Experimental approaches to identify small RNA

Nowadays the identification of small RNAs (sRNAs) and characterization of their role within regulatory networks takes a prominent place in deciphering complex bacterial phenotypes. Compared to the study of other components of bacterial cells, this is a relatively new but fast-growing research field. Although reports on new sRNAs appear regularly, some sRNAs are already subject of research for a longer time. One of such sRNAs is MicA, a sRNA best described for its role in outer membrane remodeling, but probably having a much broader function than anticipated. An overview of what we have learnt from MicA led to the conclusion that even for this well-described sRNA, we still do not have the overall picture. More general, the story of MicA might become an experimental lead for unraveling the many sRNAs with unknown functions. In this review, three important topics in the sRNA field are covered, exemplified from the perspective of MicA: (i) identification of new sRNAs, (ii) target identification and unraveling the biological function, (iii) structural analysis. The complex mechanisms of action of MicA deliver some original insights in the sRNA field which includes the existence of dimer formation or simultaneous cis and trans regulation, and might further inspire the understanding of the function of other sRNAs</p

Design of multiplex assays for molecular subtyping of pathogens with the Luminex xMAP technology

&lt;p&gt;Multiplex assays are a powerful tool for molecular subtyping of pathogens, which is crucial for rapid diagnosis, surveillance and identification and containment of outbreaks. The Luminex xMAP technology allows detection of up to 500 different analytes per sample in a high-throughput format through a liquid bead suspension array. Different types of Luminex assays can be envisaged. Each assay has its advantages and limitations, some of which are related to the inherent design of the primers and probes used. Software that predicts melting temperatures, hybridization structures and specificity for different types of oligonucleotides, and thereby simulating the multiplex assays in silico can facilitate the design of multiplex assays by reducing time and cost of experimentation.We have compared two commercial software packages for the development and in silico simulation of a ligation dependent amplification (LDA) assay and a direct hybridization assay for the subtyping of a pathogen. The main findings, limitations and challenges will be discussed.&lt;/p&gt;</p

Development of a portable on-site applicable metagenomic data generation workflow for enhanced pathogen and antimicrobial resistance surveillanceAbstract

Rapid, accurate and comprehensive diagnostics are essential for outbreak prevention and pathogen surveillance. Real-time, on-site metagenomics on miniaturized devices, such as Oxford Nanopore Technologies MinION sequencing, could provide a promising approach. However, current sample preparation protocols often require substantial equipment and dedicated laboratories, limiting their use. In this study, we developed a rapid on-site applicable DNA extraction and library preparation approach for nanopore sequencing, using portable devices. The optimized method consists of a portable mechanical lysis approach followed by magnetic bead-based DNA purification and automated sequencing library preparation, and resulted in a throughput comparable to a current optimal, laboratory-based protocol using enzymatic digestion to lyse cells. By using spike-in reference communities, we compared the on-site method with other workflows, and demonstrated reliable taxonomic profiling, despite method-specific biases. We also demonstrated the added value of long-read sequencing by recovering reads containing full-length antimicrobial resistance genes, and attributing them to a host species based on the additional genomic information they contain. Our method may provide a rapid, widely-applicable approach for microbial detection and surveillance in a variety of on-site settings

Metagenomic Characterization of Multiple Genetically Modified Bacillus Contaminations in Commercial Microbial Fermentation Products

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&nbsp;products.</p

Using a combination of short and long read sequencing to investigate the diversity in plasmid- and chromosomally encoded extended-spectrum-beta-lactamases (ESBL) in clinical Shigella and Salmonella isolates in Belgium

