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Purpose: The presence of cereulide, an emetic toxin produced by Bacillus cereus, in fried rice samples is critical evidence of food poisoning even in situations where B. cereus could not be detected. This study aims to develop a screening method for analyzing cereulide in fried rice using the QuEChERS procedure and liquid chromatography-tandem mass spectrometry (LC-MS/MS)., Methods: Cereulide was identified and quantified in fried rice samples using the QuEChERS extraction method and LC-MS/MS. The accuracies of the methods were determined by analyzing fortified blank samples at two concentrations (10 and 50 µg/kg) conducted on three samples daily for five days., Results: The QuEChERS procedure removed matrix compounds from fried rice. Characteristic MS/MS spectra enabled the identification of cereulide. As the matrix effects in seven fried rice samples were within ± 6%, an external solvent calibration curve could be used for quantification. This method exhibited good accuracy ranging from 88 to 89%. The relative standard deviations for both repeatability and intra-laboratory reproducibility were < 4%. These standard deviations satisfied the criteria of the Japanese validation guidelines for residues (MHLW 2010, Director Notice, Syoku-An No. 1224-1). The limit of quantification was 2 μg/kg. The applicability of this method was confirmed using the analysis of cereulide in fried rice samples incubated with emetic Bacillus cereus., Conclusions: The QuEChERS extraction procedure described herein showed substantial promise as a reliable screening tool for cereulide in fried rice sample., (© 2024. The Author(s).)

The rise in cyanobacterial blooms due to eutrophication and climate change has increased cyanotoxin presence in water. Most current water treatment plants do not effectively remove these toxins, posing a potential risk to public health. This study introduces a water treatment approach using nanostructured beads containing magnetic nanoparticles (MNPs) for easy removal from liquid suspension, coated with different adsorbent materials to eliminate cyanotoxins. Thirteen particle types were produced using activated carbon, CMK-3 mesoporous carbon, graphene, chitosan, 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO)-oxidised cellulose nanofibers (TOCNF), esterified pectin, and calcined lignin as an adsorbent component. The particles' effectiveness for detoxification of microcystin-LR (MC-LR), cylindrospermopsin (CYN), and anatoxin-A (ATX-A) was assessed in an aqueous solution. Two particle compositions presented the best adsorption characteristics for the most common cyanotoxins. In the conditions tested, mesoporous carbon nanostructured particles, P1-CMK3, provide good removal of MC-LR and Merck-activated carbon nanostructured particles, P9-MAC, can remove ATX-A and CYN with high and fair efficacy, respectively. Additionally, in vitro toxicity of water treated with each particle type was evaluated in cultured cell lines, revealing no alteration of viability in human renal, neuronal, hepatic, and intestinal cells. Although further research is needed to fully characterise this new water treatment approach, it appears to be a safe, practical, and effective method for eliminating cyanotoxins from water.

Intestinal stem cells (ISCs) sustain epithelial renewal by dynamically altering behaviors of proliferation and differentiation in response to various nutrition and stress inputs. However, how ISCs integrate bioactive substance morin cues to protect against heat-stable enterotoxin b (STb) produced by Escherichia coli remains an uncertain question with implications for treating bacterial diarrhea. Our recent work showed that oral mulberry leaf-derived morin improved the growth performance in STb-challenged mice. Furthermore, morin supplementation reinstated the impaired small-intestinal epithelial structure and barrier function by stimulating ISC proliferation and differentiation as well as supporting intestinal organoid expansion ex vivo. Importantly, the Wnt/β-catenin pathway, an ISC fate commitment signal, was reactivated by morin to restore the jejunal crypt-villus architecture in response to STb stimulation. Mechanically, the extracellular morin-initiated β-catenin axis is dependent or partially dependent on the Wnt membrane receptor Frizzled7 (FZD7). Our data reveal an unexpected role of leaf-derived morin, which represents molecular signaling targeting the FZD7 platform instrumental for controlling ISC regeneration upon STb injury.

The present study was aimed to detect C. perfringens and identify its toxins in mutton samples collected from Lahore City in the Punjab Province of Pakistan. A total of 40 samples of minced and non‑minced mutton were collected from local butcher and retail shops representing four areas of the city. The samples were subjected to ELISA for the detection of C. perfringens alpha, beta and epsilon toxins. The samples were simultaneously processed for bacterial isolation. The isolates were confirmed both by biochemical testing and a multiplex PCR targeting alpha, beta and epsilon toxin genes of C. perfringens. While 10% (4/40) of the samples were positive for C. perfringens alpha toxins, 17.5% (7/40) of the samples were positive for the alpha toxin gene. The present study indicated that the samples collected from the local butcher shops were contaminated with C. perfringens and its toxins. Interestingly, no such contamination was detected in any of the samples collected from retail meat shops. In conclusion, improper hygienic conditions at butcher shops could lead to the contamination of mutton with C. perfringens and its toxins.

Cyanobacteria are photosynthetic microorganisms that are able to produce a large number of secondary metabolites. In freshwaters, under favorable conditions, they can rapidly multiply, forming blooms, and can release their toxic/bioactive metabolites in water. Among them, anabaenopeptins (APs) are a less studied class of cyclic bioactive cyanopeptides. The occurrence and structural variety of APs in cyanobacterial blooms and cultured strains from Greek freshwaters were investigated. Cyanobacterial extracts were analyzed with LC-qTRAP MS/MS using information-dependent acquisition in enhanced ion product mode in order to obtain the fragmentation mass spectra of APs. Thirteen APs were detected, and their possible structures were annotated based on the elucidation of fragmentation spectra, including three novel ones. APs were present in the majority of bloom samples (91%) collected from nine Greek lakes during different time periods. A large variety of APs was observed, with up to eight congeners co-occurring in the same sample. AP F (87%), Oscillamide Y (87%) and AP B (65%) were the most frequently detected congeners. Thirty cyanobacterial strain cultures were also analyzed. APs were only detected in one strain ( Microcystis ichtyoblabe ). The results contribute to a better understanding of APs produced by freshwater cyanobacteria and expand the range of structurally characterized APs.

In June 2020, a large-scale food poisoning outbreak involving about 3000 elementary and junior high school students occurred in Yashio, Saitama, Japan. A school lunch was the only food stuff ingested by all of the patients. Escherichia coli serotype O7:H4 carrying the astA gene for enteroaggregative E. coli (EAggEC) heat-stable enterotoxin 1 (EAST1) was detected in faecal specimens from the patients, and sample inspection revealed its presence in a seaweed salad and red seaweed (Gigartina tenella) as one of the raw materials. Analysis of the antibiotic sensitivity of the isolates revealed resistance to ampicillin and cefotaxime. All isolates were confirmed to be of the same origin by pulsed-field gel electrophoresis after digestion with the restriction enzyme XbaI, and single nucleotide polymorphism analysis using whole genome sequencing. To our knowledge, this is the first report of a large-scale food poisoning caused by E. coli O7:H4, which lacks well-characterized virulence genes other than astA.

