Multilocus sequence typing (MLST)-Introduction, Principle, Test Requirements, Procedure, Result-Interpretation, Application, and Keynotes

Introduction

Multilocus Sequence Typing (MLST) is a molecular typing technique used in microbiology and genetics to classify and differentiate bacterial strains based on their genetic diversity. MLST is particularly valuable for studying the epidemiology and evolution of bacterial pathogens. Here is an introduction to the key concepts and principles of MLST:

  1. Genetic Diversity and Bacterial Typing: Bacteria within a single species can exhibit significant genetic diversity due to mutations and horizontal gene transfer. To understand this diversity and classify bacterial strains, scientists use typing methods like MLST.
  2. Loci Selection: MLST relies on the sequencing of specific housekeeping genes. These genes are typically essential for the bacterium’s survival and are relatively stable over time. The choice of loci varies depending on the bacterial species under investigation.
  3. Sequencing: DNA sequences of the selected housekeeping genes are determined for each bacterial isolate in the study. This involves amplifying the target genes using polymerase chain reaction (PCR) and then sequencing the amplified DNA.
  4. Allelic Profiles: Each unique sequence variant of a housekeeping gene is assigned an allele number. For example, if a specific allele of a gene is found, it might be designated as “allele 1,” and if a different variant is present in another strain, it might be designated as “allele 2.” These allele numbers create an allelic profile for each strain.
  5. Sequence Types (STs): To fully characterize a bacterial strain, its allelic profile is determined across all the chosen housekeeping genes. A unique combination of allele numbers defines a Sequence Type (ST). For example, a strain with allele numbers 1-2-3-4-5-6-7 might be identified as “ST1234567.”
  6. Databases: MLST data are often stored in publicly accessible databases maintained by research institutions or public health agencies. These databases help researchers compare strains from different sources and geographic locations, allowing for the tracking of outbreaks and the study of bacterial population genetics.
  7. Applications:
    • Epidemiology: MLST is widely used in outbreak investigations to determine the source and transmission of bacterial infections.
    • Phylogenetics: MLST data can be used to construct phylogenetic trees, providing insights into the evolutionary relationships among strains.
    • Vaccine Development: MLST helps identify prevalent strains and assess the effectiveness of vaccines against specific sequence types.
    • Antibiotic Resistance: It can be used to monitor the spread of antibiotic resistance genes among bacterial populations.
  8. Advantages:
    • Highly reproducible and portable method.
    • Provides standardized nomenclature for strains.
    • Allows for inter-laboratory comparisons.
    • Can be applied to a wide range of bacterial species.
  9. Limitations:
    • Requires specialized equipment for DNA sequencing.
    • May not provide fine-grained discrimination in highly clonal populations.
    • Limited to the selected loci and may miss important genomic variations.

Principle

The principle of Multilocus Sequence Typing (MLST) is based on analyzing the genetic diversity of bacteria by sequencing multiple housekeeping genes. MLST is a standardized and highly reproducible method used to classify and differentiate bacterial strains within a species. Here are the key principles of MLST:

  1. Selection of Housekeeping Genes: MLST begins with the careful selection of a set of housekeeping genes. Housekeeping genes are essential for the basic functions and survival of the bacterium. These genes tend to evolve relatively slowly and are less prone to horizontal gene transfer, making them ideal for phylogenetic analysis.
  2. DNA Amplification: For each bacterial isolate or strain being studied, the chosen housekeeping genes are amplified using the polymerase chain reaction (PCR). PCR primers specific to each target gene are used to selectively amplify DNA fragments from these loci.
  3. Sequencing: The PCR products are then subjected to DNA sequencing to determine the nucleotide sequence of each gene. Modern sequencing technologies, such as Sanger sequencing or next-generation sequencing, can be employed for this purpose.
  4. Allele Assignment: Once the sequences are obtained, each unique sequence variant of a housekeeping gene is assigned an allele number. For instance, if a specific sequence variant is found in one gene, it may be labeled as “allele 1,” and a different variant in the same gene would be “allele 2.”
  5. Sequence Type (ST) Determination: To fully characterize a bacterial strain, the allelic profiles of all the selected housekeeping genes are combined. A unique combination of allele numbers defines a Sequence Type (ST) for that strain. For example, if a strain has allele numbers 1-2-3-4-5-6-7 for the seven selected genes, it would be designated as “ST1234567.”
  6. Database Comparison: The STs generated from different bacterial isolates are compared and stored in a database. Publicly accessible MLST databases are maintained by research institutions, public health agencies, or collaborative networks. These databases enable researchers to compare strains from various sources and geographic locations.
  7. Analysis and Applications: The ST data can be used for various applications, including:
    • Epidemiology: Tracking the spread of bacterial strains during outbreaks and identifying potential sources of infection.
    • Phylogenetics: Constructing phylogenetic trees to understand the evolutionary relationships among strains.
    • Vaccine Development: Assessing the effectiveness of vaccines against specific sequence types.
    • Antibiotic Resistance: Investigating the distribution of antibiotic resistance genes among bacterial populations.
  8. Standardization: One of the key principles of MLST is standardization. The use of predetermined housekeeping genes, allele numbering, and publicly accessible databases ensures that MLST results are comparable between different laboratories and researchers.

