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Multi-Locus Sequence Typing

Multi-Locus Sequence Typing

Overview

CD Genomics offers a precise Multi-Locus Sequence Typing (MLST) service that utilizes both Sanger and next-generation sequencing technologies to deliver detailed bacterial strain differentiation. By sequencing multiple housekeeping genes, we generate unique allele profiles that enable accurate strain identification and in-depth evolutionary analysis. Our service provides high-resolution, reproducible data and is backed by extensive experience in handling various samples.

Our Advantages:

Custom MLST typing schemes designed for different strains.
Comprehensive Microbial Identification Services.
High-quality Gel Electrophoresis Images.
High-resolution and Highly Reproducible Data.
Extensive Experience in Handling Various Samples.

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What is the MLST Approach

MLST is a sophisticated molecular technique essential for the accurate identification and differentiation of bacterial strains. It involves the sequencing of internal fragments from multiple housekeeping genes—commonly seven—each spanning 400-600 base pairs. These housekeeping genes are crucial for fundamental cellular functions and exhibit minimal genetic variation within a species. Using primers specific to these genes, MLST performs PCR amplification and sequencing to determine allele numbers at designated loci. This process generates unique allele profiles or sequence types (STs) for each strain. By analyzing these profiles through methods like pairwise difference matrices and constructing phylogenetic trees, MLST enables a detailed understanding of genetic relationships and evolutionary trajectories among bacterial strains.

Key Features of MLST

Standardized Framework: MLST uses a structured method to sequence housekeeping genes, assigning unique identifiers to alleles for consistent and comparable bacterial typing.
Sequence-Based Typing: It relies on nucleotide sequences rather than fragment lengths, offering precise and reproducible strain identification.
Allelic Profiles: The Sequence Type (ST) represents a strain's allelic profile across multiple loci, facilitating evolutionary and epidemiological analyses.

Why is MLST Used

The utilization of MLST provides several essential benefits:

High Resolution and Precision: MLST provides detailed strain differentiation, essential for pinpointing outbreaks and mapping bacterial evolution with fine genetic distinctions.
Reproducibility: By adhering to uniform protocols and leveraging centralized databases, MLST delivers reliable and comparable results across various research settings.
Application Flexibility: MLST's adaptability to numerous bacterial species makes it a valuable tool for diverse research applications, from clinical diagnostics to environmental studies.

Differences Between MLST and MLSA

While both MLST and Multi-Locus Sequence Analysis (MLSA) revolve around the examination of multiple gene loci, their objectives and applications diverge significantly:

Purpose and Scope:

MLST: This method is principally employed for the typing and differentiation of bacterial strains within a species. By analyzing sequence variations in predefined housekeeping genes, MLST can accurately distinguish between different strains.
MLSA: Conversely, MLSA is predominantly utilized for constructing phylogenetic relationships and examining evolutionary lineages among diverse species or higher taxonomic groups.

Gene Selection:

MLST: Typically focuses on a fixed set of housekeeping genes, which are conserved and essential across various strains, serving fundamental cellular functions.
MLSA: Encompasses a more extensive array of genes, which may include both highly conserved and more variable genes, depending on the phylogenetic questions being addressed.

Application:

MLST: Predominantly applied in epidemiological studies, including outbreak investigations and detailed strain differentiation. Its high resolution makes it an invaluable tool for understanding the transmission and spread of bacterial pathogens.
MLSA: More suited for in-depth phylogenetic studies, MLSA facilitates the reconstruction of evolutionary histories and the mapping of genetic relationships among broader taxonomic categories.

Applications of MLST

The applications of MLST include, but are not limited to, the following areas:

Epidemiology: Tracking and monitoring bacterial outbreaks, identifying sources of infections, and assessing the spread of pathogenic strains.
Microbial Ecology: Studying the distribution and diversity of microbial populations in environmental samples.
Pharmacology: Investigating resistance patterns and susceptibility to antibiotics among different bacterial strains.

Service Specifications

Introduction to Our MLST Service

At CD Genomics, our MLST service leverages both first-generation (Sanger) and NGS technologies. While Sanger sequencing focuses on amplifying and sequencing specific housekeeping genes for strain differentiation, NGS advances this approach by sequencing the entire bacterial genome. This shift to NGS not only enhances the precision of MLST but also expands its capabilities, allowing for additional analyses such as virulence and resistance profiling, thereby providing a comprehensive view of genetic variations and improving strain identification and evolutionary studies.

MLST Workflow

The Workflow of Multi-Locus Sequence Typing.

Bioinformatics Analysis

Identify polymorphic sites
Population Structure Analysis
Correlation Analysis
Allelic Diversity Analysis
Phylogenetic Analysis
Recombination Analysis
Functional Analysis (e.g., Virulence, Antibiotic Resistance)

Note: The above content includes only a portion of the bioinformatics analysis. For more information or to customize the analysis, please contact us directly.

