Strain-level characterization of foodborne pathogens without culture enrichment for outbreak investigation using shotgun metagenomics facilitated with nanopore adaptive sampling.

Introduction: Shotgun metagenomics has previously proven effective in the investigation of foodborne outbreaks by providing rapid and comprehensive insights into the microbial contaminant. However, culture enrichment of the sample has remained a prerequisite, despite the potential impact on pathogen detection resulting from the growth competition. To circumvent the need for culture enrichment, we explored the use of adaptive sampling using various databases for a targeted nanopore sequencing, compared to shotgun metagenomics alone. Methods: The adaptive sampling method was first tested on DNA of mashed potatoes mixed with DNA of a Staphylococcus aureus strain previously associated with a foodborne outbreak. The selective sequencing was used to either deplete the potato sequencing reads or enrich for the pathogen sequencing reads, and compared to a shotgun sequencing. Then, living S. aureus were spiked at 105 CFU into 25 g of mashed potatoes. Three DNA extraction kits were tested, in combination with enrichment using adaptive sampling, following whole genome amplification. After data analysis, the possibility to characterize the contaminant with the different sequencing and extraction methods, without culture enrichment, was assessed. Results: Overall, the adaptive sampling outperformed the shotgun sequencing. While the use of a host removal DNA extraction kit and targeted sequencing using a database of foodborne pathogens allowed rapid detection of the pathogen, the most complete characterization was achieved when using solely a database of S. aureus combined with a conventional DNA extraction kit, enabling accurate placement of the strain on a phylogenetic tree alongside outbreak cases. Discussion: This method shows great potential for strain-level analysis of foodborne outbreaks without the need for culture enrichment, thereby enabling faster investigations and facilitating precise pathogen characterization. The integration of adaptive sampling with metagenomics presents a valuable strategy for more efficient and targeted analysis of microbial communities in foodborne outbreaks, contributing to improved food safety and public health.

Retrospective surveillance of viable Bacillus cereus group contaminations in commercial food and feed vitamin B2 products sold on the Belgian market using whole-genome sequencing.

Bacillus cereus is a spore-forming bacterium that occurs as a contaminant in food and feed, occasionally resulting in food poisoning through the production of various toxins. In this study, we retrospectively characterized viable B. cereus sensu lato (s.l.) isolates originating from commercial vitamin B2 feed and food additives collected between 2016 and 2022 by the Belgian Federal Agency for the Safety of the Food Chain from products sold on the Belgian market. In total, 75 collected product samples were cultured on a general medium and, in case of bacterial growth, two isolates per product sample were collected and characterized using whole-genome sequencing (WGS) and subsequently characterized in terms of sequence type (ST), virulence gene profile, antimicrobial resistance (AMR) gene profile, plasmid content, and phylogenomic relationships. Viable B. cereus was identified in 18 of the 75 (24%) tested products, resulting in 36 WGS datasets, which were classified into eleven different STs, with ST165 (n = 10) and ST32 (n = 8) being the most common. All isolates carried multiple genes encoding virulence factors, including cytotoxin K-2 (52.78%) and cereulide (22.22%). Most isolates were predicted to be resistant to beta-lactam antibiotics (100%) and fosfomycin (88.89%), and a subset was predicted to be resistant to streptothricin (30.56%). Phylogenomic analysis revealed that some isolates obtained from different products were closely related or even identical indicating a likely common origin, whereas for some products the two isolates obtained did not show any close relationship to each other or other isolates found in other products. This study reveals that potentially pathogenic and drug-resistant B. cereus s.l. can be present in food and feed vitamin B2 additives that are commercially available, and that more research is warranted to assess whether their presence in these types of products poses a threat to consumers.

Validation of a Targeted LC-MS/MS Method for Cereulide and Application in Food and Faeces.

Cereulide is an emetic toxin produced by some strains of Bacillus cereus. This bacterial toxin, a cyclic 1.2 kDa dodecadepsipeptide, is stable to heat and acids and causes nausea and vomiting when ingested via contaminated food. This work aimed to develop and validate a targeted analytical method applying liquid chromatography-tandem mass spectrometry (LC-MS/MS) to quantify this toxin in food and human faeces. Samples were extracted with acetonitrile in the presence of 13C6-cereulide, a labelled internal standard, and purified by centrifugation and filtration. The limits of quantification were 0.5 and 0.3 µg kg-1 for food and faeces, respectively. The linearity of the method was very good, with calculated R2 values above 0.995. The mean recovery of the method was within the acceptable range of 70.0%-120.0%, the repeatability was not higher than 7.3%, and the highest intra-laboratory reproducibility was 8.9%. The estimated range for the expanded measurement uncertainty was between 5.1% and 18.0%. The LC-MS/MS method was used to analyse one food sample (rice) from a Belgian foodborne outbreak and five faecal samples from patients with clinical symptoms after consumption of the contaminated rice. The levels of cereulide were 12.22 µg g-1 for food and between 6.32 and 773.37 ng g-1 for faecal samples.