The aim of this study was to purify Clostridium perfringens type D epsilon toxin and produce and purify anti-epsilon chicken immunoglobulin Y (IgY). A single-step ion exchange chromatography resulted in a high-yield and high-purity toxin, while ion exchange chromatography followed by gel filtration resulted in the highest purity of the toxin, but at a lower yield. Purified and inactivated epsilon toxin were then administered in chickens via four inoculations and IgY was obtained at a high purity and yield, with an antibody titer of 50 IU/mL and high levels of avidity (73.2%). In summary, C. perfringens type D epsilon toxin and chicken anti-epsilon IgY were successfully produced and purified, and may be used for the diagnosis of enterotoxemia caused by the epsilon toxin, as well as in potency tests of existing and future vaccines against enterotoxemia., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The Gram-negative bacteria Photorhabdus lives in a symbiotic relationship with the insect-pathogenic Heterorhabditis nematodes and produces numerous hydrolytic enzymes, secondary metabolites and protein toxins. Seven Photorhabdus strains were previously isolated from the Heterorhabditis nematodes collected from different geographical regions of India. The strains IARI-SGMG3, IARI-SGHR2, IARI-SGHR4, IARI-SGMS1 and IARI-SGGJ2 were identified as P. akhurstii, whereas IARI-SGLDK1 and IARI-SGHP1 were identified as P. laumondii subsp. laumondii and P. laumondii subsp. clarkeii, respectively. A new and previously unreported 35 kDa molecular weight protein toxin 'Galtox' was identified from these Photorhabdus strains. The nucleotide sequences of the toxin gene from seven Photorhabdus strains were PCR amplified, sequenced, cloned into pET protein expression vector, and the protein toxin was expressed and purified. The Galtox sequence from various strains showed variations in sequence and toxicity against Galleria mellonella. The injection of purified Galtox protein into the 4th instar larvae showed median lethal dose (LD 50 ) values of 2.39-26.08 ng toxin/g G. mellonella bodyweight after 48 h. The protein injection killed the insects quickly and exhibited a median lethal time (LT 50 ) of 12-60 h when injected at the rate of 3.1-31.2 ng toxin/g G. mellonella bodyweight. Galtox protein sequence analysis indicated similarity to several bacterial toxin-related protein domains, such as 6rgnA domain of Bordetella membrane targeting toxin BteA, 6gy6 domain of Xenorhabdus α-Xenorhabdolysins, 4mu6A and 4xa9a domains similar to effector protein LegC3 from Legionella pneumophila and 1cv8.1 domain of staphylococcal cysteine proteinase staphopain B. The mode of action of Galtox needs to be understood to enable its use for the management of agricultural insect-pests., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Programmed cell death (PCD) is an essential process for the immune system's development and homeostasis, enabling the remotion of infected or unnecessary cells. There are several PCD's types, depending on the molecular mechanisms, such as non-inflammatory or pro-inflammatory. Hemocytes are the main component of cellular immunity in bivalve mollusks. Numerous infectious microorganisms produce toxins that impair hemocytes functions, but there is little knowledge on the role of PCD in these cells. This study aims to evaluate in vitro whether marine toxins induce a particular type of PCD in hemocytes of the bivalve mollusk Crassostrea gigas during 4 h at 25°C. Hemocytes were incubated with two types of marine toxins: non-proteinaceous toxins from microalgae (saxitoxin, STX; gonyautoxins 2 and 3, GTX2/3; okadaic acid/dynophysistoxin-1, OA/DTX-1; brevetoxins 2 and 3, PbTx-2,-3; brevetoxin 2, PbTx-2), and proteinaceous extracts from bacteria ( Vibrio parahaemolyticus , Vp; V. campbellii , Vc). Also, we used the apoptosis inducers, staurosporine (STP), and camptothecin (CPT). STP, CPT, STX, and GTX 2/3, provoked high hemocyte mortality characterized by apoptosis hallmarks such as phosphatidylserine translocation into the outer leaflet of the cell membrane, exacerbated chromatin condensation, DNA oligonucleosomal fragments, and variation in gene expression levels of apoptotic caspases 2, 3, 7, and 8. The mixture of PbTx-2,-3 also showed many apoptosis features; however, they did not show apoptotic DNA oligonucleosomal fragments. Likewise, PbTx-2, OA/DTX-1, and proteinaceous extracts from bacteria Vp, and Vc, induced a minor degree of cell death with high gene expression of the pro-inflammatory initiator caspase-1, which could indicate a process of pyroptosis-like PCD. Hemocytes could carry out both PCD types simultaneously. Therefore, marine toxins trigger PCD's signaling pathways in C. gigas hemocytes, depending on the toxin's nature, which appears to be highly conserved both structurally and functionally., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Estrada, Núñez-Vázquez, Palacios, Ascencio, Guzmán-Villanueva and Contreras.)

In the attempt to improve the purification yield of native toxin A (TcdA) and toxin B (TcdB) from Clostridioides difficile (C. difficile) , we systematically evaluated culture parameters for their influence on toxin production. In this study, we showed that culturing C. difficile in a tryptone-yeast extract medium buffered in PBS (pH 7.5) that contained 5 mM ZnCl 2 and 10 mM glucose supported the highest TcdB production, measured by the sandwich ELISA. These culture conditions were scalable into 5 L and 15 L dialysis tube cultures, and we were able to reach a TcdB concentration of 29.5 µg/mL of culture. Furthermore, we established a purification protocol for TcdA and TcdB using FPLC column chromatography, reaching purities of >99% for both toxins with a yield around 25% relative to the starting material. Finally, by screening the melting temperatures of TcdA and TcdB in various buffer conditions using differential scanning fluorimetry, we found optimal conditions for improving the protein stability during storage. The results of this study present a complete protocol for obtaining high amounts of highly purified native TcdA and TcdB from C. difficile.

Vacuolar myelinopathy is a fatal neurological disease that was initially discovered during a mysterious mass mortality of bald eagles in Arkansas in the United States. The cause of this wildlife disease has eluded scientists for decades while its occurrence has continued to spread throughout freshwater reservoirs in the southeastern United States. Recent studies have demonstrated that vacuolar myelinopathy is induced by consumption of the epiphytic cyanobacterial species Aetokthonos hydrillicola growing on aquatic vegetation, primarily the invasive Hydrilla verticillata Here, we describe the identification, biosynthetic gene cluster, and biological activity of aetokthonotoxin, a pentabrominated biindole alkaloid that is produced by the cyanobacterium A. hydrillicola We identify this cyanobacterial neurotoxin as the causal agent of vacuolar myelinopathy and discuss environmental factors-especially bromide availability-that promote toxin production., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

We investigated the efficiency of the Verigene Enteric Pathogens Nucleic Acid Test (Verigene EP test), which is an automated microarray-based assay system that enables rapid and simultaneous genetic detection of gastrointestinal pathogens and toxins, including those in the Campylobacter Group, Salmonella species, Shigella species, the Vibrio Group, Yersinia enterocolitica, Shiga toxin 1 and 2, norovirus GI/GII, and rotavirus A. Three clinical laboratories evaluated the Verigene EP test, using 268 stool samples for bacterial and toxin genes and 167 samples for viral genes. Culture-based reference methods were used for the detection of bacteria and toxins, while a different molecular assay was used for viral detection. The overall concordance rate between the Verigene EP test and the reference methods for the 1940 assays was 99.0%. The overall sensitivity and specificity of the Verigene EP test were 97.0% and 99.3%, respectively. Of the 19 samples with discordant results, 13 samples were false positives and six were false negatives. The Verigene EP test simultaneously detected two targets in 11 samples; overall, the test demonstrated high efficiency in detecting crucial diarrheagenic pathogens, indicating its suitability for clinical practice.

Cytolethal distending toxins (CDTs) produced by P. alcalifaciens are considered as potential virulence factors. A loop-mediated isothermal amplification (LAMP) method for the detection of cdtA, cdtB, and cdtC genes was established which showed high specificity and strong sensitivity. The LAMP assay showed a detection threshold was 3.13 pg/μl within 40 min., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Bacterial type VI secretion systems (T6SSs) inject toxic effectors into adjacent eukaryotic and prokaryotic cells. It is generally thought that this process requires physical contact between the two cells. Here, we provide evidence of contact-independent killing by a T6SS-secreted effector. We show that the pathogen Yersinia pseudotuberculosis uses a T6SS (T6SS-3) to secrete a nuclease effector that kills other bacteria in vitro and facilitates gut colonization in mice. The effector (Tce1) is a small protein that acts as a Ca 2+ - and Mg 2+ -dependent DNase, and its toxicity is inhibited by a cognate immunity protein, Tci1. As expected, T6SS-3 mediates canonical, contact-dependent killing by directly injecting Tce1 into adjacent cells. In addition, T6SS-3 also mediates killing of neighboring cells in the absence of cell-to-cell contact, by secreting Tce1 into the extracellular milieu. Efficient contact-independent entry of Tce1 into target cells requires proteins OmpF and BtuB in the outer membrane of target cells. The discovery of a contact-independent, long-range T6SS toxin delivery provides a new perspective for understanding the physiological roles of T6SS in competition. However, the mechanisms mediating contact-independent uptake of Tce1 by target cells remain unclear.