Test Requirements

Performing Multilocus Sequence Typing (MLST) requires several key components and laboratory resources to ensure accurate and reliable results. Here are the main test requirements for MLST:

  1. Bacterial Isolates: MLST typically begins with the collection of bacterial isolates. These isolates should be pure cultures of the bacterial strains you wish to type. They can come from clinical samples, environmental samples, or any source of interest.
  2. Selection of Housekeeping Genes: Choose a set of housekeeping genes that are appropriate for the bacterial species you are studying. Housekeeping genes should be conserved across the species, play essential roles in the bacterium’s basic functions, and exhibit relatively low levels of genetic variation.
  3. PCR Primers: Design and procure specific PCR primers for each selected housekeeping gene. These primers are essential for amplifying the target DNA regions during the PCR step.
  4. PCR Equipment: You’ll need access to a PCR machine (thermocycler) to carry out the DNA amplification of the selected housekeeping genes.
  5. DNA Extraction Kit: Obtain a DNA extraction kit or reagents to extract genomic DNA from bacterial isolates. High-quality DNA extraction is crucial for obtaining clean and pure DNA samples for sequencing.
  6. Sequencing Equipment: You’ll require access to DNA sequencing equipment, such as Sanger sequencers or next-generation sequencing (NGS) platforms, depending on your laboratory’s capabilities and the scale of your MLST project.
  7. Sequencing Primers: Design and procure sequencing primers for each of the housekeeping genes to be sequenced. These primers should complement the PCR primers and enable sequencing of the target genes.
  8. Sequencing Reagents: Ensure you have the necessary sequencing reagents, including DNA polymerases, dNTPs (deoxynucleotide triphosphates), and fluorescent dye terminators for Sanger sequencing or appropriate chemistry for NGS.
  9. Data Analysis Software: Acquire bioinformatics software for the analysis of DNA sequence data. This software is used to align sequences, identify alleles, assign allele numbers, and determine Sequence Types (STs).
  10. MLST Databases: Access publicly available MLST databases that are specific to your bacterial species or genus. These databases are essential for comparing your STs to existing data and for epidemiological and phylogenetic analysis.
  11. Quality Control Standards: Implement quality control measures to ensure the accuracy and reliability of your MLST results. This may include running positive and negative controls in your PCR and sequencing reactions.
  12. Laboratory Personnel: Skilled laboratory personnel with expertise in molecular biology techniques, PCR, DNA sequencing, and bioinformatics are required to perform MLST experiments and data analysis.
  13. Biosafety Measures: Adhere to appropriate biosafety protocols when handling bacterial isolates to prevent contamination and ensure the safety of laboratory personnel.
  14. Documentation: Keep detailed records of sample information, primer sequences, PCR conditions, sequencing protocols, and data analysis procedures to maintain the traceability and reproducibility of your MLST results.
  15. Ethical Considerations: Ensure that the collection and use of bacterial isolates comply with ethical guidelines and any necessary approvals or permits.

Procedure

Multilocus Sequence Typing (MLST) is a standardized molecular typing technique used to characterize and classify bacterial strains based on the sequence variation in multiple housekeeping genes. Here is a general procedure for performing MLST:

1. Select Housekeeping Genes:

  • Choose a set of housekeeping genes that are suitable for your target bacterial species. These genes should be conserved across strains, play essential roles in the bacterium’s basic functions, and exhibit relatively low levels of genetic variation.

