins that end up in filter feeding shellfish are species of the marine mixotrophic dinoflagellate genus Dinophysis. Intraspecific toxin contents of Dinophysis spp. vary a lot, but the regulating factors of toxin content are still poorly understood. Dinophysis spp. have been shown to sequester and use chloroplasts from their ciliate prey, and with this rare mode of nutrition, irradiance and food availability could play a key role in the regulation of toxins contents and production. We investigated toxin contents, production and excretion of a Dinophysis acuta culture under different irradiances, food availabilities and growth phases. The newly isolated strain of D. acuta contained okadaic acid (OA), pectenotoxins-2 (PTX-2) and a novel dinophysistoxin (DTX) that we tentatively describe as DTX-1b isomer. We found that all three toxins were excreted to the surrounding seawater, and for OA and DTX-1b as much as 90% could be found in extracellular toxin pools. For PTX-2 somewhat less was excreted, but often >50% was found extracellularly. This was the case both in steady-state exponential growth and in food limited, stationary growth, and we emphasize the need to include extracellular toxins in future studies of DSP toxins. Cellular toxin contents were largely unaffected by irradiance, but toxins accumulated both intra- and extracellularly when starvation reduced growth rates of D. acuta. Toxin production rates were highest during exponential growth, but continued at decreased rates when cell division ceased, indicating that toxin production is not directly associated with ingestion of prey. Finally, we explore the potential of these new discoveries to shed light on the ecological role of DSP toxins.     

North Atlantic right whales,Eubalaena glacialis,exposed to paralytic shellfish poisoning (PSP) toxins via a zooplankton vector,Calanus finmarchicus

The seriously endangered north Atlantic right whale (Eubalaena glacialis) is regularly exposed to the neurotoxins responsible for paralytic shellfish poisoning (PSP) through feeding on contaminated zooplankton acting as a vector of these dinoflagellate toxins. This chronic exposure occurs during several months each summer while the whales are present on their late summer feeding ground in Grand Manan Basin in the lower Bay of Fundy. Based on estimated ingestion rates, we suggest that these toxins could affect respiratory capabilities, feeding behavior, and ultimately the reproductive condition of the whale population.     

Physiologic Stresses Reveal a Salmonella Persister State and TA Family Toxins Modulate Tolerance to These Stresses

Bacterial persister cells are considered a basis for chronic infections and relapse caused by bacterial pathogens. Persisters are phenotypic variants characterized by low metabolic activity and slow or no replication. This low metabolic state increases pathogen tolerance to antibiotics and host immune defenses that target actively growing cells. In this study we demonstrate that within a population of Salmonella enterica serotype Typhimurium, a small percentage of bacteria are reversibly tolerant to specific stressors that mimic the macrophage host environment. Numerous studies show that Toxin-Antitoxin (TA) systems contribute to persister states, based on toxin inhibition of bacterial metabolism or growth. To identify toxins that may promote a persister state in response to host-associated stressors, we analyzed the six TA loci specific to S. enterica serotypes that cause systemic infection in mammals, including five RelBE family members and one VapBC member. Deletion of TA loci increased or decreased tolerance depending on the stress conditions. Similarly, exogenous expression of toxins had mixed effects on bacterial survival in response to stress. In macrophages, S. Typhimurium induced expression of three of the toxins examined. These observations indicate that distinct toxin family members have protective capabilities for specific stressors but also suggest that TA loci have both positive and negative effects on tolerance.     

Biotechnological significance of toxic marine dinoflagellates

Dinoflagellates are microalgae that are associated with the production of many marine toxins. These toxins poison fish, other wildlife and humans. Dinoflagellate-associated human poisonings include paralytic shellfish poisoning, diarrhetic shellfish poisoning, neurotoxic shellfish poisoning, and ciguatera fish poisoning. Dinoflagellate toxins and bioactives are of increasing interest because of their commercial impact, influence on safety of seafood, and potential medical and other applications. This review discusses biotechnological methods of identifying toxic dinoflagellates and detecting their toxins. Potential applications of the toxins are discussed. A lack of sufficient quantities of toxins for investigational purposes remains a significant limitation. Producing quantities of dinoflagellate bioactives requires an ability to mass culture them. Considerations relating to bioreactor culture of generally fragile and slow-growing dinoflagellates are discussed. Production and processing of dinoflagellates to extract bioactives, require attention to biosafety considerations as outlined in this review.     

