[2] MLST directly measures the DNA sequence variations in a set of housekeeping genes and characterizes strains by their unique allelic profiles.
In the data collection step, definitive identification of variation is obtained by nucleotide sequence determination of gene fragments.
To strike the balance between the acceptable identification power, time and cost for the strain typing, about seven to eight house-keeping genes are commonly used in the laboratories.
For Vibrio vulnificus, the housekeeping genes used are glucose-6-phosphate isomerase (glp), DNA gyrase, subunit B (gyrB), malate-lactate dehydrogenase (mdh), methionyl-tRNA synthetase (metG), phosphoribosylaminoimidazole synthetase (purM), threonine dehydrogenase (dtdS), diaminopimelate decarboxylase (lysA), transhydrogenase alpha subunit (pntA), dihydroorotase (pyrC) and tryptophanase (tnaA).
[citation needed] Despite MLST providing high discriminatory power, the accumulation of nucleotide changes in housekeeping genes is a relatively slow process and the allelic profile of a bacterial isolate is sufficiently stable over time for the method to be ideal for global epidemiology.
The relatedness of isolates is displayed as a dendrogram constructed using the matrix of pairwise differences between their allelic profiles, eBURST or a minimum spanning tree (MST).
Several molecular typing schemes have been proposed to determine the relatedness of pathogens such as pulsed-field gel electrophoresis (PFGE), ribotyping, and PCR-based fingerprinting.
Despite PFGE being considered by many researchers as the “gold standard”, many strains are not typable by this technique due to the degradation of the DNA during the process (gel smears).
MLST is automated, combines advances in high throughput sequencing and bioinformatics with established population genetics techniques.
The application of MLST is huge, and provides a resource for the scientific, public health, and veterinary communities as well as the food industry.
Campylobacter is the common causative agent for bacterial infectious intestinal diseases, usually arising from undercooked poultry or unpasteurised milk.
In addition, Campylobacter genomes are genetically diverse and unstable with frequent inter- and intragenomic recombination, together with phase variation, which complicates the interpretation of data from many typing methods.
To improve the level of discriminatory power between the major invasive lineages, seven loci are now being used and have been accepted by many laboratories as the method of choice for characterizing meningococcal isolates.
MLST has successfully provided a reliable method for characterization of clones within other bacterial species in which the rates of clonal diversification are generally lower.
The Cronobacter MLST was initially applied to distinguish between C. sakazakii and C. malonaticus because 16S rDNA sequencing is not always accurate enough, and biotyping is too subjective.
[10] The Cronobacter MLST scheme uses 7 alleles; atpD, fusA, glnS, gltB, gyrB, infB and ppsA giving a concatenated sequence of 3036 bp for phylogenetic analysis (MLSA) and comparative genomics.
[12] The method has revealed a strong association between one genetic lineage, sequence type 4 (ST4), and cases of neonatal meningitis.,[13] The Cronobacter MLST site is at http://www.pubMLST.org/cronobacter.
Due to the sequence conservation in housekeeping genes, MLST sometimes lacks the discriminatory power to differentiate bacterial strains, which limits its use in epidemiological investigations.
To improve the discriminatory power of MLST, a multi-virulence-locus sequence typing (MVLST) approach has been developed using Listeria monocytogenes .
For example, whole-genome sequencing of numerous isolates has revealed the single MLST lineage ST258 of Klebsiella pneumoniae comprises two distinct genetic clades,[16] providing additional information about the evolution and spread of these multi-drug resistant organisms, and disproving the previous hypothesis of a single clonal origin for ST258.
To assist the gathering and formatting of the utilized sequences a simple and free plug-in for Firefox has been developed (link Archived 2014-02-22 at the Wayback Machine).