Multi-Locus Sequence Typing (MLST)

MLST targets 6-8 conserved housekeeping genes (typically 400-500 bp internal fragments) that are under stabilizing selection for metabolic function. These loci evolve slowly via neutral mutation accumulation, providing a stable phylogenetic signal. The specific gene set varies by species—e.g., Staphylococcus aureus uses 7 loci (arcC, aroE, glpF, gmk, pta, tpi, yqiL), while Escherichia coli uses a different set (adk, fumC, gyrB, icd, mdh, purA, recA).

For each locus, every unique nucleotide sequence is assigned a distinct allele number by a central curator. The ordered combination of allele numbers across all loci forms the allelic profile, which is assigned a unique Sequence Type (ST). Identical STs indicate clonal relatedness, while single-locus variants (SLVs) suggest recent evolutionary divergence. This digital, integer-based nomenclature enables unambiguous global comparison.

eBURST (Based Upon Related Sequence Types) is a clustering algorithm that groups related STs into clonal complexes (CCs). The algorithm identifies a founder genotype—the ST with the most single-locus variants—and links all STs that share alleles at 6 of 7 loci. This reveals the population structure and evolutionary descent of bacterial lineages, distinguishing epidemic clones from sporadic isolates.

MLST relies on curated, publicly accessible databases (e.g., PubMLST.org, EnteroBase) that maintain allele sequences, ST profiles, and isolate metadata. Each new allele is verified by a curator before assignment, ensuring nomenclature stability. These databases enable real-time global surveillance—an isolate sequenced in one country can be instantly compared against thousands of historical records to identify outbreak clusters.

An extension of traditional 7-gene MLST, wgMLST expands the scheme to include hundreds or thousands of loci across the core genome. This provides strain-level resolution far beyond classical MLST, enabling fine-grained outbreak tracing. Tools like chewBBACA and EnteroBase's cgMLST define species-specific core-genome schemes, while whole-genome approaches capture accessory genome variation for maximum discriminatory power.

MLST is the gold standard for global molecular epidemiology. It enables:

• Outbreak detection: Identifying clusters of identical STs across time and geography
• Source tracking: Tracing foodborne pathogens back to reservoirs
• Population genetics: Inferring recombination rates and selection pressures
• Antimicrobial resistance monitoring: Associating resistance phenotypes with specific clonal lineages The portability of ST designations makes MLST indispensable for cross-jurisdictional public health collaboration.

A high-resolution alternative to MLST that sequences the entire bacterial genome rather than a subset of housekeeping genes. WGS enables core genome MLST (cgMLST) , which expands the analysis from 7 loci to thousands, providing superior discriminatory power for outbreak investigations. While MLST assigns a sequence type (ST), WGS can distinguish between nearly identical isolates within the same ST, revealing single nucleotide polymorphism (SNP) differences critical for transmission chain reconstruction.

A legacy molecular typing method that preceded MLST as the gold standard for bacterial subtyping. PFGE separates large DNA fragments generated by restriction enzyme digestion using an alternating electric field, producing a unique banding pattern or 'fingerprint.' Unlike MLST, which provides unambiguous, portable sequence data, PFGE patterns are analog and difficult to compare between laboratories, leading to its gradual replacement by sequence-based methods in public health networks like PulseNet.

A computational method for inferring clonal complexes (CCs) and evolutionary relationships from MLST data. eBURST groups sequence types that share a defined number of identical alleles, typically 6 out of 7 loci, into a single clonal complex. The algorithm identifies the predicted founder ST — the genotype with the highest number of single-locus variants — and constructs a radial spanning tree that visualizes the microevolutionary descent of isolates within a bacterial population.

The foundational design principle of MLST schemes, requiring the identification of 7 core loci that are:

• Ubiquitous: Present in all strains of the species
• Conserved: Under stabilizing selection for essential metabolic functions
• Diverse: Contain sufficient neutral variation to discriminate between strains Typical targets include genes like aroE, dnaN, and recA, which encode proteins for shikimate metabolism, DNA replication, and homologous recombination, respectively. The ~450-500 bp internal fragment sequenced for each gene captures maximal variation while remaining amplifiable with universal primers.

An extension of traditional MLST that indexes 53 genes encoding ribosomal protein subunits instead of housekeeping metabolic enzymes. rMLST provides a universal, domain-level typing framework applicable across the entire bacterial kingdom because ribosomal proteins are universally conserved single-copy genes. This approach bridges the resolution gap between 16S rRNA gene phylogeny and species-specific MLST schemes, enabling cross-genera comparisons and metagenomic strain tracking directly from shotgun sequencing data.