Mono-ADP-ribosyltransferase (mART) toxins are secreted by several pathogenic bacteria that disrupt vital host cell processes in deadly diseases like cholera and whooping cough. In the last two decades, the discovery of mART toxins has helped uncover the mechanisms of disease employed by pathogens impacting agriculture, aquaculture, and human health. Due to the current abundance of mARTs in bacterial genomes, and an unprecedented availability of genomic sequence data, mART toxins are amenable to discovery using an in silico strategy involving a series of sequence pattern filters and structural predictions. In this work, a bioinformatics approach was used to discover six bacterial mART sequences, one of which was a functional mART toxin encoded by the plant pathogen, Erwinia amylovora , called Vorin. Using a yeast growth-deficiency assay, we show that wild-type Vorin inhibited yeast cell growth, while catalytic variants reversed the growth-defective phenotype. Quantitative mass spectrometry analysis revealed that Vorin may cause eukaryotic host cell death by suppressing the initiation of autophagic processes. The genomic neighbourhood of Vorin indicated that it is a Type-VI-secreted effector, and co-expression experiments showed that Vorin is neutralized by binding of a cognate immunity protein, VorinI. We demonstrate that Vorin may also act as an antibacterial effector, since bacterial expression of Vorin was not achieved in the absence of VorinI. Vorin is the newest member of the mART family; further characterization of the Vorin/VorinI complex may help refine inhibitor design for mART toxins from other deadly pathogens.

Necrotic enteritis (NE) is a devastating enteric disease caused by Clostridium perfringens type A/G, which affects global poultry industry by compromising the performance, health, and welfare of chickens. The causative main virulent factor responsible for NE pathogenesis has been shifted from a phospholipase C portion of an α-toxin, to an NE B-like (NetB) toxin, a plasmid-encoded pore-forming heptameric protein, in NE development. Therefore, the ability to detect NetB toxin will enable early diagnosis of field NE. Because the NetB protein can only be detected by western blot analysis with polyclonal anti-NetB antiserum, we developed a NetB-specific monoclonal antibody (mAb)-based capture enzyme-linked immunosorbent assay (ELISA). Twenty mAbs reacting with Escherichia coli-expressed NetB protein were selected, isotyped, and conjugated with horseradish peroxidase for antibody pair tests. Multiple mAb pairs were found to detect E. coli NetB protein and native NetB protein secreted by netB-positive C. perfringens isolates. The developed capture (sandwich) ELISA could be useful to identify in vitro production of native NetB protein secreted from netB-positive field C. perfringens isolates and to conduct a large field test of commercial chickens undergoing NE infection. Here, we first report that native NetB toxin can be detected in C. perfringens NetB-specific mAb-based capture ELISA.

Tripartite α-pore-forming toxins are constructed of three proteins (A, B and C) and are found in many bacterial pathogens. While structures of the B and C components from Gram-negative bacteria have been described, the structure of the A component of a Gram-negative α-pore-forming toxin has so far proved elusive. SmhA, the A component from the opportunistic human pathogen Serratia marcescens, has been cloned, overexpressed and purified. Crystals were grown of selenomethionine-derivatized protein and anomalous data were collected. Phases were calculated and an initial electron-density map was produced., (open access.)

Vacuolating cytotoxin A (VacA) is a highly polymorphic virulence protein produced by the human gastric pathogen Helicobacter pylori which can cause gastritis, peptic ulcer and gastric cancer. Here, we present an optimized protein preparation of the mature full-length VacA variants (m1-and m2-types) and their 33-kDa N-terminal and 55/59-kDa C-terminal domains as biologically active recombinant proteins fused with an N-terminal His (6) tag. All recombinant VacA constructs were over-expressed in Escherichia coli as insoluble inclusions which were soluble when phosphate buffer (pH 7.4) was supplemented with 5-6 M urea. Upon immobilized-Ni 2+ affinity purification under 5-M urea denaturing conditions, homogenous products (>95% purity) of 55/59-kDa domains were consistently obtained while only ~80% purity of both mature VacA variants and the 33-kDa truncate was achieved, thus requiring additional purification by size-exclusion chromatography. After successive refolding via optimized stepwise dialysis, all refolded VacA proteins were proven to possess both cytotoxic and vacuolating activity against cultured human gastric epithelial cells albeit the activity observed for VacA-m2 was lower than the m1-type variant. Such an optimized protocol described herein was effective for production of high-purity recombinant VacA proteins in large amounts (~30-40 mg per liter culture) that would pave the way for further studies on sequence-structure and function relationships of different VacA variants., (Copyright © 2020 Elsevier Inc. All rights reserved.)

An acute hepatopancreatic necrosis disease (AHPND) causes serious losses to the global shrimp industry. The etiologic agent of AHPND is Vibrio spp. carrying a large plasmid which encodes a binary toxin, PirAB. Currently, AHPND is diagnosed by PCR based methods that detect the presences of both pirA and pirB genes. However, the bacterial strains containing the pirA and pirB genes do not always express the binary toxin, resulting in mis-estimation of the virulence of bacterial strains containing pirA and pirB genes. Thus, the immuno based assay (i.e. ELISA) is a promising approach to detect PirA Vp and PirB Vp . In the present study, a total of forty monoclonal antibodies clones (mAb) against PirA Vp (20 mAbs) and PirB Vp (20 mAbs) were screened by western blot analysis to select four mAb clones that show the strongest immunoreactivity in indirect ELISA (iELISA). The four selected mAbs (i.e. 1B9 and 5E9 against PirA Vp ; 7B7 and 7B9 against PirB Vp ) detected specifically Vibrio spp. causing AHPND. In addition, four selected mAbs were able to detect either PirA Vp or PirB Vp down to 0.008 ng/μl. A double blind assay using thirty AHPND-infected and six SPF shrimp Penaeus vannamei were analyzed by iELISA to determine the detection sensitivity of the assay. The results showed that iELISA was able to accurately detect 29 out of 30 AHPND infected shrimp. These finding indicated that iELISA is a reliable method to detect PirA Vp and PirB Vp toxins in infected shrimp and will be a useful tool in AHPND diagnosis and in studying the role of binary toxins in AHPND pathogenesis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Enterotoxigenic Bacteroides fragilis is a causative agent of colitis and secrets enterotoxin (BFT), leading to the disease. Sulfiredoxin (Srx)-1 serves to protect from oxidative damages. Although BFT can generate reactive oxygen species in intestinal epithelial cells (IECs), no Srx-1 expression has been reported in ETBF infection. In this study, we explored the effects of ETBF-produced BFT on Srx-1 induction in IECs. Treatment of IECs with BFT resulted in increased expression of Srx-1 in a time-dependent manner. BFT treatment also activated transcriptional signals including Nrf2, AP-1 and NF-κB, and the Srx-1 induction was dependent on the activation of Nrf2 signals. Nrf2 activation was assessed using immunoblot and Nrf2-DNA binding activity and the specificity was confirmed by supershift and competition assays. Suppression of NF-κB or AP-1 signals did not affect the upregulation of Srx-1 expression. Nrf2-dependent Srx-1 expression was associated with the activation of p38 mitogen-activated protein kinases (MAPKs) in IECs. Furthermore, suppression of Srx-1 significantly enhanced apoptosis while overexpression of Srx-1 significantly attenuated apoptosis during exposure to BFT. These results imply that a signaling cascade involving p38 and Nrf2 is essential for Srx-1 upregulation in IECs stimulated with BFT. Following this upregulation, Srx-1 may control the apoptosis in BFT-exposed IECs.