Traceability of Amanita fuliginea poisoning using DNA barcoding and UPLC-MS/MS

Amanita fuliginea is a lethal poisonous mushroom found in Japan and southern China. The primary toxins are α-amanitin (α-AMA) and β-amanitin (β-AMA). There is a lack of systematic and comprehensive investigations on the traceability of A. fuliginea poisoning due to technological limitations. This study aimed to examine whether A. fuliginea poisoning incidents could be traced using DNA barcoding and ultraperformance liquid chromatography coupled with triple quadrupole mass spectrometry (UPLC-MS/MS). We collected A. fuliginea specimens and prepared cooked and cooked plus simulated gastric fluid (SGF)-treated samples. We then performed DNA barcoding of internal transcribed spacer regions for species identification and UPLC-MS/MS for toxin level determination. Our results indicate that under the experimental conditions used herein, DNA barcoding can be used for molecular identification of mushroom samples that are cooked and/or cooked plus SGF-treated for less than 30 min; UPLC-MS/MS can be used for toxin analysis of cooked and SGF-treated (0–1440 min) samples. This is the first time that DNA barcoding and UPLC-MS/MS have been combined for studying the toxicological traceability of A. fuliginea using simulated gastric contents or vomit in northern China. Our data provide support for the treatment of clinical mushroom poisoning cases.     

Implementation of a Permeable Membrane Insert-based Infection System to Study the Effects of Secreted Bacterial Toxins on Mammalian Host Cells

Many bacterial pathogens secrete potent toxins to aid in the destruction of host tissue, to initiate signaling changes in host cells or to manipulate immune system responses during the course of infection. Though methods have been developed to successfully purify and produce many of these important virulence factors, there are still many bacterial toxins whose unique structure or extensive post-translational modifications make them difficult to purify and study in in vitro systems. Furthermore, even when pure toxin can be obtained, there are many challenges associated with studying the specific effects of a toxin under relevant physiological conditions. Most in vitro cell culture models designed to assess the effects of secreted bacterial toxins on host cells involve incubating host cells with a one-time dose of toxin. Such methods poorly approximate what host cells actually experience during an infection, where toxin is continually produced by bacterial cells and allowed to accumulate gradually during the course of infection. This protocol describes the design of a permeable membrane insert-based bacterial infection system to study the effects of Streptolysin S, a potent toxin produced by Group A Streptococcus, on human epithelial keratinocytes. This system more closely mimics the natural physiological environment during an infection than methods where pure toxin or bacterial supernatants are directly applied to host cells. Importantly, this method also eliminates the bias of host responses that are due to direct contact between the bacteria and host cells. This system has been utilized to effectively assess the effects of Streptolysin S (SLS) on host membrane integrity, cellular viability, and cellular signaling responses. This technique can be readily applied to the study of other secreted virulence factors on a variety of mammalian host cell types to investigate the specific role of a secreted bacterial factor during the course of infection.     

Erratum to Bacterial diversity in toxic<Emphasis Type="Italic"> Alexandrium tamarense</Emphasis> blooms off the Orkney Isles and the Firth of Forth

