Commentary: Next-Generation Epidemiology: Using Real-Time Core Genome Multilocus Sequence Typing To Support Infection Control Policy

Abstract

M ultilocus sequencing typing (MLST), introduced by Maiden and colleagues in 1998, has come to represent one of the most widely used and successful molecular methods for strainlevel classification of bacterial isolates (1). MLST schemes work by indexing sequence variation in a small set of housekeeping genes to a numerical classification system. Each novel sequence occurring at any of the selected loci is named as a unique numbered allele, and the profile of a given isolate is specified by the numbers representing the allelic composition. Each combination of alleles is assigned a unique sequence type (ST), and STs, in turn, can be grouped into higher levels of classification (2, 3). The ability of MLST to resolve closely related strains is limited in principle by the absolute sequence diversity present in the loci covered by the scheme and by the manner in which this sequence diversity distributes across the population to be classified. As MLST schemes require conserved PCR primer binding sites, the targets must also be sufficiently conserved so that the method works robustly, and this puts limits on the resolution of classical MLST (3). MLST schemes have now been developed for Ͼ100 genera and species, and continuously updated databases are maintained by the University of Oxford, United Kingdom (pubmlst.org), Imperial College London, London, United Kingdom (http://www.mlst.net/), and others.Classical MLST analysis, developed to be compatible with Sanger sequencing methods, is based on 400-to-500-nucleotide segments of (usually) seven genes, encompassing Ͻ0.2% of the bacterial genome in many cases (3). Advances in whole-genome sequencing (WGS) and the availability of a large number of assembled bacterial genomes have made possible new approaches to strain-and clone-level epidemiologic tracking of isolates, including the ability to apply MLST schemes on a genome-wide scale. Inference of clonal relationships from the comparison of wholegenome sequences for the purpose of epidemiologic investigations can involve a bit more subtlety than classical MLST analysis, however, and there are abundant opportunities for drawing incorrect conclusions. Perhaps the simplest of the many available approaches is that of direct-alignment-based (single nucleotide polymorphism [SNP]-based) phylogenetic analysis, often built on the underlying statistical assumptions of independent individual substitution events. Direct-alignment-based phylogenetic analyses have been used to study the population structures of bacterial outbreaks and to construct transmission maps in a variety of epidemiologic investigations (4-6). However, single recombination events, mobile genetic element insertions, or other horizontal genetic transfer events can result in multiple SNPs or more-complex rearrangements over a contiguously exchanged or interrupted region. Consequently, genome-wide direct-alignment methods, in addition to being computationally expensive, may result in the generation of incorrect tree topologies due to such rearrangements (7). Alternative...

Drug repurposing screens and synergistic drug‐combinations for infectious diseases

“…Improvement of the current antimicrobial susceptibility testing methods or invention of new methods is needed to meet the challenge of drug‐resistant bacteria. Recent advances include the use of matrix‐assisted laser desorption/ionization time of flight MS and next generation sequencing that enables rapid identification of proteins and plasmids of clinically relevant multidrug‐resistant bacteria in a real time and high‐throughput fashion (Conlan et al , 2014; Dekker and Frank, 2016; Youn et al , 2016). The new and future generations of antimicrobial susceptibility testing methods should be able to rapidly screen hundreds of approved drugs in a concentration–response manner with individual compounds and with hundreds of drug combinations.…”

“…The overuse of broad‐spectrum antibiotics actually contributes to development of drug resistance in pathogens as well as in many non‐harmful or less pathogenic bacteria. To avoid this unnecessary generation of resistance, effective and narrow‐spectrum antibiotics might be the first choice for treatment of infections if the pathogens can be diagnosed quickly with new methods such as the bacterial genome sequencing technology (Dekker and Frank, 2016). …”

“…Only a short time (8 ‐ 10 h) is needed for determination of effective antimicrobial agents and effective drug combinations in the bacterial growth assay with an absorbance assay format (Sun et al , 2016b). New methods for rapid diagnosis of pathogens (10 h or less) such as genome sequencing of pathogens are also needed (van Belkum and Dunne, 2013; Dekker and Frank, 2016). The data should be quickly analysed to reveal the nature of pathogens and the information of drug susceptibility for a particular pathogen.…”

Genomic epidemiology of multidrug‐resistant Gram‐negative organisms

“…Another challenge in standardizing WGS for clinical applications is agreement in the field regarding best practices and common analytical frameworks. Likely, the optimal framework will depend on the question at hand, where computationally friendly pipelines like whole‐genome multi‐locus sequence typing (WG‐MLST) might be preferable for real‐time analyses, while more sophisticated phylogenetic approaches that take full advantage of genomic data can be applied in retrospective analyses . A related issue is coming to a consensus as to whether the establishment of concrete variant thresholds is appropriate for evaluating whether two patients are plausibly linked by transmission .…”

“…Likely, the optimal framework will depend on the question at hand, where computationally friendly pipelines like whole-genome multi-locus sequence typing (WG-MLST) might be preferable for real-time analyses, while more sophisticated phylogenetic approaches that take full advantage of genomic data can be applied in retrospective analyses. 46,47,50,51 A related issue is coming to a consensus as to whether the establishment of concrete variant thresholds is appropriate for evaluating whether two patients are plausibly linked by transmission. 16,19,49,52 Owing to the aforementioned issue of increased variation due to prolonged asymptomatic colonization of patients, it is unlikely that a hard variant cutoff that is not overly conservative will work in all situations.…”