2. Isolate Bacterial DNA:

  • Obtain pure cultures of the bacterial strains you want to type.
  • Use a DNA extraction kit or laboratory protocols to extract genomic DNA from the bacterial isolates. Ensure the DNA extraction is of high quality and free from contaminants.

3. PCR Amplification:

  • Design specific PCR primer pairs for each of the selected housekeeping genes.
  • Set up separate PCR reactions for each gene, amplifying DNA fragments of the target genes. Each reaction should include template DNA, PCR primers, DNA polymerase, and reaction buffer.
  • Perform PCR amplification in a thermocycler, following the appropriate cycling conditions for each gene. Amplify all selected genes in parallel.

4. PCR Cleanup (Optional):

  • Purify the PCR products to remove excess primers and unincorporated nucleotides if necessary. You can use a PCR cleanup kit or enzymatic methods for this step.

5. DNA Sequencing:

  • Perform DNA sequencing for each PCR product separately. You may use Sanger sequencing or next-generation sequencing (NGS) platforms, depending on your laboratory’s capabilities and the scale of your project.
  • Use sequencing primers specific to each gene for sequencing reactions.

6. Sequence Data Analysis:

  • Analyze the obtained sequence data using bioinformatics software. This includes:
    • Sequence alignment: Align the sequences of each gene to their respective reference sequences.
    • Allele identification: Determine the allele numbers for each gene based on the sequence variations.
    • Sequence Type (ST) assignment: Combine the allele numbers for all genes to define the ST for each bacterial isolate.

7. Database Comparison:

  • Compare the STs of your bacterial isolates to publicly available MLST databases specific to your bacterial species or genus. This step helps identify previously known and new sequence types.

8. Data Interpretation:

  • Interpret the results based on the STs and genetic diversity observed. You can construct phylogenetic trees, calculate genetic distances, and assess relationships between strains.

9. Reporting and Documentation:

  • Document all experimental details, including primer sequences, PCR conditions, sequencing protocols, and results.
  • Report the STs and any additional relevant genetic information for each bacterial isolate.

10. Data Sharing:

  • If applicable, submit your MLST data to relevant MLST databases to contribute to the global knowledge of bacterial strain diversity.

11. Quality Control:

  • Implement quality control measures to ensure the accuracy and reproducibility of your MLST results. This includes using positive and negative controls and performing repeat sequencing when necessary.

12. Interpretation and Application:

  • Use the MLST data for various applications, such as epidemiological studies, population genetics research, and understanding the evolutionary history of bacterial strains.

Result-Interpretation

Interpreting the results of Multilocus Sequence Typing (MLST) involves analyzing the allelic profiles and Sequence Types (STs) generated for each bacterial strain. The interpretation of MLST results can provide insights into the genetic diversity, relatedness, and epidemiology of bacterial populations. Here’s how to interpret MLST results:

  1. Allelic Profiles:
    • The allelic profile of a bacterial strain consists of a series of allele numbers corresponding to the chosen housekeeping genes.
    • For example, an allelic profile might look like this: 1-2-3-4-5-6-7, where each number represents the allele found at a specific locus.
  2. Sequence Type (ST):
    • The ST is a unique identifier for each bacterial strain based on its allelic profile.
    • For example, if the allelic profile is 1-2-3-4-5-6-7, it corresponds to a specific ST, such as ST1234567.
    • Each ST is specific to a particular bacterial isolate and represents its genetic fingerprint within the MLST database.
  3. Comparative Analysis:
    • Compare the STs of different bacterial strains within your dataset or against existing data in MLST databases.
    • Similar STs suggest a closer genetic relationship, while distinct STs indicate genetic diversity.
  4. Phylogenetic Analysis:
    • Construct phylogenetic trees using the STs and genetic distances between them. This can reveal evolutionary relationships among strains.
    • Clusters of closely related STs may indicate recent common ancestry.
  5. Epidemiological Insights:
    • MLST results can be used in epidemiological investigations to trace the source of an outbreak or track the spread of specific bacterial strains.
    • Identical or highly similar STs among isolates from different geographic locations or sources may suggest a common origin or transmission route.
  6. Population Structure:
    • MLST can help identify the population structure of a bacterial species.
    • Clonal complexes (CCs) may be defined, consisting of closely related STs that share several alleles. CCs represent groups of strains with recent common ancestry.
  7. Diversity and Evolution:
    • High diversity in STs and alleles may indicate a diverse bacterial population with multiple sources of genetic variation.
    • Low diversity may suggest a clonal population with limited genetic diversity.
  8. Vaccine and Antibiotic Resistance:
    • MLST can be used to assess the distribution of specific STs associated with vaccine targets or antibiotic resistance genes.
    • It aids in monitoring the effectiveness of vaccines and tracking the spread of antibiotic resistance.
  9. Data Sharing:
    • Consider sharing your MLST data with relevant databases to contribute to the global knowledge of bacterial strain diversity.
  10. Documentation:
  • Ensure that all MLST results, including allelic profiles, STs, and any additional genetic information, are properly documented for future reference and analysis.