Toxin-antitoxin (TA) systems are ubiquitously found in bacteria and are related to cell maintenance and survival under environmental stresses such as heat shock, nutrient starvation, and antibiotic treatment. Here, we report for the first time the crystal structure of the Staphylococcus aureus TA complex YoeB Sa1 -YefM Sa1 at a resolution of 1.7 Å. This structure reveals a heterotetramer with a 2:2 stoichiometry between YoeB Sa1 and YefM Sa1 . The N-terminal regions of the YefM Sa1 antitoxin form a homodimer characteristic of a hydrophobic core, and the C-terminal extended region of each YefM Sa1 protomer makes contact with each YoeB Sa 1 monomer. The binding stoichiometry of YoeB Sa1 and YefM Sa1 is different from that of YoeB and YefM of E. coli (YoeB Ec and YefM Ec ), which is the only structural homologue among YoeB-YefM families; however, the structures of individual YoeB Sa1 and YefM Sa1 subunits in the complex are highly similar to the corresponding structures in E. coli. In addition, docking simulation with a minimal RNA substrate provides structural insight into the guanosine specificity of YoeB Sa1 for cleavage in the active site, which is distinct from the specificity of YoeB Ec for adenosine rather than guanosine. Given the previous finding that YoeB Sa1 exhibits fatal toxicity without inducing persister cells, the structure of the YoeB Sa1 -YefM Sa1 complex will contribute to the design of a new category of anti-staphylococcal agents that disrupt the YoeB Sa1 -YefM Sa1 complex and increase YoeB Sa1 toxicity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Photorhabdus luminescens is an entomopathogenic rod-shaped bacterium infected with insect nematodes of the Heterorhabditidae family. It kills insects through the secretion of high molecular weight toxin complexes. In this study, Plutella xylostella larvae were orally administered P. luminescens for bioassay. After incubation in Luria-Bertani (LB) medium for a sufficiently long period, the mortality rates of P. xylostella observed after diluting the fermentation broth 50 times and diluting the supernatant 5 times were 18.89% and 91.11%, respectively. Retentates measuring more than 70 kDa showed 88% mortality after ultrafiltration (UF) membrane treatment. Thus, the supernatant of P. luminescens had insecticidal activity, and the main insecticidal toxin complexes had a molecular weight exceeding 70 kDa. The L 9 (3 4 ) Taguchi orthogonal experimental optimized medium mode-predicted insecticidal activity levels were 84% and 119% in the 50-fold diluted fermentation broth and 5-fold diluted supernatant, respectively. Moreover, the insecticidal activity was improved to 92.2% in the 100-fold diluted fermentation broth and to 97.8% in the 10-fold diluted supernatant in the experiments. All combinations tested showed clear indications of lethality, including swelling, vesicle formation, cytoplasm vacuolization, and brush border membrane lysis. Thus, these results promote the use of P. luminescens 0805-P2R as a potent biopesticide to effectively control P. xylostella.

Background: The cobas® omni Utility Channel enables users to integrate lab-developed tests (LDTs) on the cobas® 6800 System to perform molecular diagnostics with high-throughput capacity and full automation. At present, there are no CE- or FDA-approved tests for stool pathogens on this system. To assess the performance of stool as a matrix, we evaluated the analytical and clinical performance of an LDT for detection of Clostridioides difficile (C. difficile) toxin B using the Utility Channel (C.diff&#95;UTC)., Methods: A 10% stool suspension prepared from liquid stool samples diluted in phosphate buffered saline was used for analysis. Limit of detection (LoD) was determined in six dilutions with 126 replicates/dilution. Clinical evaluation was performed using 514 predetermined patient stool samples from two study sites in Germany. The C.diff&#95;UTC was compared with LC 480 amplification and an LDT or the R-BioPharm C. difficile assay. Discrepant results were further analyzed using the GeneXpert C. difficile assay., Results: Limit of detection was 23.48 cfu/mL (95% Confidence Interval [CI]: 19.14-31.01) with inter-run variation of <2 cycle thresholds at 3 × and 10 × LoD. No cross-reactivity was observed with a panel of fecal organisms and pathogens. Bioinformatic analysis showed coverage of the major C. difficile toxinotypes by the primer/probe set. Clinical evaluation revealed sensitivity of 96.7% (95% CI: 88.7-99.6) and specificity of 99.3% (95% CI: 98.0-99.9) compared with the reference method; inhibition rate was 3.5% (18/514)., Conclusion: Using a predesigned primer/probe set, the C.diff&#95;UTC assay features analytical performance and clinical sensitivity and specificity comparable to currently available nucleic acid amplification tests (NAATs) and is suitable for high-throughput testing. This was a proof-of-concept study, indicating the cobas Utility Channel could likely be adapted for other clinically relevant stool pathogens in outbreak scenarios., Competing Interests: Declaration of Competing Interest UE and ML received speaker honoraria and related travel expenses from Roche Diagnostics. JF and KC are employees of Roche Molecular Systems. All other authors had no conflicts of interest., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Pseudomonas entomophila is a highly pathogenic bacterium that infects insects. It is also used as a suitable model pathogen to analyze Drosophila's innate immunity. P. entomophila's virulence is largely derived from Monalysin, a β-barrel pore-forming toxin that damages Drosophila tissues, inducing necrotic cell death. Here we report the first and efficient purification of endogenous Monalysin and its characterization. Monalysin is successfully purified as a pro-form, and trypsin treatment results in a cleaved mature form of purified Monalysin which kills Drosophila cell lines and adult flies. Electrophysiological measurement of Monalysin in a lipid membrane with an on-chip device confirms that Monalysin forms a pore, in a cleavage-dependent manner. This analysis also provides a pore-size estimate of Monalysin using current amplitude for a single pore and suggests lipid preferences for the insertion. Atomic Force Microscope (AFM) analysis displays its structure in a solution and shows that active-Monalysin is stable and composed of an 8-mer complex; this observation is consistent with mass spectrometry data. AFM analysis also shows the 8-mer structure of active-Monalysin in a lipid bilayer, and real-time imaging demonstrates the moment at which Monalysin is inserted into the lipid membrane. These results collectively suggest that endogenous Monalysin is indeed a pore-forming toxin composed of a rigid structure before pore formation in the lipid membrane. The endogenous Monalysin characterized in this study could be a desirable tool for analyzing host defense mechanisms against entomopathogenic bacteria producing damage-inducing toxins., (Copyright © 2020 Nonaka, Salim, Kamiya, Hori, Nainu, Asri, Masyita, Nishiuchi, Takeuchi, Kodera and Kuraishi.)

Various bacteria from the Bacillus species have been used as pesticides against mosquito larvae for more than a decade. The prolonged use of these bacterial species by little alteration within their genome, using various permutations and combinations of mosquito-cidal toxins, has proven unsuccessful in controlling the mosquito population. In our current study we report Enterococcus sp. to be exhibiting similar kind of mosquito-cidal toxins alike those which are present in the mainly used Bacillus strains. Three Enterococcus species were isolated on a rich media selective for gram- positive bacteria from the mid-gut of dead mosquito larvae which were collected from the wild locations within and around the city of Mumbai, India. Their surface morphologies were studied by Scanning Electron Microscopy (SEM) and their identity was confirmed using the standard 16S rRNA sequencing method. Upon performing several repetitive toxicity assays of these three strains on the laboratory cultured third instar stage of Culex quinquefasciatus larvae, showed differential toxicities from a minimum of 20% (LC 50 : 59.6 CFU/ml), intermediate 35% (LC 50 : 48.4 CFU/ml) and a maximum of 60% (LC 50 : 35.7 CFU/ml). To justify the data in all the three similar strains of Enterococcus durans, we followed the differential proteomics using LCMS 6540 UHD Accurate Mass QTOF and differential metabolomics approach using both LCMS 6540 UHD Accurate Mass QTOF and 1 H-NMR. The presence and significance of the obtained toxins were studied to elucidate the plausible reason for showing differential toxicities. This work helped in identifying Enterococcus durans as a new, potential and alternative strain to the Bacillus species in terms of mosquito larvicidal toxicity against Culex quinquefasciatus.