Engraftment of Allotransplanted Tumor Cells in Adult rag2 Mutant Xenopus tropicalis.

Modeling human genetic diseases and cancer in lab animals has been greatly aided by the emergence of genetic engineering tools such as TALENs and CRISPR/Cas9. We have previously demonstrated the ease with which genetically engineered Xenopus models (GEXM) can be generated via injection of early embryos with Cas9 recombinant protein loaded with sgRNAs targeting single or multiple tumor suppressor genes. What has been lacking so far is the possibility to propagate and characterize the induced cancers via transplantation. Here, we describe the generation of a rag2 knockout line in Xenopus tropicalis that is deficient in functional T and B cells. This line was validated by means of allografting experiments with primary tp53-/- and apc+/-/tp53-/- donor tumors. In addition, we optimized available protocols for the sub-lethal irradiation of wild-type X. tropicalis froglets. Irradiated animals also allowed the stable, albeit transient, engraftment of transplanted X. tropicalis tumor cells. The novel rag2-/- line and the irradiated wild-type froglets will further expand the experimental toolbox in the diploid amphibian X. tropicalis and help to establish it as a versatile and relevant model for exploring human cancer.

CRISPR-SID: Identifying EZH2 as a druggable target for desmoid tumors via in vivo dependency mapping.

Cancer precision medicine implies identification of tumor-specific vulnerabilities associated with defined oncogenic pathways. Desmoid tumors are soft-tissue neoplasms strictly driven by Wnt signaling network hyperactivation. Despite this clearly defined genetic etiology and the strict and unique implication of the Wnt/ß-catenin pathway, no specific molecular targets for these tumors have been identified. To address this caveat, we developed fast, efficient, and penetrant genetic Xenopus tropicalis desmoid tumor models to identify and characterize drug targets. We used multiplexed CRISPR/Cas9 genome editing in these models to simultaneously target a tumor suppressor gene (apc) and candidate dependency genes. Our methodology CRISPR/Cas9 selection-mediated identification of dependencies (CRISPR-SID) uses calculated deviations between experimentally observed gene editing outcomes and deep-learning-predicted double-strand break repair patterns to identify genes under negative selection during tumorigenesis. This revealed EZH2 and SUZ12, both encoding polycomb repressive complex 2 components, and the transcription factor CREB3L1 as genetic dependencies for desmoid tumors. In vivo EZH2 inhibition by Tazemetostat induced partial regression of established autochthonous tumors. In vitro models of patient desmoid tumor cells revealed a direct effect of Tazemetostat on Wnt pathway activity. CRISPR-SID represents a potent approach for in vivo mapping of tumor vulnerabilities and drug target identification.

Targeted Genome Engineering in Xenopus Using the Transcription Activator-Like Effector Nuclease (TALEN) Technology.

Targeted genome engineering technologies are revolutionizing the field of functional genomics and have been extensively used in a variety of model organisms, including X. tropicalis and X. laevis. The original methods based on Zn-finger proteins coupled to endonuclease domains were initially replaced by the more efficient and straightforward transcription activator-like effector nucleases (TALENs), adapted from plant pathogenic Xanthomonas species. Although functional genomics are more recently dominated by the even faster and more convenient CRISPR/Cas9 technology, the use of TALENs may still be preferred in a number of cases. We have successfully implemented this technology in Xenopus and in this chapter we describe our working protocol for targeted genome editing in X. tropicalis using TALENs.

TALENs and CRISPR/Cas9 fuel genetically engineered clinically relevant Xenopus tropicalis tumor models.

The targeted nuclease revolution (TALENs, CRISPR/Cas9) now allows Xenopus researchers to rapidly generate custom on-demand genetic knockout models. These novel methods to perform reverse genetics are unprecedented and are fueling a wide array of human disease models within the aquatic diploid model organism Xenopus tropicalis (X. tropicalis). This emerging technology review focuses on the tools to rapidly generate genetically engineered X. tropicalis models (GEXM), with a focus on establishment of genuine genetic and clinically relevant cancer models. We believe that due to particular advantageous characteristics, outlined within this review, GEXM will become a valuable alternative animal model for modeling human cancer. Furthermore, we provide perspectives of how GEXM will be used as a platform for elucidation of novel therapeutic targets and for preclinical drug validation. Finally, we also discuss some future prospects on how the recent expansions and adaptations of the CRISPR/Cas9 toolbox might influence and push forward X. tropicalis cancer research.

