First report of foodborne botulism due to Clostridium botulinum type A(B) from vegetarian home-canned pate in Hanoi, Vietnam.

Even one case of foodborne botulism constitutes a public health emergency. We report a series of cases with delayed treatment due to delayed diagnosis. Clostridium botulinum type A(B) was isolated from vegetarian home-canned pate, but not from stool samples. These are the first recorded cases of foodborne botulism in Hanoi.

Chip-based sensing for release of unprocessed cell surface proteins in vitro and in serum and its (patho)physiological relevance.

To study the possibility that certain components of eukaryotic plasma membranes are released under certain (patho)physiological conditions, a chip-based sensor was developed for the detection of cell surface proteins, which are anchored at the outer leaflet of eukaryotic plasma membranes by a covalently attached glycolipid, exclusively, and might be prone to spontaneous or regulated release on the basis of their amphiphilic character. For this, unprocessed, full-length glycosylphosphatidylinositol-anchored proteins (GPI-AP), together with associated phospholipids, were specifically captured and detected by a chip- and microfluidic channel-based sensor, leading to changes in phase and amplitude of surface acoustic waves (SAW) propagating over the chip surface. Unprocessed GPI-AP in complex with lipids were found to be released from rat adipocyte plasma membranes immobilized on the chip, which was dependent on the flow rate and composition of the buffer stream. The complexes were identified in the incubation medium of primary rat adipocytes, in correlation to the cell size, and in rat as well as human serum. With rats, the measured changes in SAW phase shift, reflecting specific mass/size or amount of the unprocessed GPI-AP in complex with lipids, and SAW amplitude, reflecting their viscoelasticity, enabled the differentiation between the lean and obese (high-fat diet) state, and the normal (Wistar) and hyperinsulinemic (Zucker fatty) as well as hyperinsulinemic hyperglycemic (Zucker diabetic fatty) state. Thus chip-based sensing for complexes of unprocessed GPI-AP and lipids reveals the inherently labile anchorage of GPI-AP at plasma membranes and their susceptibility for release in response to (intrinsic/extrinsic) cues of metabolic relevance and may, therefore, be useful for monitoring of (pre-)diabetic disease states.

An Integrative Approach to Computational Modelling of the Gene Regulatory Network Controlling Clostridium botulinum Type A1 Toxin Production.

Clostridium botulinum produces botulinum neurotoxins (BoNTs), highly potent substances responsible for botulism. Currently, mathematical models of C. botulinum growth and toxigenesis are largely aimed at risk assessment and do not include explicit genetic information beyond group level but integrate many component processes, such as signalling, membrane permeability and metabolic activity. In this paper we present a scheme for modelling neurotoxin production in C. botulinum Group I type A1, based on the integration of diverse information coming from experimental results available in the literature. Experiments show that production of BoNTs depends on the growth-phase and is under the control of positive and negative regulatory elements at the intracellular level. Toxins are released as large protein complexes and are associated with non-toxic components. Here, we systematically review and integrate those regulatory elements previously described in the literature for C. botulinum Group I type A1 into a population dynamics model, to build the very first computational model of toxin production at the molecular level. We conduct a validation of our model against several items of published experimental data for different wild type and mutant strains of C. botulinum Group I type A1. The result of this process underscores the potential of mathematical modelling at the cellular level, as a means of creating opportunities in developing new strategies that could be used to prevent botulism; and potentially contribute to improved methods for the production of toxin that is used for therapeutics.

Reoccurrence of botulinum neurotoxin subtype A3 inducing food-borne botulism, Slovakia, 2015.

A case of food-borne botulism occurred in Slovakia in 2015. Clostridium botulinum type A was isolated from three nearly empty commercial hummus tubes. The product, which was sold in Slovakia and the Czech Republic, was withdrawn from the market and a warning was issued immediately through the European Commission's Rapid Alert System for Food and Feed (RASFF). Further investigation revealed the presence of botulinum neurotoxin (BoNT) subtype BoNT/A3, a very rare subtype implicated in only one previous outbreak (Loch Maree in Scotland, 1922). It is the most divergent subtype of BoNT/A with 15.4% difference at the amino acid level compared with the prototype BoNT/A1. This makes it more prone to evading immunological and PCR-based detection. It is recommended that testing laboratories are advised that this subtype has been associated with food-borne botulism for the second time since the first outbreak almost 100 years ago, and to validate their immunological or PCR-based methods against this divergent subtype.