Clostridium perfringens type G is one of the pathogens involved in enteric diseases in poultry. NetB, a pore-forming toxin, is considered the main virulence factor responsible for necrotic enteritis during C. perfringens infection. We carried out a field study involving 14 farms to evaluate the occurrence of netB- positive C. perfringens and the impact of infection in Italian poultry flocks. Environmental samples ( n = 117) and 50 carcasses were screened by microbiologic and molecular methods. Microbiologic investigations yielded 82 C. perfringens isolates. DNA was extracted from all samples and screened for α-toxin and NetB encoding genes by real-time PCR. The C. perfringens α-toxin gene was detected in 151 of 167 extracts (90.4%), and 31 of 151 (20.5%) were netB gene positive also. Sixteen isolates from a turkey flock with mild enteric disorders were also netB positive, demonstrating their occurrence not only in broiler but also in turkey flocks. A pulsed-field gel electrophoresis protocol was optimized to evaluate the diversity among isolates and revealed high genetic heterogeneity. The complete NetB toxin-coding gene of 2 C. perfringens isolates from turkey and broiler flocks were analyzed and showed very high relatedness with analogous sequences worldwide.

Storage procedures are known to affect the detectability of Clostridium difficile toxins in equine and human feces. We assessed the impact of different storage conditions on the detectability of C. difficile toxins in swine feces. Specimens were inoculated with toxins, 112 ng/g of toxin A (TcdA) and 16 ng/g of toxin B (TcdB) and subjected to the following 3 storage treatments: 4°C, -30°C, repetitive freezing at -30°C and thawing. Toxin determination was assessed at 1, 2, 7, 14, and 21 d with ELISA. A decrease in concentrations of TcdA with time was observed for samples stored at 4°C and repetitive freezing-thawing ( p ≤0.05). On day 14, storage at 4°C resulted in decreased TcdA concentration as opposed to storage at -30°C and repetitive freezing-thawing ( p ≤0.05). On day 21, storage at 4°C resulted in decreased TcdA detectability compared with storage at -30°C ( p ≤0.05). The TcdB concentration was unaffected. These results on toxin detectability in swine feces should be carefully considered in in vitro studies on toxigenic C. difficile. Our results also offer valuable information for microbiologists and veterinarians monitoring the presence of virulent C. difficile in pigs.

The Clostridium perfringens alpha toxin (CPA), encoded by the plc gene, is the causative pathogen of gas gangrene, which is a lethal infection. In this study, we used an E. coli system for the efficient production of recombinant proteins and developed a bicistronic design (BCD) expression construct consisting of two copies of the C-terminal (247-370) domain of the alpha toxin (CPA-C) in the first cistron, followed by Cholera Toxin B (CTB) linked with another two copies of CPA-C in the second cistron that is controlled by a single promoter. Rabbits were immunized twice with purified proteins (rCPA-C rCTB-CPA-C) produced in the BCD expression system, with an inactivated recombinant E. coli vaccine (RE), C. perfringens formaldehyde-inactivated alpha toxoid (FA-CPA) and C. perfringensl-lysine/formaldehyde alpha toxoid (LF-CPA) vaccines. Following the second vaccination, 0.1 mL of pooled sera of the RE-vaccinated rabbits could neutralize 12× mouse LD 100 (100% lethal dose) of CPA, while that of the rCPA-C rCTB-CPA-C-vaccinated rabbits could neutralize 6× mouse LD 100 of CPA. Antibody titers against CPA were also assessed by ELISA, reaching titers as high as 1:2048000 in the RE group; this was significantly higher compared to the C. perfringens alpha toxoid vaccinated groups (FA-CPA and LF-CPA). Rabbits from all vaccinated groups were completely protected from a 2× rabbit LD 100 of CPA challenge. These results demonstrate that the recombinant proteins are able to induce a strong immune responses, indicating that they may be potentially utilized as targets for novel vaccines specifically against the C. perfringens alpha toxin., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Harmful algal blooms formed by colony-forming cyanobacteria deteriorate water resources by producing cyanotoxins, which frequently occur at high intracellular concentrations. We aimed to localize toxic microcystins (MCs) and bioactive anabaenopeptins (APs) at the subcellular level under noninvasive conditions. Since both metabolites are synthesized nonribosomally, the relaxed specificity of key enzymes catalyzing substrate activation allowed chemical labeling through a standard copper-catalyzed click chemistry reaction. The genera Planktothrix and Microcystis specifically incorporated unnatural amino acids such as N-propargyloxy-carbonyl-L-lysine or O-propargyl-L-tyrosine, resulting in modified AP or MC peptides carrying the incorporated alkyne moiety. The labeled cells were quantitatively differentiated from the unlabeled control cells. MCs and APs occurred intracellularly as distinct entities showing a cell-wide distribution but a lowered spatial overlap with natural autofluorescence. Using the immunofluorescence technique, colocalization with markers of individual organelles was utilized to relate the distribution of labeled MCs to cellular compartments, e.g., using RbcL and FtsZ (cytosol) and PsbA (thylakoids). The colocalization correlation coefficients calculated pairwise between organelles and autofluorescence were highly positive as opposed to the relatively low positive indices derived from labeled MCs. The lower correlation coefficients imply that only a portion of the labeled MC molecules were related spatially to the organelles in the cell.

Here, two monoclonal antibodies (MAbs) specific to different epitopes on ToxB, a toxin produced by Vibrio parahaemolyticus that causes acute hepatopancreatic necrosis disease (VP AHPND ), were employed to develop a rapid strip test. One MAb was conjugated to colloidal gold to bind to ToxB at the application pad, and another MAb was used to capture colloidal gold MAb-protein complexes at the test line (T) on the nitrocellulose strip. To validate test performance, a downstream control line (C) of goat anti-mouse immunoglobulin G antibody was used to capture the free colloidal gold conjugate MAb. The sample in the application buffer could be applied directly to the application well, and the test result was obtained within 15 min. The sensitivity of the kit is approximately 6.25 µg/ml of toxin, which was equivalent to the toxin produced by approximately 10 7  cfu/ml of bacteria. This kit is convenient and easy to use since it can be used to identify VP AHPND directly using a single colony of bacteria grown on agar culture plates. Because of its high specificity and simplicity, as well as not being reliant on sophisticated equipment or specialized skills, this strip test could be used by farmers for surveillance for ToxB-producing bacteria., (© 2019 John Wiley & Sons Ltd.)

Myxobacteria are an example of how single-cell individuals can transition into multicellular life by an aggregation strategy. For these and all organisms that consist of social groups of cells, discrimination against, and exclusion of, nonself is critical. In myxobacteria, TraA is a polymorphic cell surface receptor that identifies kin by homotypic binding, and in so doing exchanges outer membrane (OM) proteins and lipids between cells with compatible receptors. However, TraA variability alone is not sufficient to discriminate against all cells, as traA allele diversity is not necessarily high among local strains. To increase discrimination ability, myxobacteria include polymorphic OM lipoprotein toxins called SitA in their delivered cargo, which poison recipient cells that lack the cognate, allele-specific SitI immunity protein. We previously characterized 3 SitAI toxin/immunity pairs that belong to 2 families. Here, we discover 4 additional SitA families. Each family is unique in sequence, but share the characteristic features of SitA: OM-associated toxins delivered by TraA. We demonstrate that, within a SitA family, C-terminal nuclease domains are polymorphic and often modular. Remarkably, sitA loci are strikingly numerous and diverse, with most genomes possessing >30 and up to 83 distinct sitAI loci. Interestingly, all SitA protein families are serially transferred between cells, allowing a SitA inhibitor cell to poison multiple targets, including cells that never made direct contact. The expansive suites of sitAI loci thus serve as identify barcodes to exquisitely discriminate against nonself to ensure populations are genetically homogenous to conduct cooperative behaviors., Competing Interests: The authors declare no competing interest.