Application

Multilocus Sequence Typing (MLST) is a powerful molecular tool with a wide range of applications in microbiology and epidemiology. It is commonly used to characterize and differentiate bacterial strains based on genetic diversity. Here are some key applications of MLST:

  1. Epidemiological Surveillance:
    • MLST is extensively used in epidemiological studies to track the spread of infectious diseases and identify sources of outbreaks.
    • It helps determine whether cases of infection are linked to a common source or if they are caused by genetically distinct strains.
  2. Outbreak Investigation:
    • MLST aids in identifying the source of an outbreak by comparing the genetic profiles (Sequence Types or STs) of bacterial isolates.
    • It can be crucial in public health responses to foodborne, nosocomial, or community-acquired outbreaks.
  3. Phylogenetic Analysis:
    • MLST data can be used to construct phylogenetic trees, revealing the evolutionary relationships between different bacterial strains.
    • It helps in understanding the evolutionary history and relatedness of bacterial populations.
  4. Population Genetics:
    • MLST allows for the analysis of population structure within a bacterial species.
    • It helps researchers identify clonal complexes (CCs) and assess the genetic diversity within a species.
  5. Vaccine Development:
    • MLST can assist in vaccine development by identifying prevalent strains and determining whether vaccine candidates cover a wide range of Sequence Types.
    • It helps monitor vaccine effectiveness by tracking changes in bacterial populations over time.
  6. Antibiotic Resistance Surveillance:
    • MLST can be used to study the distribution of antibiotic resistance genes and their association with specific STs.
    • It aids in understanding the emergence and spread of antibiotic-resistant strains.
  7. Source Attribution:
    • MLST can be applied in source attribution studies to identify potential reservoirs or sources of bacterial infections.
    • It helps determine whether bacteria are of human, animal, or environmental origin.
  8. Clinical Microbiology:
    • In clinical settings, MLST can assist in identifying specific bacterial strains responsible for infections.
    • It can be used to track the persistence of particular strains within healthcare facilities.
  9. Public Health Surveillance:
    • MLST data contribute to public health surveillance efforts, providing valuable information for disease control and prevention strategies.
  10. Research on Emerging Diseases:
    • MLST is used in the study of emerging and re-emerging infectious diseases to understand the genetic diversity and evolution of newly identified pathogens.
  11. Bacterial Taxonomy and Species Identification:
    • MLST can be used to delineate species boundaries and refine bacterial taxonomy by examining the genetic relationships among strains.
  12. Pharmaceutical and Biotechnology Applications:
    • In the pharmaceutical and biotechnology industries, MLST can be used to monitor and ensure the quality and purity of bacterial strains used in bioprocessing and fermentation.
  13. Environmental Microbiology:
    • MLST can help identify and characterize bacterial populations in environmental samples, such as water, soil, and air, for ecological and environmental studies.