An atypical outbreak of food-borne botulism due to Clostridium botulinum types B and E from ham.

An outbreak of human botulism was due to consumption of ham containing botulinum neurotoxins B and E. A Clostridium botulinum type E strain isolated from ham was assigned to a new subtype (E12) based on bont/E gene sequencing and belongs to a new multilocus sequence subtype, as analyzed by whole-genome sequencing.

Two-component signal transduction system CBO0787/CBO0786 represses transcription from botulinum neurotoxin promoters in Clostridium botulinum ATCC 3502.

Blocking neurotransmission, botulinum neurotoxin is the most poisonous biological substance known to mankind. Despite its infamy as the scourge of the food industry, the neurotoxin is increasingly used as a pharmaceutical to treat an expanding range of muscle disorders. Whilst neurotoxin expression by the spore-forming bacterium Clostridium botulinum appears tightly regulated, to date only positive regulatory elements, such as the alternative sigma factor BotR, have been implicated in this control. The identification of negative regulators has proven to be elusive. Here, we show that the two-component signal transduction system CBO0787/CBO0786 negatively regulates botulinum neurotoxin expression. Single insertional inactivation of cbo0787 encoding a sensor histidine kinase, or of cbo0786 encoding a response regulator, resulted in significantly elevated neurotoxin gene expression levels and increased neurotoxin production. Recombinant CBO0786 regulator was shown to bind to the conserved -10 site of the core promoters of the ha and ntnh-botA operons, which encode the toxin structural and accessory proteins. Increasing concentration of CBO0786 inhibited BotR-directed transcription from the ha and ntnh-botA promoters, demonstrating direct transcriptional repression of the ha and ntnh-botA operons by CBO0786. Thus, we propose that CBO0786 represses neurotoxin gene expression by blocking BotR-directed transcription from the neurotoxin promoters. This is the first evidence of a negative regulator controlling botulinum neurotoxin production. Understanding the neurotoxin regulatory mechanisms is a major target of the food and pharmaceutical industries alike.

Large Outbreak of Botulism Associated with a Church Potluck Meal--Ohio, 2015.

On April 21, 2015, the Fairfield Medical Center (FMC) and Fairfield Department of Health contacted the Ohio Department of Health (ODH) about a patient suspected of having botulism in Fairfield County, Ohio. Botulism is a severe, potentially fatal neuroparalytic illness.* A single case is a public health emergency, because it can signal an outbreak. Within 2 hours of health department notification, four more patients with similar clinical features arrived at FMC's emergency department. Later that afternoon, one patient died of respiratory failure shortly after arriving at the emergency department. All affected persons had eaten at the same widely attended church potluck meal on April 19. CDC's Strategic National Stockpile sent 50 doses of botulinum antitoxin to Ohio. FMC, the Fairfield Department of Health, ODH, and CDC rapidly responded to confirm the diagnosis, identify and treat additional patients, and determine the source.

Distinguishing highly-related outbreak-associated Clostridium botulinum type A(B) strains.

BACKGROUND: In the United States, most Clostridium botulinum type A strains isolated during laboratory investigations of human botulism demonstrate the presence of an expressed type A botulinum neurotoxin (BoNT/A) gene and an unexpressed BoNT/B gene. These strains are designated type A(B). The most common pulsed-field gel electrophoresis (PFGE) pattern in the C. botulinum PulseNet database is composed of A(B) strains. The purpose of this study was to evaluate the ability of genome sequencing and multi-loci variable number of tandem repeat analysis (MLVA) to differentiate such strains. RESULTS: The genome sequences of type A(B) strains evaluated in this study are closely related and cluster together compared to other available C. botulinum Group I genomes. In silico multilocus sequence typing (MLST) analysis (7-loci) was unable to differentiate any of the type A(B) strains isolated from seven different outbreak investigations evaluated in this study. A 15-locus MLVA scheme demonstrated an improved ability to differentiate these strains, however, repeat unit variation among the strains was restricted to only two loci. Reference-free single nucleotide polymorphism (SNP) analysis demonstrated the ability to differentiate strains from all of the outbreaks examined and a non-outbreak associated strain. CONCLUSIONS: This study confirms that type A(B) strains that share the same PFGE pattern also share closely-related genome sequences. The lack of a complete type A(B) strain representative genome sequence hinders the ability to assemble genomes by reference mapping and analysis of SNPs at pre-identified sites. However, compared to other methods evaluated in this study, a reference-free SNP analysis demonstrated optimal subtyping utility for type A(B) strains using de novo assembled genome sequences.

