MLST

Multilocus sequence typing (MLST) is a technique in molecular biology for the typing of multiple loci. The procedure characterizes isolates of microbial species using the DNA sequences of internal fragments of multiple housekeeping genes. Approximately 450-500 bp internal fragments of each gene are used, as these can be accurately sequenced on both strands using an automated DNA sequencer. For each housekeeping gene, the different sequences present within a bacterial species are assigned as distinct alleles and, for each isolate, the alleles at each of the loci define the allelic profile or sequence type (ST).

The first MLST scheme to be developed was for Neisseria meningitidis, the causative agent of meningococcal meningitis and septicaemia. Since its introduction for the research of evolutionary history, MLST has been used not only for human pathogens but also for plant pathogens.

MLST directly measures the DNA sequence variations in a set of housekeeping genes and characterizes strains by their unique allelic profiles. The principle of MLST is simple: the technique involves PCR amplification followed by DNA sequencing. Nucleotide differences between strains can be checked at a variable number of genes depending on the degree of discrimination desired.

The workflow of MLST involves: 1) data collection, 2) data analysis and 3) multilocus sequence analysis. In the data collection step, definitive identification of variation is obtained by nucleotide sequence determination of gene fragments. In the data analysis step, all unique sequences are assigned allele numbers and combined into an allelic profile and assigned a sequence type (ST). If new alleles and STs are found, they are stored in the database after verification. In the final analysis step of MLST, the relatedness of isolates are made by comparing allelic profiles. Researchers do epidemiological and phylogenetical studies by comparing STs of different clonal complexes. A huge set of data is produced during the sequencing and identification process so bioinformatic techniques are used to arrange, manage, analyze and merge all of the biological data.

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. Quoting Staphylococcus aureus as an example, seven housekeeping genes are used in MLST typing. These genes include carbamate kinase (arcC), shikimate dehydrogenase (aroE), glycerol kinase (glpF), guanylate kinase (gmk), phosphate acetyltransferase (pta), triosephosphate isomerase (tpi) and acetyl coenzyme A acetyltransferase (yqiL) as specified by the MLST website. However, it is not uncommon for up to ten housekeeping genes to be used. 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 dehyrogenase (dtdS), diaminopimelate decarboxylase (lysA), transhydrogenase alpha subunit (pntA), dihydroorotase (pyrC) and tryptophanase (tnaA). Thus both the number and type of housekeeping genes interrogated by MLST may differ from species to species.

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