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In recent years, there has been an increase in the number of reported cases of Mycobacterium microti infection in various animals, which can interfere with the ante-mortem diagnosis of animal tuberculosis caused by Mycobacterium bovis. In this study, whole genome sequencing (WGS) was used to search for protein-coding genes to distinguish M. microti from M. bovis. In addition, the population structure of the available M. microti genomic WGS datasets is described, including three novel Belgian isolates from infections in alpacas. Candidate genes were identified by examining the presence of the regions of difference and by a pan-genome analysis of the available WGS data. A total of 80 genes showed presence-absence variation between the two species, including genes encoding Proline-Glutamate (PE), Proline-Proline-Glutamate (PPE), and Polymorphic GC-Rich Sequence (PE-PGRS) proteins involved in virulence and host interaction. Filtering based on predicted subcellular localization, sequence homology and predicted antigenicity resulted in 28 proteins out of 80 that were predicted to be potential antigens. As synthetic peptides are less costly and variable than recombinant proteins, an in silico approach was performed to identify linear and discontinuous B-cell epitopes in the selected proteins. From the 28 proteins, 157 B-cell epitope-based peptides were identified that discriminated between M. bovis and M. microti species. Although confirmation by in vitro testing is still required, these candidate synthetic peptides containing B-cell epitopes could potentially be used in serological tests to differentiate cases of M. bovis from M. microti infection, thus reducing misdiagnosis in animal tuberculosis surveillance. [ABSTRACT FROM AUTHOR]

Infections due to antimicrobial-resistant bacteria have become a major threat to global health. Some patients may carry resistant bacteria in their gut microbiota. Specific risk factors may trigger the conversion of these carriages into infections in hospitalized patients. Preventively eradicating these carriages has been postulated as a promising preventive intervention. However, previous attempts at such eradication using oral antibiotics or probiotics have led to discouraging results. Phage therapy, the therapeutic use of bacteriophage viruses, might represent a worthy alternative in this context. Taking inspiration from this clinical challenge, we built Gut-On-A-Chip (GOAC) models, which are tridimensional cell culture models mimicking a simplified gut section. These were used to better understand bacterial dynamics under phage pressure using two relevant species: Pseudomonas aeruginosa and Escherichia coli. Model mucus secretion was documented by ELISA assays. Bacterial dynamics assays were performed in GOAC triplicates monitored for 72 h under numerous conditions, such as pre-, per-, or post-bacterial timing of phage introduction, punctual versus continuous phage administration, and phage expression of mucus-binding properties. The potential genomic basis of bacterial phage resistance acquired in the model was investigated by variant sequencing. The bacterial "escape growth" rates under phage pressure were compared to static in vitro conditions. Our results suggest that there is specific bacterial prosperity in this model compared to other in vitro conditions. In E. coli assays, the introduction of a phage harboring unique mucus-binding properties could not shift this balance of power, contradicting previous findings in an in vivo mouse model and highlighting the key differences between these models. Genomic modifications were correlated with bacterial phage resistance acquisition in some but not all instances, suggesting that alternate ways are needed to evade phage predation, which warrants further investigation. [ABSTRACT FROM AUTHOR]

Infectious bronchopneumonia has a major economic impact, causing high morbidity and mortality rates in cattle production systems worldwide (1). Furthermore, it is the main indication for antimicrobial use in calves [...]

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. [ABSTRACT FROM AUTHOR]

Genetically modified microorganisms (GMM) are frequently employed for manufacturing microbial fermentation products such as food enzymes or vitamins. Although the fermentation product is required to be pure, GMM contaminations have repeatedly been reported in numerous commercial microbial fermentation produce types, leading to several rapid alerts at the European level. The aim of this study was to investigate the added value of shotgun metagenomic high-throughput sequencing to confirm and extend the results of classical analysis methods for the genomic characterization of unauthorized GMM. By combining short- and long-read metagenomic sequencing, two transgenic constructs were characterized, with insertions of alpha-amylase genes originating from B. amyloliquefaciens and B. licheniformis, respectively, and a transgenic construct with a protease gene insertion originating from B. velezensis, which were all present in all four investigated samples. Additionally, the samples were contaminated with up to three unculturable Bacillus strains, carrying genetic modifications that may hamper their ability to sporulate. Moreover, several samples contained viable Bacillus strains. Altogether these contaminations constitute a considerable load of antimicrobial resistance genes, that may represent a potential public health risk. In conclusion, our study showcases the added value of metagenomics to investigate the quality and safety of complex commercial microbial fermentation products. [ABSTRACT FROM AUTHOR]