Infant botulism with prolonged faecal excretion of botulinum neurotoxin and Clostridium botulinum for 7 months.

In Finland in April 2010, a 3-month old baby was diagnosed with type A infant botulism. He excreted botulinum neurotoxin and/or Clostridium botulinum in his faeces until November 2010. Five months of excretion was after clinical recovery and discharge from hospital. C. botulinum isolates recovered from the household dust in the patient's home were genetically identical to those found in the infant's stool samples. Long-term faecal excretion of C. botulinum may pose a possible health risk for the parents and others in close contact with the infant.

Outbreak of type A foodborne botulism at a boarding school, Uganda, 2008.

Botulism has rarely been reported in Africa. In October 2008, botulism was reported in three Ugandan boarding-school students. All were hospitalized and one died. A cohort study was performed to assess food exposures among students, and clinical specimens and available food samples were tested for botulinum toxin. Three case-patients were identified; a homemade, oil-based condiment was eaten by all three. In the cohort study, no foods were significantly associated with illness. Botulinum toxin type A was confirmed in clinical samples. This is the first confirmed outbreak of foodborne botulism in Uganda. A homemade, oil-based condiment was the probable source. Consumption of homemade oil-based condiments is widespread in Ugandan schools, putting children at risk. Clinicians and public health authorities in Uganda should consider botulism when clusters of acute flaccid paralysis are seen. Additionally, schools should be warned of the hazard of homemade oil-based condiments, and take steps to prevent their use.

An electrochemiluminescence assay for the detection of bio threat agents in selected food matrices and in the screening of Clostridium botulinum outbreak strains associated with type A botulism.

BACKGROUND: Specific screening methods for complex food matrices are needed that enable unambiguous and sensitive detection of bio threat agents (BTAs) such as Bacillus anthracis spores and microbial toxins (e.g. staphylococcal enterotoxin B (SEB) and clostridial botulinum neurotoxins (BoNTs)). The present study describes an image-based 96-well Meso Scale Discovery (MSD) electrochemiluminescence (ECL) assay for simultaneous detection of BTAs in dairy milk products. RESULTS: The limit of detection of this ECL assay is 40 pg mL⁻¹ for BoNT/A complex, 10 pg mL⁻¹ for SEB and 40000 CFU mL⁻¹ for Bacillus anthracis spores in dairy milk products. The ECL assay was successfully applied to screen type A Clostridium botulinum outbreak strains. CONCLUSION: The results of the study indicate that this ECL assay is very sensitive, rapid (<6 h) and multiplex in nature. The ECL assay has potential for use as an in vitro screening method for BTAs over other comparable immunoassays.

Reversal of the substrate specificity of CMP N-glycosidase to dCMP.

MilB is a CMP hydrolase involved in the early steps of biosynthesis of the antifungal compound mildiomycin. An enzyme from the bacimethrin biosynthetic pathway, BcmB, is closely related to MilB in both sequence and function. These two enzymes belong to the nucleoside 2'-deoxyribosyltransferase (NDT) superfamily. NDTs catalyze N-glycosidic bond cleavage of 2'-deoxynucleosides via a covalent 2-deoxyribosyl-enzyme intermediate. Conservation of key active site residues suggests that members of the NDT superfamily share a common mechanism; however, the enzymes differ in their substrate preferences. Substrates vary in the type of nucleobase, the presence or absence of a 2'-hydroxyl group, and the presence or absence of a 5'-phosphate group. We have determined the structures of MilB and BcmB and compared them to previously determined structures of NDT superfamily members. The comparisons reveal how these enzymes differentiate between ribosyl and deoxyribosyl nucleotides or nucleosides and among different nucleobases. The 1.6 Å structure of the MilB-CMP complex reveals an active site feature that is not obvious from comparisons of sequence alone. MilB and BcmB that prefer substrates containing 2'-ribosyl groups have a phenylalanine positioned in the active site, whereas NDT family members with a preference for 2'-deoxyribosyl groups have a tyrosine residue. Further studies show that the phenylalanine is critical for the specificity of MilB and BcmB toward CMP, and mutation of this phenylalanine residue to tyrosine results in a 1000-fold reversal of substrate specificity from CMP to dCMP.

Targeting botulinum neurotoxin persistence by the ubiquitin-proteasome system.