For antimicrobial resistance (AMR) surveillance, it is important not only to detect AMR genes, but also to determine their plasmidic or chromosomal location, as this will impact their spread differently. Whole-genome sequencing (WGS) is increasingly used for AMR surveillance. However, determining the genetic context of AMR genes using only short-read sequencing is complicated. The combination with long-read sequencing offers a potential solution, as it allows hybrid assemblies. Nevertheless, its use in surveillance has so far been limited. This study aimed to demonstrate its added value for AMR surveillance based on a case study of extended-spectrum beta-lactamases (ESBLs). ESBL genes have been reported to occur also on plasmids. To gain insight into the diversity and genetic context of ESBL genes detected in clinical isolates received by the Belgian National Reference Center between 2013 and 2018, 100 ESBL-producing Shigella and 31 ESBL-producing Salmonella were sequenced with MiSeq and a representative selection of 20 Shigella and six Salmonella isolates additionally with MinION technology, allowing hybrid assembly. The bla CTX-M-15 gene was found to be responsible for a rapid rise in the ESBL Shigella phenotype from 2017. This gene was mostly detected on multi-resistance-carrying IncFII plasmids. Based on clustering, these plasmids were determined to be distinct from the circulating plasmids before 2017. They were spread to different Shigella species and within Shigella sonnei between multiple genotypes. Another similar IncFII plasmid was detected after 2017 containing bla CTX-M-27 for which only clonal expansion occurred. Matches of up to 99 % to plasmids of various bacterial hosts from all over the world were found, but global alignments indicated that direct or recent ESBL-plasmid transfers did not occur. It is most likely that travellers introduced these in Belgium and subsequently spread them domestically. However, a clear link to a specific country could not be made. Moreover, integration of bla CTX-M in the chromosome of two Shigella isolates was determined for the first time, and shown to be related to ISEcp1. In contrast, in Salmonella, ESBL genes were only found on plasmids, of which bla CTX-M-55 and IncHI2 were the most prevalent, respectively. No matching ESBL plasmids or cassettes were detected between clinical Shigella and Salmonella isolates. The hybrid assembly data allowed us to check the accuracy of plasmid prediction tools. MOB-suite showed the highest accuracy. However, these tools cannot replace the accuracy of long-read and hybrid assemblies. This study illustrates the added value of hybrid assemblies for AMR surveillance and shows that a strategy where even just representative isolates of a collection used for hybrid assemblies could improve international AMR surveillance as it allows plasmid tracking

Shifting national surveillance of Shigella infections toward geno-serotyping by the development of a tailored Luminex assay and NGS workflow.

&lt;p&gt;The phylogenetically closely related Shigella species and enteroinvasive Escherichia coli (EIEC) are responsible for millions of episodes of bacterial dysenteriae worldwide. Given its distinct epidemiology and public health relevance, only Shigellae are subject to mandatory reporting and follow-up by public health authorities. However, many clinical laboratories struggle to differentiate non-EIEC, EIEC, and Shigella in their current workflows, leading to inaccuracies in surveillance and rising numbers of misidentified E.&amp;nbsp;coli samples at the National Reference Centre (NRC). In this paper, we describe two novel tools to enhance Shigella surveillance. First, we developed a low-cost Luminex-based multiplex assay combining five genetic markers for species identification with 11 markers for serotype prediction for S.&amp;nbsp;sonnei and S.&amp;nbsp;flexneri isolates. Using a test panel of 254 clinical samples, this assay has a sensitivity of 100% in differentiation of EIEC/Shigella pathotype from non-EIEC strains, and 68.7% success rate in distinction of Shigella and EIEC. A novel, and particularly successful marker was a Shigella-specific deletion in the spermidine acetyltransferase gene speG, reflecting its metabolic decay. For Shigella serotype prediction, the multiplex assay scored a sensitivity and specificity of 96.6% and 98.4%, respectively. All discrepancies were analyzed with whole-genome sequencing and shown to be related to causative mutations (stop codons, indels, and promoter mutations) in glycosyltransferase genes. This observation spurred the development of an in silico workflow which extracts the Shigella serotype from Next-Generation Sequencing (NGS) data, taking into account gene functionality. Both tools will be implemented in the workflow of the NRC, and will play a major role in the shift from phenotypic to genotyping-based surveillance of shigellosis in Belgium.&lt;/p&gt;</p