Intranasal administration delivers molecules directly to the brain bypassing the blood-brain barrier. Three distinct routes of access have been identified; olfactory, trigeminal and via the paranasal sub-mucosa of the posterior sinuses. Consequently, environmental toxins may access the CNS directly to induce inflammatory and degenerative disease. They may also activate bacterial species of the nasal mucosal microbiome to release both immune-deviating cell wall antigens and transportable neurotoxins with local direct access to the CNS. Evidence is reviewed that toxins of the nasal bacterial microbiota may be directly implicated in the inflammatory and degenerative pathogenesis of multiple sclerosis., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

Background: Clinical practice guidelines define Clostridium difficile infections (CDI) as diarrhea (≥3 unformed stools in 24 h) with either a positive C difficile stool test or detection of pseudomembranous colitis. Diagnostic modalities such as toxigenic culture and nucleic acid amplification testing can identify the presence of toxigenic C difficile in stools. But these tests are confounded by the presence of asymptomatic colonization of toxigenic C difficile and lead to overdiagnosis of CDI. The presence of two large toxins, toxin A and B (TcdA and TcdB) is necessary for pathogenicity. Detection of toxins using toxin enzyme immunoassay is difficult as it has low sensitivity and moderate specificity. Raman spectroscopy (RS) is a novel technology that is used to detect bacteria and their toxins. RS does not require any reagents for detection such as antibodies, enzymes, primers, or stains. We hypothesize that RS is a sensitive method to detect C difficile toxins in stool and will solve the problem of overdiagnosis of CDI., Materials and Methods: CDI negative stool samples were spiked with concentrations (1 ng/mL, 100 pg/mL, 1 pg/mL, and 0.1 pg/mL) of TcdA and TcdB. RS was performed on air-dried smeared samples of stool supernatant on a mirror-polished stainless-steel slide. As RS of feces is difficult because of confounding background material and autofluorescence, samples were photo-bleached before spectral acquisition to reduce autofluorescence. Raman spectra were obtained, background corrected, and vector normalized. The data were split into training (70%) and test (30%) datasets. The machine learning methods used on the training data set were Support Vector Machine with Linear and Radial Kernels, Random Forest, Stochastic Gradient Boosting Machine, and Principle Component Analysis-Linear Discriminant Analysis. Results were validated using a test data set. The best model was chosen, and its accuracy, sensitivity, and specificity were determined., Results: In our preliminary results, at all concentrations (1 ng/mL, 100 pg/mL, 1 pg/mL, and 0.1 pg/mL), TcdA or TcdB spiked stool was distinguished from unspiked stool by all models with accuracies ranging from 64% to 77%. Gradient Boosting Machine, Principle Component Analysis-Linear Discriminant Analysis, and Support Vector Machine Linear Kernel performed best with sensitivities ranging from 69% to 90% and specificities ranging from 43% to 78%., Conclusions: Using RS, we successfully detected TcdA and TcdB in stool samples albeit with moderate-to-high sensitivity and low-to-moderate specificity. Sensitivity and specificity could be further increased with the implementation of deep learning methods, which require large sample sizes. In terms of sensitivity, RS performs better than toxin enzyme immunoassay and has the potential to rapidly detect C difficile toxins in stool at clinically relevant concentrations and thereby help mitigate overdiagnosis of CDI., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Objectives: Clostridioides (previously Clostridium) difficile is a major growing cause of nosocomial diarrhoea known as C. difficile infection (CDI). This study investigated the prevalence and antimicrobial resistance patterns of C. difficile isolated from patients suffering from diarrhoea in Iran between 2016-2018., Methods: A total of 151 stool specimens were collected and were screened for the presence of C. difficile. Specimens were examined for toxins by culture, enzyme immunoassay (EIA) and PCR. Antimicrobial susceptibility testing was performed for 12 antibiotics (metronidazole, vancomycin, clindamycin, tetracycline, erythromycin, ciprofloxacin, levofloxacin, moxifloxacin, fusidic acid, piperacillin, piperacillin/tazobactam and rifampicin) by the disk diffusion method according to the guidelines of the CLSI, EUCAST and CA-SFM., Results: Of 151 stool specimens, 66 (43.7%) were positive for C. difficile by PCR, whereas 2 (1.3%) were only positive for C. difficile toxins based on EIA. A total of 292 clostridial isolates were obtained from specimens by culture, of which 133 (45.5%) were finally confirmed as C. difficile by PCR. Of 121 isolates resistant to at least one antibiotic, 107 (88.4%) were resistant to three or more antimicrobials and thus were defined as multidrug-resistant (MDR). Different and diverse resistance patterns to the antimicrobial drugs were seen among the isolates., Conclusion: This is the first report of the isolation of C. difficile from different governmental hospitals of Iran and indicates that CDI might be an important nosocomial infection in different hospital wards. Moreover, this study provides a comprehensive picture of the MDR phenotype characteristics of C. difficile isolates in Iran., (Copyright © 2019 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.)

Aims: To determine if presence of the Bacteroides fragilis toxin ( bft ) gene, a molecular marker of colonic carriage of entertoxigenic Bacteroides fragilis (ETBF) in humans, was associated with a finding of small intestinal adenocarcinomas (SIA) in sheep in New Zealand. Methods: Samples of jejunal tissue were collected from the site of tumours and from grossly normal adjacent tissue in 20 sheep, in different consignments, diagnosed with SIA based on gross examination of viscera following slaughter. Two jejunal samples were also collected from a control sheep in the same consignment that had no gross evidence of SIA. A PCR assay was used to detect the presence of the bft gene in the samples. Results: Of the sheep with SIA, the bft gene was amplified from one or both samples from 7/20 (35%) sheep, and in sheep that had no gross evidence of SIA the bft gene was amplified from at least one sample in 11/20 (55%) sheep (RR 0.61; 95% CI = 0.30-1.25; p = 0.34). Of 11 positive samples analysed, ETBF subtype bft -1 was detected in one, bft -2 was detected in 10, and none were bft -3. Conclusions and Clinical Relevance: There was a high prevalence of detection of the bft gene in both SIA-affected and non-affected sheep, but there was no apparent association between carriage of ETBF, evidenced by detection of the bft gene, and the presence of SIA. ETBF are increasingly implicated in the aetiology of human colorectal cancer, raising the possibility that sheep may provide a zoonotic reservoir of this potentially carcinogenic bacterium. Abbreviation: Bft : Bacteroides fragilis toxin; ETBF: Enterotoxigenic Bacteroides fragilis ; SIA: Small intestinal adenocarcinoma.

Existing research for using the protective antigen (PA) of Bacillus anthracis as a vaccine component shows that protection against anthrax may be obtained using fragments of this protein. The aim of the research is to check whether the selected protein fragment of the protective antigen (domain 4) encoded by an appropriate nucleotide sequence of gene pag of B. anthracis , was expressed in the bacterial system of E. coli . In order to examine the selected sequence of the pag gene, a PCR reaction and a highly effective TOPO cloning strategy were used, followed by purification of the recombinant proteins and their detection by a western-blot method. In the planning of the PA4 antigen expression a higher level of effectiveness in production of small protein - domain 4 - was anticipated. As a result, the 139 amino acids protein fragment of B. anthracis PA (domain 4) was isolated. The research may have found the basis for in vivo research aimed at finding potential anthrax vaccine components.

Efforts to develop a vaccine for the elimination of malaria include the use of carrier proteins to assemble monomeric antigens into nanoparticles to maximize immunogenicity. Recombinant ExoProtein A (EPA) is a detoxified form of Pseudomonas aeruginosa Exotoxin A which has been used as a carrier in the conjugate vaccine field. A pilot-scale process developed for purification of EPA yielded product that consistently approached a preset upper limit for host cell protein (HCP) content per human dose. To minimize the risk of bulk material exceeding the specification, the purification process was redeveloped using mixed-mode chromatography resins. Purified EPA derived from the primary and redeveloped processes were comparable following full biochemical and biophysical characterization. However, using a process specific immunoassay, the HCP content was shown to decrease from a range of 0.14-0.24% w/w of total protein to below the level of detection with the revised process. The improved process reproducibly yields EPA with highly similar quality characteristics as the original process but with an improved profile for the HCP content., (Published by Elsevier Ltd.)

