Abstract
BackgroundMulti-locus sequence typing (MLST) is a portable, broadly applicable method for classifying bacterial isolates at an intra-species level. This methodology provides clinical and scientific investigators with a standardized means of monitoring evolution within bacterial populations. MLST uses the DNA sequences from a set of genes such that each unique combination of sequences defines an isolate’s sequence type. In order to reliably determine the sequence of a typing gene, matching sequence reads for both strands of the gene must be obtained. This study assesses the ability of both the standard, and an alternative set of, Streptococcus pneumoniae MLST primers to completely sequence, in both directions, the required typing alleles.ResultsThe results demonstrated that for five (aroE, recP, spi, xpt, ddl) of the seven S. pneumoniae typing alleles, the standard primers were unable to obtain the complete forward and reverse sequences. This is due to the standard primers annealing too closely to the target regions, and current sequencing technology failing to sequence the bases that are too close to the primer. The alternative primer set described here, which includes a combination of primers proposed by the CDC and several designed as part of this study, addresses this limitation by annealing to highly conserved segments further from the target region. This primer set was subsequently employed to sequence type 105 S. pneumoniae isolates collected by the Canadian Immunization Monitoring Program ACTive (IMPACT) over a period of 18 years.ConclusionsThe inability of several of the standard S. pneumoniae MLST primers to fully sequence the required region was consistently observed and is the result of a shift in sequencing technology occurring after the original primers were designed. The results presented here introduce clear documentation describing this phenomenon into the literature, and provide additional guidance, through the introduction of a widely validated set of alternative primers, to research groups seeking to undertake S. pneumoniae MLST based studies.
Highlights
Multi-locus sequence typing (MLST) is a portable, broadly applicable method for classifying bacterial isolates at an intra-species level
Similar to the Multi-locus enzyme electrophoresis (MLEE), pulse field gel electrophoresis (PFGE) classifies individual strains based on the gel electrophoretic mobility of bacterial components: in this case the relative mobility of DNA fragments which have been obtained through restriction enzyme digestion [9]
The purpose of this study is to systematically identify the primers unable to obtain the correct sequence, describe an alternative set of primers, and introduce documentation to the literature offering additional guidance to groups undertaking S. pneumoniae MLST studies
Summary
Multi-locus sequence typing (MLST) is a portable, broadly applicable method for classifying bacterial isolates at an intra-species level. Many globally significant bacterial pathogens such as Streptococcus pneumoniae and Neisseria meningitidis are readily able to incorporate environmental genetic material into their genomes allowing for rapid genetic variation and interchange of immunogenic components; including those on which serotyping is based [5] This phenomenon has been observed recently with S. pneumoniae capsular typing following the introduction of the seven-valent pneumococcal conjugate vaccine (PCV7) [6]. Multi-locus enzyme electrophoresis (MLEE) is another typing method, and is based on the relative electrophoretic mobility of a set of ubiquitously present bacterial enzymes [8] This approach is not dependent on a single immunogenic component and as such is less influenced by horizontal exchange or positive selection events. PFGE has been widely used for typing and has been considered a gold standard for some epidemiological studies, there have been challenges in standardizing protocols between different research groups [10]
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