Shifting national surveillance of Shigella infections toward geno‐serotyping by the development of a tailored Luminex assay and NGS workflow

&lt;p&gt;The phylogenetically closely related Shigella species and enteroinvasive Escherichia coli (EIEC) are responsible for millions of episodes of bacterial dysenteriae worldwide. Given its distinct epidemiology and public health relevance, only Shigellae are subject to mandatory reporting and follow-up by public health authorities. However, many clinical laboratories struggle to differentiate non-EIEC, EIEC, and Shigella in their current workflows, leading to inaccuracies in surveillance and rising numbers of misidentified E.&amp;nbsp;coli samples at the National Reference Centre (NRC). In this paper, we describe two novel tools to enhance Shigella surveillance. First, we developed a low-cost Luminex-based multiplex assay combining five genetic markers for species identification with 11 markers for serotype prediction for S.&amp;nbsp;sonnei and S.&amp;nbsp;flexneri isolates. Using a test panel of 254 clinical samples, this assay has a sensitivity of 100% in differentiation of EIEC/Shigella pathotype from non-EIEC strains, and 68.7% success rate in distinction of Shigella and EIEC. A novel, and particularly successful marker was a Shigella-specific deletion in the spermidine acetyltransferase gene speG, reflecting its metabolic decay. For Shigella serotype prediction, the multiplex assay scored a sensitivity and specificity of 96.6% and 98.4%, respectively. All discrepancies were analyzed with whole-genome sequencing and shown to be related to causative mutations (stop codons, indels, and promoter mutations) in glycosyltransferase genes. This observation spurred the development of an in silico workflow which extracts the Shigella serotype from Next-Generation Sequencing (NGS) data, taking into account gene functionality. Both tools will be implemented in the workflow of the NRC, and will play a major role in the shift from phenotypic to genotyping-based surveillance of shigellosis in Belgium.&lt;/p&gt;</p

Strain-level characterization without culture enrichment? Easing and accelerating outbreak investigation using shotgun metagenomics facilitated with nanopore adaptive sampling

Introduction Traditionally, the investigation of suspect food samples during an outbreak investigation requires the samples to be enriched in order to reach detectable levels. However, a bias can occur during the enrichment of the food due to competition between the various living organisms or the cultivation parameters. Moreover, the current methods require a subsequent but not always successful isolation. In part due to these two drawbacks, not all outbreaks can be resolved. Shotgun metagenomics is a sequencing method involving the analysis of the genetic material directly extracted from a sample, without the need for an isolation of the bacteria. By sequencing and analysing the DNA of the sample, it can identify the presence of various pathogens to the strain level. Moreover, adaptive sampling is a new tool proposed during nanopore sequencing to target certain DNA strands to be sequenced preferably. The combination of metagenomics and adaptive sampling could allow to circumvent the isolation but also the enrichment of the food&nbsp;sample. Materials and Methods As a proof-of-concept, we explored the use of adaptive sampling using various databases (depletion of the food matrix i.e. potato, enrichment of a selection of foodborne pathogens, enrichment of a database of Staphylococcus aureus) compared to shotgun metagenomics without adaptive sampling on DNA of mashed potatoes spiked with DNA of S. aureus at a level of 0.5%. The strain was previously associated with a foodborne outbreak. Additionally, the living S. aureus strain was spiked into mashed potatoes at a level of 105 CFU per 25 grams and three DNA extraction kits were tested, in combination with two databases for adaptive sampling, following whole genome amplification to increase the amount of genetic material to meet sequencing standards. The strain-level data analysis was performed as previously described (Buytaers et al.&nbsp;2021). Discussion Our results showed that the combination of whole genome amplification, metagenomics and adaptive sampling using a database of S. aureus genomes outperformed shotgun metagenomics and adaptive sampling using other databases. It allowed strain-level analysis of foodborne outbreaks without the need for culture enrichment, thereby enabling faster investigations and facilitating precise pathogen characterization, contributing to improved food safety and public&nbsp;health.</p