Background: Linezolid is a critically important antibiotic used to treat human infections caused by MRSA and VRE. While linezolid is not licensed for food-producing animals, linezolid-resistant (LR) isolates have been reported in European countries, including Belgium.Objectives: To: (i) assess LR occurrence in staphylococci and enterococci isolated from different Belgian food-producing animals in 2019 through selective monitoring; and (ii) investigate the genomes and relatedness of these isolates.Methods: Faecal samples (n = 1325) and nasal swab samples (n = 148) were analysed with a protocol designed to select LR bacteria, including a 44-48 h incubation period. The presence of LR chromosomal mutations, transferable LR genes and their genetic organizations and other resistance genes, as well as LR isolate relatedness (from this study and the NCBI database) were assessed through WGS.Results: The LR rate differed widely between animal host species, with the highest rates occurring in nasal samples from pigs and sows (25.7% and 20.5%, respectively) and faecal samples from veal calves (16.4%). WGS results showed that LR determinants are present in a large diversity of isolates circulating in the agricultural sector, with some isolates closely related to human isolates, posing a human health risk.Conclusions: LR dedicated monitoring with WGS analysis could help to better understand the spread of LR. Cross-selection of LR transferable genes through other antibiotic use should be considered in future action plans aimed at combatting antimicrobial resistance and in future objectives for the rational use of antibiotics in a One Health perspective. [ABSTRACT FROM AUTHOR]

Despite their presence being unauthorized on the European market, contaminations with genetically modified (GM) microorganisms have repeatedly been reported in diverse commercial microbial fermentation produce types. Several of these contaminations are related to a GM Bacillus velezensis used to synthesize a food enzyme protease, for which genomic characterization remains currently incomplete, and it is unknown whether these contaminations have a common origin. In this study, GM B. velezensis isolates from multiple food enzyme products were characterized by short- and long-read whole-genome sequencing (WGS), demonstrating that they harbor a free recombinant pUB110-derived plasmid carrying antimicrobial resistance genes. Additionally, single-nucleotide polymorphism (SNP) and whole-genome based comparative analyses showed that the isolates likely originate from the same parental GM strain. This study highlights the added value of a hybrid WGS approach for accurate genomic characterization of GMM (e.g., genomic location of the transgenic construct), and of SNP-based phylogenomic analysis for source-tracking of GMM. [ABSTRACT FROM AUTHOR]

Rapid, accurate bacterial identification in biological samples is an important task for microbiology laboratories, for which 16S rRNA gene Sanger sequencing of cultured isolates is frequently used. In contrast, next-generation sequencing does not require intermediate culturing steps and can be directly applied on communities, but its performance has not been extensively evaluated. We present a comparative evaluation of second (Illumina) and third (Oxford Nanopore Technologies (ONT)) generation sequencing technologies for 16S targeted genomics using a well-characterized reference sample. Different 16S gene regions were amplified and sequenced using the Illumina MiSeq, and analyzed with Mothur. Correct classification was variable, depending on the region amplified. Using a majority vote over all regions, most false positives could be eliminated at the genus level but not the species level. Alternatively, the entire 16S gene was amplified and sequenced using the ONT MinION, and analyzed with Mothur, EPI2ME, and GraphMap. Although >99% of reads were correctly classified at the genus level, up to ≈40% were misclassified at the species level. Both technologies, therefore, allow reliable identification of bacterial genera, but can potentially misguide identification of bacterial species, and constitute viable alternatives to Sanger sequencing for rapid analysis of mixed samples without requiring any culturing steps. [ABSTRACT FROM AUTHOR]

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 B 2 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 B 2 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., 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 © 2023 Bogaerts, Fraiture, Huwaert, Van Nieuwenhuysen, Jacobs, Van Hoorde, De Keersmaecker, Roosens and Vanneste.)