Botulinum neurotoxins (BoNTs) are the most potent natural toxins known. The effects of BoNT serotype A (BoNT/A) can last several months, whereas the effects of BoNT serotype E (BoNT/E), which shares the same synaptic target, synaptosomal-associated protein 25 (SNAP25), last only several weeks. The long-lasting effects or persistence of BoNT/A, although desirable for therapeutic applications, presents a challenge for medical treatment of BoNT intoxication. Although the mechanisms for BoNT toxicity are well known, little is known about the mechanisms that govern the persistence of the toxins. We show that the recombinant catalytic light chain (LC) of BoNT/E is ubiquitylated and rapidly degraded in cells. In contrast, BoNT/A LC is considerably more stable. Differential susceptibility of the catalytic LCs to ubiquitin-dependent proteolysis therefore might explain the differential persistence of BoNT serotypes. In this regard we show that TRAF2, a RING finger protein implicated in ubiquitylation, selectively associates with BoNT/E LC and promotes its proteasomal degradation. Given these data, we asked whether BoNT/A LC could be targeted for rapid proteasomal degradation by redirecting it to characterized ubiquitin ligase domains. We describe chimeric SNAP25-based ubiquitin ligases that target BoNT/A LC for degradation, reducing its duration in a cellular model for toxin persistence.

The effect of pH on growth of Clostridium botulinum type A and expression of bontA and botR during different growth stages.

In this study, the effects of pH on the growth, relative expressions of bontA and botR genes, and neurotoxin formation of foodborne pathogens Clostridium botulinum type A were systematically studied throughout its growth stage. As in the previous reports, no C. botulinum growth was observed at extremely acidic pH. However, the effect of alkaline pH on the growth and neurotoxin production of C. botulinum was first revealed in this study. The maximum growth rate at pH 9.0 was similar to that at other pH values, although the lag phase at pH 9.0 was 16 h longer than that at pH 8.0. The peak of bontA mRNA expression at pH 9.0 was only 15.5% compared with that at pH 7.0. However, the neurotoxin concentration quantified in the cultures did not differ significantly. BotR is a known regulatory protein of bontA. The quantitative relationship between bontA and botR at different growth stages was first determined in this study. The mRNA levels of bontA were found to be positively correlated with those of botR, and the ratio of the mRNA transcript varied with pH. All these findings provide important physiological information on C. botulinum and thereby contribute to the improvement of food safety.

Facile and rapid DNA extraction and purification from food matrices using IFAST (immiscible filtration assisted by surface tension).

Extraction and purification of DNA is a prerequisite to detection and analytical techniques. While DNA sample preparation methods have improved over the last few decades, current methods are still time consuming and labor intensive. Here we demonstrate a technology termed IFAST (Immiscible Filtration Assisted by Surface Tension), that relies on immiscible phase filtration to reduce the time and effort required to purify DNA. IFAST replaces the multiple wash and centrifugation steps required by traditional DNA sample preparation methods with a single step. To operate, DNA from lysed cells is bound to paramagnetic particles (PMPs) and drawn through an immiscible fluid phase barrier (i.e. oil) by an external handheld magnet. Purified DNA is then eluted from the PMPs. Here, detection of Clostridium botulinum type A (BoNT/A) in food matrices (milk, orange juice), a bioterrorism concern, was used as a model system to establish IFAST's utility in detection assays. Data validated that the DNA purified by IFAST was functional as a qPCR template to amplify the bont/A gene. The sensitivity limit of IFAST was comparable to the commercially available Invitrogen ChargeSwitch® method. Notably, pathogen detection via IFAST required only 8.5 µL of sample and was accomplished in five-fold less time. The simplicity, rapidity and portability of IFAST offer significant advantages when compared to existing DNA sample preparation methods.

Two cases of type A infant botulism in Grenoble, France: no honey for infants.

We report two severe cases of infant botulism diagnosed at Grenoble University Hospital, France, respectively in 2006 and 2009. Both cases were characterized by a delay in diagnosis, severe neurological manifestations and extended period of hospitalization in intensive care unit, but a complete recovery. Infant botulism is a rare but life-threatening disease. It primarily affects infants, and the main risk factor is honey ingestion. Diagnosis should be systematically evoked by pediatricians in infants suffering from constipation, fatigue, muscle weakness, difficult feeding and altered cry, but before the onset of generalized flaccid paralysis, so as to administer specific treatment (BabyBIG®, a human derived botulinum antitoxin) at an early stage of the disease when it is most effective. In conclusion, parents should be aware of the role of honey as a source of spores of Clostridium botulinum and therefore infant botulism in the first year of life.