The genetic diversity of the bacterial community associated with Alexandrium tamarense blooms was studied in blooms of the toxic dinoflagellates in the waters around the Orkney Isles and the Firth of Forth (Scotland). For toxin and molecular analysis of the bacterial communities associated with the toxic bloom, water samples were taken in 1998 and 1999 from A. tamarense blooms. The bacterial community structure, as determined by DGGE (denaturing gradient gel electrophoresis) showed clear differences between all three investigated size fractions (dinoflagellate-associated bacteria, attached bacteria and free-living bacteria), with high diversity within each sample. DNA sequence analysis of the dominant and most frequent DGGE bands revealed the dominance of α Proteobacteria, mainly of the Roseobacter clade, with similarities of 91–99%. Moreover, DGGE bands occurring at the same position in the gel throughout in most samples corroborate the presence of several specific α Proteobacteria of the Roseobacter clade. Overall, 500 bacteria were isolated from the bloom and partly phylogenetically analysed. They were members of two prokaryotic phyla, the Proteobacteria and the Bacteroidetes, related to Proteobacteria of the α and γ subdivisions (Alteromonas, Pseudoalteromonas and Colwellia). All bacteria were tested for the production of sodium channel blocking (SCB) toxins using mouse neuroblastoma assay. No production of SCB toxins was found and high performance liquid chromatography (HPLC) analysis confirmed these results. The content of total paralytic shellfish poisoning (PSP) toxin in the water samples, as measured within the toxic dinoflagellate blooms using HPLC, ranged from 53 to 2191 ng PSP l^?1 in 1998 and from 0 to 478 ng PSP l^?1 in 1999. Changes in PSP toxin content were not accompanied by changes of DGGE band patterns. We therefore presume that the bacterial groups identified in this study were not exclusively associated with toxic A. tamarense, but were generally associated with the phytoplankton.     

Impacts of Florida red tides on coastal communities

Over the last few decades, scientific research has helped to describe the disease neurotoxic shellfish poisoning (NSP) by identifying the causative organism, Karenia brevis, and by characterizing the disease-causing toxins, a suite of polyether toxins called brevetoxins. In addition to causing disease in exposed human populations, K. brevis blooms and associated management responses have been linked to other effects on coastal communities. Some of these effects are negative, such as the loss of tourism dollars and the increased burden on local health care services caused by increases in human disease incidence. However, some of the effects are positive, such as the significant improvement in detecting brevetoxins in environmental samples and clinical specimens. This review discusses the health and economic effects from K. brevis blooms on Florida coastal communities and the current efforts to identify the data needed to assess social and cultural effects.     

Design,synthesis, in silico studies and in vitro evaluation of isatin-pyridine oximes hybrids as novel acetylcholinesterase reactivators

Organophosphorus poisoning caused by some pesticides and nerve agents is a life-threating condition that must be swiftly addressed to avoid casualties. Despite the availability of medical countermeasures, the clinically available compounds lack a broad spectrum, are not effective towards all organophosphorus toxins, and have poor pharmacokinetics properties to allow them crossing the blood-brain barrier, hampering cholinesterase reactivation at the central nervous system. In this work, we designed and synthesised novel isatin derivatives, linked to a pyridinium 4-oxime moiety by an alkyl chain with improved calculated properties, and tested their reactivation potency against paraoxon- and NEMP-inhibited acetylcholinesterase in comparison to the standard antidote pralidoxime. Our results showed that these compounds displayed comparable in vitro reactivation also pointed by the in silico studies, suggesting that they are promising compounds to tackle organophosphorus poisoning.     

The seasonal dynamics of diarrhetic shellfish poisoning toxins in the digestive gland of <Emphasis Type="Italic">Crenomytilus grayanus</Emphasis> (Dunker, 1853) analyzed by high-performance liquid chromatography with fluorometric detection

The seasonal dynamics of diarrhetic shellfish poisoning (DSP) toxins in the digestive gland of the mussel Crenomytilus grayanus (Dunker, 1853) from Peter the Great Bay in the Sea of Japan have been studied. Toxins in the form of 4-bromomethyl-7-methoxycoumarin derivatives have been analyzed using high-performance liquid chromatography (HPLC) with fluorometric detection. This method allows the determination of the quantitative and qualitative composition of individual toxins. All the C. grayanus samples contained DSP toxins, but their concentration varied substantially during the year. The highest concentration was recorded from the mussels collected in May; the lowest concentration was recorded in February. The toxins were represented predominantly by dinophysistoxin-2; in July-September and December, okadaic acid was detected. An assumption has been made about the relationship between the potential producers of DSP toxins in the waters of the Peter the Great Bay and the dynamics of these toxins in tissues of C. grayanus. The HPLC method, as an alternative to the enzyme-linked immunosorbent assay, was used to assess the quantitative and qualitative composition of DSP toxins in seafood for the first time in the Russian Federation. This method can be recommended for the routine monitoring of toxins conducted by the controlling organizations of the Russian Federal Service for Veterinary and Phytosanitary Surveillance.