Keynotes

Multilocus Sequence Typing (MLST) is a valuable molecular technique used for characterizing and differentiating bacterial strains based on the sequence variation in multiple housekeeping genes. Here are some keynotes and important points to remember about MLST:

  1. Standardization: MLST is highly standardized, with predefined sets of housekeeping genes and allele numbering systems for specific bacterial species or genera. This standardization allows for consistent and comparable results between laboratories.
  2. Housekeeping Genes: MLST focuses on housekeeping genes, which are typically essential for bacterial survival and are less prone to horizontal gene transfer. These genes provide a stable genetic basis for strain typing.
  3. Allele Numbers: Each unique sequence variant of a housekeeping gene is assigned an allele number. The combination of allele numbers across multiple genes defines a Sequence Type (ST) for a bacterial strain.
  4. Sequence Types (STs): STs are unique identifiers for bacterial strains and can be used for strain classification and comparison. Identical STs indicate closely related strains.
  5. Databases: Publicly accessible MLST databases are maintained by research institutions and public health agencies. Researchers can compare their STs to these databases to identify known strains and contribute new data.
  6. Epidemiological Tool: MLST is a powerful tool for epidemiological surveillance, outbreak investigations, and source attribution. It helps track the spread of bacterial infections and identify sources of outbreaks.
  7. Phylogenetic Analysis: MLST data can be used to construct phylogenetic trees, allowing researchers to study the evolutionary relationships among bacterial strains.
  8. Population Genetics: MLST facilitates the study of population genetics within a bacterial species, including the identification of clonal complexes (CCs) and genetic diversity.
  9. Applications: MLST has diverse applications, including epidemiology, vaccine development, antibiotic resistance monitoring, clinical microbiology, and environmental microbiology.
  10. Quality Control: Quality control measures are essential to ensure the accuracy and reproducibility of MLST results. This includes using positive and negative controls in PCR and sequencing reactions.
  11. Data Sharing: Contributing MLST data to public databases enhances the global understanding of bacterial diversity and evolution. Data sharing is encouraged in the scientific community.
  12. Biosafety: Adherence to biosafety protocols is crucial when handling bacterial isolates to prevent contamination and ensure the safety of laboratory personnel.
  13. Interpretation: Interpreting MLST results involves comparing STs, constructing phylogenetic trees, and considering the genetic relatedness and diversity of bacterial strains.
  14. Evolutionary Insights: MLST provides insights into the evolutionary history of bacterial populations and can be used to study the emergence and spread of new strains.
  15. Future Relevance: MLST remains relevant as new bacterial species and strains are characterized, and it continues to evolve with advances in sequencing technologies and bioinformatics.

Further Readings

  1. MLST Databases and Resources:
    • PubMLST: A comprehensive repository of MLST data for various bacterial species, along with analysis tools and guidance.
    • MLST.net: MLST resources, databases, and links to software and publications.
  2. Books:
    • “Multilocus Sequence Typing” by Keith A. Jolley and Martin C. J. Maiden: This book provides an in-depth exploration of the principles, techniques, and applications of MLST.
    • “Molecular Epidemiology: Applications in Epidemiology and Microbiology” by Mark Achtman: Includes a chapter on MLST and its use in molecular epidemiology.
  3. Review Articles:
    • “Multilocus Sequence Typing: A Tool for Global Epidemiology” by Martin C. J. Maiden et al. in Trends in Microbiology (2013).
    • “Multilocus Sequence Typing (MLST) for Characterization of Enteric Pathogens” by P. P. Evers et al. in Journal of Clinical Microbiology (2015).
  4. Scientific Journals:
    • Explore scientific journals related to microbiology, epidemiology, and genomics, as they often feature research articles and studies that utilize MLST.
  5. Online Courses and Tutorials:
    • Look for online courses and tutorials on genomics, microbiology, and molecular epidemiology that cover MLST as a topic.
  6. University Research Websites:
    • University websites often host research publications and resources related to MLST, particularly in the microbiology and molecular biology departments.
  7. Professional Conferences:
    • Attend microbiology or epidemiology conferences and workshops where experts may present their research on MLST and related topics.
  8. PubMed:
    • Use PubMed, a database of scientific articles, to search for recent research papers, reviews, and studies related to MLST in various bacterial species.
  9. Collaborate with Experts:
    • If you have specific questions or research interests in MLST, consider reaching out to experts in the field for guidance and collaboration.
  10. Bioinformatics Tools:
    • Explore bioinformatics tools and software used for MLST analysis. Many of these tools come with documentation and resources that can enhance your understanding.

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