Bioinformatics at Biosafe Ltd is optimized for the analysis of safety aspects of microbial genomes.

Bioinformatics at Biosafe Ltd is focused on analyzing the safety aspects of microbial genomes on behalf of companies who develop microbial products into the food chain. They may be production organisms for amino acids, enzymes or vitamins, or live micro-organisms. Many of the products will be evaluated by
the European Food Safety Authority (EFSA). Whatever the purpose, safety means the absence of acquired antimicrobial resistance genes, genes for toxins and virulence factors. We occasionally also describe the genetic modifications in detail or search for secondary metabolite biosynthesis clusters. The genome is used for unequivocal taxonomic identification at the species level, which is essential in granting the QPS (qualified presumption of safety) status for a microorganism. Sometimes this analysis leads to the description of a new species.

The use of whole genome sequence (WGS) – what and when?

Whole genome refers to both chromosome(s) and plasmids. If it is unclear whether or not the microorganism carries plasmids this should be explored. The DNA extraction and sequencing method will be selected accordingly.
According to The Guidance on microorganisms used as feed additives or as production organisms (EFSA FEEDAP Panel, 2018), WGS is required for bacteria and yeasts. For fungi, WGS should be used for identification if available.

Bioinformatics and sequence analysis

Identification

Proper taxonomic classification of a microbe is the basis of safety analysis. The most important level of classification is the species level, which is also the basis for assigning a microbe a Qualified Presumption of Safety status (QPS). EFSA maintains and develops a list of QPS microorganisms for food or feed use. Currently the list contains many bacteria and yeasts but excludes e.g. filamentous fungi. Whenever a new species is introduced by an application submitted to the Commission and forwarded to EFSA, the QPS working group can assess its safety and conclude on its possible inclusion in the QPS list. The assessment is based on taxonomic identification, body of knowledge, possible pathogenicity (or other safety concerns) and the use of the microorganism. The QPS concept is also applicable to genetically modified microorganisms used as production strains.

Sometimes it is important to identify the species at the strain level – such as in the case of E. coli K-12. Although E. coli is not QPS, K-12 strains have a long history of safe use and are generally considered as safe, provided that genes of concern are not introduced in the genome.

The increase of genome sequence information has brought a lot of confusion for taxonomic classification in some genera, such as Bacillus, Lactobacillus and Streptomyces. It has been proposed, for instance, that Bacillus subtilis subsp. inaquosorum should be recognized as a novel taxon distinct from Bacillus subtilis. Novel species can sometimes be particularly problematic for the interpretation of MIC values and may require extra scientific justifications.

The most widely used means for taxonomic classification, 16S rRNA gene sequence analysis, is often not sufficient for species identification. Biosafe uses multiple approaches to taxonomic classification purposes, such as average nucleotide identity (ANI), phylogenomics or housekeeping genes.

Genome screening for antimicrobial resistance genes and genes for toxins and virulence factors

The genome should be interrogated searched for genes conferring resistance to antimicrobials relevant for their use in humans and animals (Critically or Highly important antimicrobials, WHO). Biosafe Ltd uses several databases to screen the genomes in an in-house pipeline. This genome-based prediction is then assessed together with the results from phenotypic testing to draw final conclusions.

Interpretation of the outcome of WGS and phenotypic testing

Biosafe Ltd was among the first ones to apply whole genome sequencing (WGS) in the microbial safety assessment for the dossiers submitted to EFSA evaluations. We are constantly improving our analysis pipeline and the clarity of the reports. Our experience on dozens of genomes gives us a good perspective to interpret the results. For example, it is much easier to detect acquired antimicrobial resistance (AMR) genes after examining many genomes of the same species.

Almost all microbial genomes have matches to genes in the AMR databases, i.e. to genes that potentially contribute to AMR. Most of these are intrinsic genes, often efflux pumps, which do not necessarily confer resistance. In the case of production microorganisms, it is important that the acquired resistance genes are not transferred into the product. In case of viable microorganisms as products, the acquired functional AMR genes are considered a hazard.

The presence of genes encoding virulence factors may trigger further phenotypic testing (cytotoxicity tests ).

Genetically modified microorganisms (GMM)

In the case of GMMs, WGS should be used to characterize the structure of the genetic modification. This is mandatory for bacteria and yeasts and recommended for filamentous fungi.

Biosafe Ltd follows in detail the EFSA FEEDAP Panel guidance (2018) to characterize the GMM. To do this, we work in close collaboration with the customer, as a proper characterization requires knowledge of the strain history and genetic modifications. Importantly, the WGS is also used to explore if genes of concern were introduced into the genome. This information is necessary for the analysis of the presence of DNA from the production strain in the fermentation products.

Sequencing

Biosafe Ltd also offers comprehensive sequencing services in collaboration with our sequencing partners. We sequence microbial genomes, plasmids or individual genes based on the particular needs. All sequencing projects are unique and designed in collaboration with the customers to answer their specific needs. The sequencing platform is also selected on a case-by-case basis.

How do we work?
We can help you during the planning of a WGS study, during genome analysis for EFSA, FSANZ or other regulatory purposes, or during the publication phase of a new microorganism. You are welcome to contact us to learn more.