Multilocus Sequence Typing (MLST)

      • Multilocus Sequence Typing (MLST) is a molecular typing method used for the characterization and classification of bacterial strains based on the nucleotide sequences of multiple housekeeping genes [1]. MLST has become a widely adopted technique in microbiology and epidemiology due to its high discriminatory power, reproducibility, and portability across different laboratories and bacterial species [2]. Here's a detailed overview of MLST:

      • Principle :

        MLST relies on the analysis of the DNA sequences of several housekeeping genes, typically 5 to 7 loci, which are conserved among bacterial species [3].


        Housekeeping genes are those involved in fundamental cellular functions and are less likely to undergo horizontal gene transfer or recombination compared to virulence or antibiotic resistance genes [4].


        By sequencing multiple housekeeping genes from bacterial isolates and comparing the sequences, MLST assigns a unique allelic profile or sequence type (ST) to each strain, allowing for the differentiation and classification of bacterial strains [1].


        Workflow [5]:


        Isolation and Culturing: Bacterial isolates are obtained from clinical specimens, environmental samples, or culture collections and cultured under appropriate conditions.


        PCR Amplification: DNA is extracted from bacterial isolates, and specific regions of the housekeeping genes of interest are amplified using polymerase chain reaction (PCR) with gene-specific primers.


        DNA Sequencing: The PCR products are sequenced using Sanger sequencing or next-generation sequencing (NGS) platforms to determine the nucleotide sequences of the target genes.


        Allele Assignment: The obtained sequences are compared to reference sequences in curated MLST databases, and allelic profiles or STs are assigned based on the sequence matches [1,6].


        Data Analysis: MLST data can be analyzed using various bioinformatics tools and software to assess genetic relatedness, population structure, and phylogenetic relationships among bacterial strains [1].


        MLST Databases [6]:  MLST databases, such as PubMLST, provide curated collections of MLST profiles for different bacterial species, along with tools for allele calling, sequence analysis, and data visualization.


        These databases facilitate the standardization and sharing of MLST data among researchers and public health agencies, enabling global surveillance of bacterial pathogens and outbreak investigations.


      • An illustration for MLTS of an unknown bacteria for a given strain 1 is cited below [11-19]:

      • Fig. MLST of an unknown bacteria and the results
        Fig. 1 MLST of an unknown sample later recognized as Escherichia coli ST type 678 by MLST (figure edited in Biorender.com)

Applications:

Epidemiological Surveillance: MLST is widely used for monitoring the spread and transmission dynamics of bacterial pathogens, including hospital-acquired infections, community outbreaks, and foodborne illnesses [7].


Population Genetics: MLST data can be used to study the genetic diversity, population structure, and evolutionary relationships of bacterial populations, providing insights into microbial evolution and adaptation [8].


Vaccine Development: MLST can inform vaccine development efforts by identifying genetically diverse strains or hypervirulent lineages that may need to be targeted by vaccines [9].


Antimicrobial Resistance Monitoring: MLST data can be correlated with antimicrobial susceptibility profiles to investigate the emergence and dissemination of antibiotic-resistant bacterial strains, for instance, analysis of resistance  caused due to Escherichia coli in China [10].


Overall, MLST is a powerful and versatile tool for bacterial typing and molecular epidemiology, contributing to our understanding of bacterial pathogenesis, transmission dynamics, and population biology. Its widespread adoption has facilitated collaborative research efforts and enhanced surveillance capabilities for bacterial diseases worldwide.

References:

  1. https://doi.org/10.1128/JCM.06094-11
  2. https://doi.org/10.1016 B978-0-12-378612-8.00117-7
  3. https://pubmlst.org/multilocus-sequence-typing
  4. https://doi.org/10.1371/journal.pcbi.1010295
  5. https://assets.thermofisher.com/TFS-Assets/LSG/Application-Notes/cms_042189.pdf
  6. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6192448/
  7. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3988353/
  8. 8. Pérez-Losada, M., Browne, E. B., Madsen, A., Wirth, T., Viscidi, R. P., & Crandall, K. A. (2006). Population genetics of microbial pathogens estimated from multilocus sequence typing (MLST) data. Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases, 6(2), 97–112. https://doi.org/10.1016/j.meegid.2005.02.003
  9. https://doi.org/10.1080/21645515.2017.1412020
  10. https://doi.org/10.3389/fpubh.2021.780700
  11. Multilocus Sequence Typing of Total Genome Sequenced Bacteria.
    Larsen MV, Cosentino S, Rasmussen S, Friis C, Hasman H, Marvig RL, Jelsbak L, Sicheritz-Pontén T, Ussery DW, Aarestrup FM and Lund O.
    J. Clin. Micobiol. 2012. 50(4): 1355-1361.
    PMID: 22238442         doi: 10.1128/JCM.06094-11
  12.  Larsen, M., Cosentino, S., Rasmussen, S., Rundsten, C., Hasman, H., Marvig, R., Jelsbak, L., Sicheritz-Pontén, T., Ussery, D., Aarestrup, F., & Lund, O. (2012). Multilocus Sequence Typing of Total Genome Sequenced Bacteria. Journal of Clinical Microbiology, 50(4), 1355-1361.
  13. Clausen, P., Aarestrup, F., & Lund, O. (2018). Rapid and precise alignment of raw reads against redundant databases with KMA. Bmc Bioinformatics,19(1), 307
  14. Bartual, S., Seifert, H., Hippler, C., Luzon, M., Wisplinghoff, H., & Rodríguez-Valera, F. (2005). Development of a multilocus sequence typing scheme for characterization of clinical isolates of Acinetobacter baumannii. Journal of Clinical Microbiology, 43(9), 4382-4390.
  15. Griffiths, D., Fawley, W., Kachrimanidou, M., Bowden, R., Crook, D., Fung, R., Golubchik, T., Harding, R., Jeffery, K., Jolley, K., Kirton, R., Peto, T., Rees, G., Stoesser, N., Vaughan, A., Walker, A., Young, B., Wilcox, M., & Dingle, K. (2010). Multilocus sequence typing of Clostridium difficile. Journal of Clinical Microbiology, 48(3), 770-778.
  16. Lemee, L., Dhalluin, A., Pestel-Caron, M., Lemeland, J., & Pons, J. (2004). Multilocus sequence typing analysis of human and animal Clostridium difficile isolates of various toxigenic types. Journal of Clinical Microbiology, 42(6), 2609-2617.
  17. Wirth, T., Falush, D., Lan, R., Colles, F., Mensa, P., Wieler, L., Karch, H., Reeves, P., Maiden, M., Ochman, H., & Achtman, M. (2006). Sex and virulence in Escherichia coli: An evolutionary perspective. Molecular Microbiology, 60(5), 1136-1151.
  18. Jaureguy, F., Landraud, L., Passet, V., Diancourt, L., Frapy, E., Guigon, G., Carbonnelle, E., Lortholary, O., Clermont, O., Denamur, E., Picard, B., Nassif, X., & Brisse, S. (2008). Phylogenetic and genomic diversity of human bacteremic Escherichia coli strains. Bmc Genomics, 9(1), 560.
  19. Camacho, C., Coulouris, G., Avagyan, V., Ma, N., Papadopoulos, J., Bealer, K., & Madden, T. (2009). BLAST+: Architecture and applications. Bmc Bioinformatics, 10(1), 421.






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