The Bioinformatics Analysis of Multi-Locus Sequence Typing.

Sample Requirement

Microbial genomic DNA: total amount > 500 ng, concentration > 10 ng/μl.
1.8 < OD260/280 < 2.0, no degradation or contamination.
Sampling kits: We provide a range of microbial sampling kits for clients, including MicroCollect™ oral sample microbial collection products and MicroCollect™ stool sample collection products.

Note: If you wish to obtain more accurate and detailed information regarding sample requirements, please feel free to contact us directly.

Deliverables

The experimental report includes information on experimental methods
Primers
PCR conditions
Gel electropherogram
MLST allele and sequence type (st)
Phylogenetic tree, etc

Demo

Partial results of our SCFA analysis service are shown below:

The Multi-Locus Sequence Typing Results Display Figure.

FAQs

Multi-Locus Sequence Typing FAQ

1. How does MLST compare to other typing methods?
- MLST surpasses traditional methods like pulse-field gel electrophoresis (PFGE) and ribotyping in terms of resolution and reproducibility. MLST employs a standardized, sequence-based approach, thereby minimizing variability across different laboratories and enhancing the consistency of results.
2. Can MLST be used for non-bacterial organisms?
- Though MLST is predominantly employed for bacterial typing, its underlying principles are versatile and can be adapted for other microorganisms, including fungi and certain protozoa. The critical factor is the selection of appropriate housekeeping genes that are representative of the target organism.
3. What are the limitations of MLST?
- Despite its strengths, MLST may be less effective for highly diverse or rapidly evolving species where significant variation exists across multiple loci. Furthermore, the utility of MLST relies heavily on the availability of comprehensive and well-maintained reference databases for accurate allele identification.
4. How often should MLST databases be updated?
- Frequent updates of MLST databases are imperative to integrate new allele types and maintain the accuracy of strain identification. Generally, the responsibility of updating these databases falls to research communities and centralized repositories, ensuring that the databases remain current and reliable.

Case Study

Whole-Genome Sequencing-Based Profiling of Antimicrobial Resistance Genes and Core-Genome Multilocus Sequence Typing of Campylobacter jejuni from Different Sources in Lithuania
Journal: International Journal of Molecular Sciences
Impact factor: 5.6
Published: 6 November 2023

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Background

Campylobacter jejuni causes acute gastroenteritis, typically from undercooked poultry or contaminated water. Severe cases might require antibiotics. AMR, driven by gene mutations and horizontal gene transfer, is a rising concern. Whole-genome sequencing (WGS) accurately identifies AMR genes and mechanisms, surpassing traditional methods. This study uses WGS to evaluate AMR and genetic diversity in C. jejuni from multiple sources.

Materials & Methods

Sample preparation:

Campylobacter jejuni
DNA extraction

Method:

Whole-Genome Sequencing
MLST

Data Analysis:

Quality control
Assembly
Annotation
Phylogenomic analysis
Resistance gene screening

Results

The genomic analysis of C. jejuni strains revealed genome sizes ranging from 1.6 to 1.86 Mb with an average G+C content of 30.32%. The pan-genome analysis identified 4204 genes, including 1215 core and 2989 accessory genes. Three clusters of C. jejuni strains were identified based on cgMLST, showing varying resistance profiles. Notably, ST-5 was predominant. WGS identified 26 AMR genes, with specific point mutations associated with resistance. Virulence genes related to motility, adherence, and stress response were found across isolates, with significant differences in gene profiles among strains from different sources.

Figure 1. Minimum spanning tree of C. jejuni isolates generated.

Figure 2. Interactive tree of life (iTOL) of 53 C. jejuni isolates based on the presence and absence of gene point mutations and sequence cluster classification.

Conclusions

Combining WGS and cgMLST techniques revealed the spread of antimicrobial-resistant C. jejuni clones and potential transmission routes between humans and animals. The analysis showed that gene mutations linked to resistance are accumulating, indicating long-term selective pressure and evolving resistance mechanisms.

References

Maiden M C J, et al. Multilocus sequence typing: a portable approach to the identification of clones within populations of pathogenic microorganisms. Proceedings of the National Academy of Sciences, 1998, 95(6): 3140-3145.
Boonsilp S, et al. A single multilocus sequence typing (MLST) scheme for seven pathogenic Leptospira species. PLoS neglected tropical diseases, 2013, 7(1): e1954.
Aksomaitiene J, Novoslavskij A, Malakauskas M. Whole-Genome Sequencing-Based Profiling of Antimicrobial Resistance Genes and Core-Genome Multilocus Sequence Typing of Campylobacter jejuni from Different Sources in Lithuania. International Journal of Molecular Sciences, 2023, 24(21): 16017.