Recombinant ExoProtein A (EPA), a detoxified form of Pseudomonas aeruginosa Exotoxin A, is used as a protein carrier in the vaccine field. A scaled manufacturing process, in which EPA was expressed in Escherichia coli, yielded a product that approached or exceeded our upper limit of E. coli host cell protein (HCP) content per human dose. The purification process was redeveloped to reduce HCP levels in the bulk product and HCP content was evaluated by orthogonal methods. Using a platform specific immunoassay, the HCP level from the original purification method was 1,830 ppm (0.18% w/w) while the revised purification process yielded the HCP below the detection limits of the assay. With a 2D/LC-MS E methodology the reference sample from the original process was found to contain 57 unique HCPs at a total level of 37,811 ppm (3.78% w/w). Two lots were tested after purification with the revised process and contained 730 and 598 ppm (0.07% and 0.06% w/w), respectively. To develop a high-throughput MS method, the samples were tested on a 1D/LC-MS/MS. The data sets from the two mass spectrometers correlated well. These improved HCP profiles support implementing the revised purification process for manufacturing the EPA protein carrier and 1D/LC-MS/MS for HCP analysis., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Introduction: Severe intestinal infections caused by V. cholerae, ETEC and EHEC have contributed to the mortality rate in developing countries. Vibrio Cholera, ETEC and EHEC bacterium with the production of CT, LT and Stx2 toxins respectively lead to severe watery and bloody diarrhea. This study aimed to investigate a trimeric vaccine candidate containing recombinant chimeric protein, encapsulate the protein in chitosan nanoparticles and assess its immunogenicity., Methods: The LSC recombinant gene was used. It is composed of LTB (L), STXB (S) and CTXB (C) subunits respectively. The LSC recombinant protein was expressed and purified and confirmed by western blotting. The purified protein was encapsulated in chitosan nanoparticles, and its size was measured. BalB/c mice were immunized in four groups through oral and injection methods by LSC protein. The antibody titer was then evaluated by ELISA, and finally, the challenge test of the toxins from all three bacteria was done on the immunized mouse., Results: After expression and purification LSC protein size of nanoparticles containing protein was measured at 104.6 nm. Nanoparticles were able to induce systemic and mucosal immune responses by generating a useful titer of IgG and IgA. The challenge results with LT, CT and Stx toxins showed that the LSC protein might partially neutralize the effect of toxins., Conclusion: LSC chimeric protein with the simultaneous three essential antigens have a protective effect against the toxins produced by ETEC, EHEC and Vibrio cholera bacteria and it can be used in vaccines to prevent Diarrhea caused by these three bacteria., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

TosA, a putative repeats-in-toxin protein that has recently gained importance as an antigenic molecule, has characteristics of nonfimbrial adhesins and can act as a virulence marker in uropathogenic Escherichia coli (UPEC) strains; however, little is known about the association of this protein with antibiotic resistance profiles in UPEC tosA + clinical strains. The aim of this study was to evaluate UPEC tosA + strains, including examining genetic diversity, associations with phylogenetic groups, resistance profiles, virulence genes, adherence assays, integrons, and extended-spectrum beta-lactamase phenotypes. Pulsed-field gel electrophoresis analysis grouped these strains into eight clusters with 62% genetic diversity. These strains were mainly associated with the multidrug-resistant profiles, together with an association with class 1 integron and the extended-spectrum beta-lactamase phenotype. Additionally, the strains exhibited a distribution of ≥96% for core-associated genes, while a variable distribution was identified for pathogenic islands-associated genes. Strong associations between UPEC tosA + strains and two phylogenetic groups (B2 and D) were identified, including resistance to β-lactam and non-β-lactam antibiotics. The UPEC tosA + clinical strains exhibited major adherence, which was related to the fitness and virulence genes. A recombinant TosA protein reacted with antibodies from the sera of urinary tract infection patients, and anti-recombinant TosA polyclonal antibodies also detected TosA expression in these strains. In conclusion, strains of UPEC tosA + belonging to phylogenetic group B2 had a high frequency of fitness and virulence genes associated with class 1 integrons and the extended-spectrum beta-lactamase phenotype, which exhibited a high adherence profile. The TosA protein is expressed during infection with UPEC and is considered an immunogenic molecule., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Immunotoxins are a class of recombinant proteins which consist of an antibody and a part of a bacterial or herbal toxin. Immunotoxins containing Pseudomonas aeruginosa exotoxin A (PEA) have been found to be very applicable in clinical trials. Many obstacles such as solubility and absorbency reduce their usability in solid tumors. The current study aims to overcome the mentioned barriers by addition and removal of functional and non-functional domains with a structural approach. In the experimental section, we took advantage of molecular dynamics simulations to predict the functionality of candidate immunotoxins which target human HER2 receptors and confirmed our findings with in vitro experiments. We found out when no changes were made to domain II of PEA, addition of solubilizing domains to immunotoxins would not reduce their targeting and anti-tumor activity, while increasing the yield of expression and stability. On the other side, when we replaced domain II with eleven amino acids of furin cleavage site (FCS), the activity of the immunotoxin was mainly affected by the FCS neighboring domains and linkers. A combination of seven beneficial point mutations in domain III was also assessed and reconfirmed that the toxicity of the immunotoxin would be reduced dramatically. The obtained results indicate that the addition or removal of domains cannot depict the activity of immunotoxins and the matter should be assessed structurally in advance., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Cyanobacteria have been shown to produce a number of bioactive compounds, including toxins. Some bioactive compounds obtained from a marine cyanobacterium Moorea producens (formerly Lyngbya majuscula ) have been recognized as drug leads; one of these compounds is aplysiatoxin. We have isolated various aplysiatoxin derivatives from a M. producens sample obtained from the Okinawan coastal area. The frozen sample was extracted with organic solvents. The ethyl acetate layer was obtained from the crude extracts via liquid-liquid partitioning, then separated by HPLC using a reversed-phase column. Finally, 1.1 mg of the compound was isolated. The chemical structure of the isolated compound was elucidated with spectroscopic methods, using HR-MS and 1D and 2D NMR techniques, and was revealed to be oscillatoxin I, a new member of the aplysiatoxin family. Oscillatoxin I showed cytotoxicity against the L1210 mouse lymphoma cell line and diatom growth-inhibition activity against the marine diatom Nitzschia amabilis .

A rapid and sensitive liquid chromatography-mass spectrometry assay was developed and used to quantify emetic cereulide peptide exotoxin, which can be related to possible Bacillus cereus contamination in monoclonal antibody (mAb) bioprocess feeds. The assay limit of detection was 0.05 ng/mL (~1 fmol injected) and limit of quantification 0.16 ng/mL (~3 fmol injected) over a standard curve with >3 orders of magnitude linear dynamic range. The assay allowed quantification of toxin removal in an established two-step mAb purification process consisting of Protein A affinity chromatography followed by multi-modal anion exchange chromatography. Toxin content was ascertained in process stream sample fractions as well as on the Protein A affinity column. An optimized analytical method allowed separation of cereulide toxin from other mAb cell culture components within 6 min. Spiking experiments showed that samples should be collected in high (80% v/v) content acetonitrile to reduce nonspecific losses of the cereulide. The majority of mAb purification process-associated cereulide was detected in the Protein A flow through fraction, whereas only residual amounts were found in wash, strip, and elution fractions. Column cleaning-in-place (CIP) procedures were evaluated to prevent carryover between affinity capture cycles. No carryover was detected between cycles, however trace amounts of cereulide were extracted from the Protein A resin. Increasing the CIP NaOH concentration from 0.1 M to 0.5 M, and contact time from 15 min to 1 h, improved removal of residual cereulide from the resin. Applicability of CIP clearance of cereulide during Protein A chromatography was confirmed with three different mAb feeds. Post Protein A polishing, via target flow through on a multi-modal anion exchange chromatography column, resulted in a product pool with no detectable cereulide. Approximately 5 logs of reduction in cereulide concentration was obtained over the two-step chromatography process. Cereulide contamination is well known and of concern in food processing, however this research may be the first LC-MS quantification of cereulide contamination, and its clearance, in biopharmaceutical mAb processing. The analytical method may also be used to rapidly screen for cereulide contamination in upstream cell culture process streams, prior to downstream product purification. This will allow appropriate measures to be taken to reduce toxin exposure to downstream bioprocess raw materials, consumables and equipment., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Epsilon toxin (Etx), a potent pore forming toxin (PFT) produced by Clostridium perfringens, is responsible for the pathogenesis of enterotoxaemia of ruminants and has been suggested to play a role in multiple sclerosis in humans. Etx is a member of the aerolysin family of β-PFTs (aβ-PFTs). While the Etx soluble monomer structure was solved in 2004, Etx pore structure has remained elusive due to the difficulty of isolating the pore complex. Here we show the cryo-electron microscopy structure of Etx pore assembled on the membrane of susceptible cells. The pore structure explains important mutant phenotypes and suggests that the double β-barrel, a common feature of the aβ-PFTs, may be an important structural element in driving efficient pore formation. These insights provide the framework for the development of novel therapeutics to prevent human and animal infections, and are relevant for nano-biotechnology applications.

