Pimlapas Leekitcharoenphon looked at the issue in her PhD project at the National Food Institute, Technical University of Denmark.
The work showed there is no simple, automated tool to analyse data about individual nucleotide differences and use findings to construct a phylogenetic tree - a diagram showing the relationship between various bacteria.
Relevant method and analytical tool
Her work evaluated what methods are best suited for different purposes and developed an online analytical tool for determining various bacteria’s type.
One study examined 18 different strains from six previous outbreaks to determine which approach is better suited for a particular purpose e.g. determining species, sub-species and serovar, or to relate sources to outbreaks.
The best method for outbreak detection of Salmonella is the analysis of single nucleotide polymorphisms (SNPs), which are differences in the DNA’s building blocks.
SnpTree, a server for online SNPs analysis, was superior in term of clonal or outbreak level because it can cluster strains from the same outbreak and differentiate it from background strains.
Leekitcharoenphon told FoodQualityNews that she tried to evaluate different WGS approaches in her thesis.
“These included 16S rRNA (use one genes), MLST tree (use 7 housekeeping genes), core genes tree (use core genes), pan-genome tree (use all genes) and single nucleotide polymorphism or SNPs (use a point mutation in the genome)," she said.
“In principle, the WGS methods differ by the source of nucleotide sequences to build the tree. They can be one gene, multiple genes, all genes or point mutation. And the resolution obtained from each method is different.”
Salmonella is the second leading cause of foodborne disease in Denmark.
The key to good results from 'real-time' monitoring and identification of outbreaks is rapid and reliable determination of bacterial type.
Leekitcharoenphon said snpTree is an online freely accessible tool to create a SNP phylogenetic tree.
“Users can upload their whole genomic samples either raw reads (fastq) or assembled genomes (fasta). After uploading and clicking submit, users will get SNP tree based on their samples," she said.
“SnpTree and the extended version called CSI phylogeny are the only friendly online tools that actually make snpTree from genomic data. The other tools only identify SNPs and do not construct the tree and most of them are very complicated for users who lack bioinformatics skill.”
The WGS methods used and the snpTree could be used for other bacterial pathogens, said Leekitcharoenphon.
“What I think would be the next improvement or challenge is sequencing without culturing or sequencing directly from the entire sample would be the future of typing of pathogens," she said.
“There have been some studies using metagenomics approach to define the microbiomes of diverse samples and environments. This is very useful particularly to overcome the low proportion of pathogen DNA in a clinical sample.
“The technology should be getting cheaper and should be very convenient to use. If the sequencing is possible but the analysis is still complicated, WGS will not become the chosen one. An analysis tool should be accessible, rapid and easy to use especially for routine diagnostic use.”