CRISPR/Cas9 mediated knockout of rb1 and rbl1 leads to rapid and penetrant retinoblastoma development in Xenopus tropicalis.

Retinoblastoma is a pediatric eye tumor in which bi-allelic inactivation of the Retinoblastoma 1 (RB1) gene is the initiating genetic lesion. Although recently curative rates of retinoblastoma have increased, there are at this time no molecular targeted therapies available. This is, in part, due to the lack of highly penetrant and rapid retinoblastoma animal models that facilitate rapid identification of targets that allow therapeutic intervention. Different mouse models are available, all based on genetic deactivation of both Rb1 and Retinoblastoma-like 1 (Rbl1), and each showing different kinetics of retinoblastoma development. Here, we show by CRISPR/Cas9 techniques that similar to the mouse, neither rb1 nor rbl1 single mosaic mutant Xenopus tropicalis develop tumors, whereas rb1/rbl1 double mosaic mutant tadpoles rapidly develop retinoblastoma. Moreover, occasionally presence of pinealoblastoma (trilateral retinoblastoma) was detected. We thus present the first CRISPR/Cas9 mediated cancer model in Xenopus tropicalis and the first genuine genetic non-mammalian retinoblastoma model. The rapid kinetics of our model paves the way for use as a pre-clinical model. Additionally, this retinoblastoma model provides unique possibilities for fast elucidation of novel drug targets by triple multiplex CRISPR/Cas9 gRNA injections (rb1 + rbl1 + modifier gene) in order to address the clinically unmet need of targeted retinoblastoma therapy.

TALEN-mediated apc mutation in Xenopus tropicalis phenocopies familial adenomatous polyposis.

Truncating mutations in the tumor suppressor gene adenomatous polyposis coli (APC) are the initiating step in the vast majority of sporadic colorectal cancers, and they underlie familial adenomatous polyposis (FAP) syndromes. Modeling of APC- driven tumor formation in the mouse has contributed substantially to our mechanistic understanding of the associated disease, but additional models are needed to explore therapeutic opportunities and overcome current limitations of mouse models. We report on a novel and penetrant genetic cancer model in Xenopus tropicalis, an aquatic tetrapod vertebrate with external development, diploid genome and short life cycle. Tadpoles and froglets derived from embryos injected with TAL effector nucleases targeting the apc gene rapidly developed intestinal hyperplasia and other neoplasms observed in FAP patients, including desmoid tumors and medulloblastomas. Bi-allelic apc mutations causing frame shifts were detected in the tumors, which displayed activation of the Wnt/ß-catenin pathway and showed increased cellular proliferation. We further demonstrate that simultaneous double bi-allelic mutation of apc and a non-relevant gene is possible in the neoplasias, opening the door for identification and characterization of effector or modifier genes in tumors expressing truncated apc. Our results demonstrate the power of modeling human cancer in Xenopus tropicalis using mosaic TALEN-mediated bi-allelic gene disruption.

TLR2 activation causes no morbidity or cardiovascular failure, despite excessive systemic nitric oxide production.

AIMS: Septic shock is the leading cause of death in intensive care units worldwide, resulting from a progressive systemic inflammatory reaction causing cardiovascular and organ failure. Nitric oxide (NO) is a potent vasodilator and inhibition of NO synthases (NOS) can increase blood pressure in septic shock. However, NOS inhibition does not improve outcome, on the contrary, and certain NO donors may even provide protection. In addition, NOS produce superoxide in case of substrate or cofactor deficiency or oxidation. We hypothesized that excessive systemic iNOS-derived NO production is insufficient to trigger cardiovascular failure and shock. METHODS AND RESULTS: We found that the systemic injection with various synthetic Toll-like receptor-2 (TLR2), TLR3, or TLR9 agonists triggered systemic NO production identical to that of lipopolysaccharide (LPS) or tumour necrosis factor. In contrast to the latter, however, these agonists did not cause hypothermia or any other signs of discomfort or morbidity, and inflammatory cytokine production was low. TLR2 stimulation with the triacylated lipopeptide Pam3CSK4 not only caused identical NO levels in circulation, but also identical iNOS expression patterns as LPS. Nevertheless, Pam3CSK4 did not cause hypotension, bradycardia, reduced blood flow, or inadequate tissue perfusion in the kidney or the liver. CONCLUSION: We demonstrate that excessive iNOS-derived NO in circulation is not necessarily linked to concomitant cardiovascular collapse, morbidity, or mortality. As such, our data indicate that the central role of iNOS-derived NO in inflammation-associated cardiovascular failure may be overestimated.