Next-generation DNA sequencing technology was applied to generate molecular data from semiarid reservoirs during well-defined seasons. Target sequences of 16S-23S rRNA ITS and cpcBA-IGS were used to reveal the taxonomic groups of cyanobacteria present in the samples, and genes coding for cyanotoxins such as microcystins (mcyE), saxitoxins (sxtA), and cylindrospermopsins (cyrJ) were investigated. The presence of saxitoxins in the environmental samples was evaluated using ELISA kit. Taxonomic analyses of high-throughput DNA sequencing data showed the dominance of the genus Microcystis in Mundaú reservoir. Furthermore, it was the most abundant genus in the dry season in Ingazeira reservoir. In the rainy season, 16S-23S rRNA ITS analysis revealed that Cylindrospermopsis raciborskii comprised 46.8% of the cyanobacterial community in Ingazeira reservoir, while the cpcBAIGS region revealed that C. raciborskii (31.8%) was the most abundant taxon followed by Sphaerospermopsis aphanizomenoides (17.3%) and Planktothrix zahidii (16.6%). Despite the presence of other potential toxin-producing genera, the detected sxtA gene belonged to C. raciborskii, while the mcyE gene belonged to Microcystis in both reservoirs. The detected mcyE gene had good correlation with MC content, while the amplification of the sxtA gene was related to the presence of STX. The cyrJ gene was not detected in these samples. Using DNA analyses, our results showed that the cyanobacterial composition of Mundaú reservoir was similar in successive dry seasons, and it varied between seasons in Ingazeira reservoir. In addition, our data suggest that some biases of analysis influenced the cyanobacterial communities seen in the NGS output of Ingazeira reservoir.

Biological treatment of cyanotoxins has gained much importance in recent decades and holds a promise to work in coordination with various physicochemical treatments. In drinking water treatment plants (DWTPs), effective removal of cyanotoxins with reduced toxicity is a primary concern. Commonly used treatments, such as ozonation, chlorination or activated carbon, undergo significant changes in their operating conditions (mainly dosage) to counter the variation in different environmental parameters, such as pH, temperature, and high cyanotoxin concentration. Presence of metal ions, natural organic matter (NOM), and other chemicals demand higher dosage and hence affect the activation energy to efficiently break down the cyanotoxin molecule. Due to these higher dose requirements, the treatment leads to the formation of toxic metabolites at a concentration high enough to break the guideline values. Biological methods of cyanotoxin removal proceed via enzymatic pathway where the protein-encoding genes are often responsible for the compound breakdown into non-toxic metabolites. However, in contrast to the chemical treatment, the biological processes advance at a much slower kinetic rate, predominantly due to a longer onset period (high lag phase). In fact, more than 90% of the studies reported on the biological degradation of the cyanotoxins attribute the biodegradation to the bacterial suspension. This suspended growth limits the mass transfer kinetics due to the presence of metal ions, NOMs and, other oxidizable matter, which further prolongs the lag phase and makes biological process toxic-free, albeit less efficient. In this context, this review attempts to bring out the importance of the attached growth mechanism, in particular, the biofilm-based treatment approaches which can enhance the biodegradation rate., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Bacillus thuringiensis (Bt) strains produce Cry (crystal) and Cyt (cytolytic) proteins belonging to the group of bacterial toxins known as pore-forming toxins (PFTs), which interact with midgut cells of target insects to create pores, disruption of ion homeostasis and eventual death. PFTs have synergistic insecticidal activities and have been used as biopesticides against agriculturally important insects. Identification of new Cyt proteins is important because of their specific toxicity towards hemipteran pests, against which the Cry proteins are not effective. We have structurally characterized a cyt (cyt1007) gene from an Indian Bt isolate SK-1007. The presence of a "Bacillus thuringiensis toxin" domain and maximum identity of 36% with Cyt2Ca in the deduced amino acid sequence indicated Cyt1007 protein to be a new member of Cyt family. Three dimensional (3D) modeling (PMDB ID: PM0081490) revealed that it adopts a typical ferredoxin-like fold, and is composed of a single domain of α/β architecture, in which a single β sheet is surrounded by two α helical layers. The putative lipid binding site and probable mode of action of Cyt1007 protein were predicted through comparative analysis with other Cyt toxins and their distant homologs Evf (Erwinia virulence factor) and VVA2 (Volvatoxin A2). Heterologous expression of cyt1007 gene as a 25 kDa protein in Escherichia coli was achieved at high levels in both soluble and insoluble fractions. Affinity chromatography-based purification yielded 83.6% purified Cyt1007 protein, which can be used for downstream applications for the investigation of its toxicity. Graphical abstract Steps in the structural characterization and heterologous expression of a new cyt gene cloned from Bacillus thuringiensis.

Mycolactone, the amphiphilic macrolide toxin secreted by Mycobacterium ulcerans , plays a significant role in the pathology and manifestations of Buruli ulcer (BU). Consequently, it follows that the toxin is a suitable target for the development of diagnostics and therapeutics for this disease. Yet, several challenges have deterred such development. For one, the lipophilic nature of the toxin makes it difficult to handle and store and contributes to variability associated with laboratory experimentation and purification yields. In this manuscript, we have attempted to incorporate our understanding of the lipophilicity of mycolactone in order to define the optimal methods for the storage, handling, and purification of this toxin. We present a systematic correlation of variability associated with measurement techniques (thin-layer chromatography (TLC), mass spectrometry (MS), and UV-Vis spectrometry), storage conditions, choice of solvents, as well as the impact of each of these on toxin function as assessed by cellular cytotoxicity. We also compared natural mycolactone extracted from bacterial culture with synthesized toxins in laboratory (solvents, buffers) and physiologically relevant (serum) matrices. Our results point to the greater stability of mycolactone in organic, as well as detergent-containing, solvents, regardless of the container material (plastic, glass, or silanized tubes). They also highlight the presence of toxin in samples that may be undetectable by any one technique, suggesting that each detection approach captures different configurations of the molecule with varying specificity and sensitivity. Most importantly, our results demonstrate for the very first time that amphiphilic mycolactone associates with host lipoproteins in serum, and that this association will likely impact our ability to study, diagnose, and treat Buruli ulcers in patients.

Current genetic detection methods require gene isolation, gene amplification and detection with a fluorescent-tagged probe. They typically require sophisticated equipment and expensive fluorescent probes, rendering them not widely available for rapid acute infection diagnoses at the point of care to ensure timely treatment of the diseases. Here we report a rapid genetic detection method that can detect the bacterial gene directly from patient stools using a piezoelectric plate sensor (PEPS) in conjunction with a continuous flow system with two temperature zones. With stools spiked with sodium dodecyl sulfate (SDS) in situ bacteria lysing and DNA denaturation occurred in the high-temperature zone whereas in situ specific detection of the denatured DNA by the PEPS occurred in the lower-temperature zone. The outcome was a rapid genetic detection method that directly detected bacterial genes from stool in < 40 min without the need of gene isolation, gene amplification, or expensive fluorescent tag but with polymerase chain reaction (PCR) sensitivity. In 40 blinded patient stools, it detected the toxin B gene of Clostridium difficile with 95% sensitivity and 95% specificity. The all-electrical, label-free nature of the detection further supports its potential as a low-cost genetic test that can be used at the point of care., (Copyright © 2019 Elsevier B.V. All rights reserved.)