MLST revisited: the gene-by-gene approach to bacterial genomics

Abstract

Multilocus sequence typing (MLST) was proposed in 1998 as a portable sequence-based method for identifying clonal relationships among bacteria. Today, in the whole-genome era of microbiology, the need for systematic, standardized descriptions of bacterial genotypic variation remains a priority. Here, to meet this need, we draw on the successes of MLST and 16S rRNA gene sequencing to propose a hierarchical gene-by-gene approach that reflects functional and evolutionary relationships and catalogues bacteria 'from domain to strain'. Our gene-based typing approach using online platforms such as the Bacterial Isolate Genome Sequence Database (BIGSdb) allows the scalable organization and analysis of whole-genome sequence data.Advances in nucleotide-sequencing technology have provided unparalleled access to the enormous genetic diversity that has accumulated in the bacterial domain during 3.5-4 billion years of evolution 1 . Numerous sets of whole-genome sequencing (WGS) data for bacterial isolates (BOX 1) are available 2 , and metagenomic studies using these technologies continue to reveal further, seemingly boundless, diversity in bacterial communities 3 . Faced with this plethora of information, microbiologists must develop structured means of describing this diversity and of linking phenotype and genotype, thereby facilitating an improved understanding of the microbiological world. Given that we have precise information on the function of only a very small proportion of bacterial genes, and no knowledge at all about most, this is a formidable, if extremely exciting, challenge.Here, we focus primarily on pathogenic bacteria, although the concepts discussed are applicable more widely to all bacteria and archaea. Bacterial pathogens played a crucial part in the development of experimental microbiology and remain the most intensively studied prokaryotes more than 100 years later 4 . Pathogens have emerged across the diversity of the bacterial -but, interestingly, not the archaeal -domain on many occasions and are both polyphyletic and highly diverse. Thus, although pathogens represent only a tiny subset of the bacterial world, the challenges faced by the clinical microbiology laboratory are representative of those faced by microbiology as a whole.Taxonomic and functional analyses are based on the observations that diversity among bacteria is not continuous and that distinct, stable types with particular properties exist 5 . These founding principles of microbiology 6 have been upheld by much subsequent research, but the study of such clusters remains largely descriptive, and the evolutionary mechanisms that led to cluster emergence and persistence remain incompletely understood 7,8 . Structuring is also evident within bacterial genomes, as diversity is unevenly distributed among genes Pre-WGS cataloguing of diversityA major advance in defining bacterial diversity was the proposal, by the late Carl Woese and colleagues, of a universal and 'natural' -that is, genealogical -classification system based on small-su...

Integration of Genomic and Other Epidemiologic Data to Investigate and Control a Cross-Institutional Outbreak ofStreptococcus pyogenes

“…The identification of core SNPs from a referencebased assembly, exclusion of SNPs that are nearby to avoid an excessive effect from single recombination events, and subsequent generation of an SNP-based phylogeny echo the techniques used in other analyses of this species (20,21). In our analysis of wgMLST data, we used a reference-free assembly method and assessed the number of shared and discordant alleles across the dataset without making assumptions on processes giving rise to allelic variation (28). The replication of clustering by different and independent approaches adds credibility to the epidemiologic inferences drawn.…”

Ribosomal protein biomarkers provide root nodule bacterial identification by MALDI-TOF MS

“…However, many of the individual peaks that form a MALDI-TOF mass spectrum correspond to ribosomal proteins (RPs), which are encoded by more than 50 genes scattered in mostly chromosomal loci. RPs are ubiquitous and among the most abundant cytosolic proteins, regardless of the cell life stage or growth conditions (Ishihama et al 2008), and have masses mostly within the 4 to 30 kDa range that is examined with MALDI-TOF MS. Ribosomal multilocus sequence typing (rMLST) has been shown to be consistent with current bacterial nomenclature schemes and to provide accurate phylogenies for diverse taxonomic groups (Jolley et al 2012;Maiden et al 2013;Yutin et al 2012). These characteristics make RPs reliable biomarkers in MALDI-TOF MS analyses to obtain species and subspecies identification of diverse bacteria including bifidobacteria (Sato et al 2011), lactobacilli Teramoto et al 2007a), rhodococci (Teramoto et al 2009), members of the Sphingomonadaceae , and strains of Staphylococcus aureus (Josten et al 2013) and Neisseria meningitidis (Suarez et al 2013).…”

Increasing importance of genomics in recognition of infectious animal diseases taking into account OIE requirements

“…Przedmiotem niniejszego opracowania jest charakterystyka ważniej-szych spośród nich. Omawiane techniki genetyczne, coraz częściej i szerzej zastępują testy opierające się na ocenie właściwościach fenotypowych drobnoustrojów i reakcjach antygen-przeciwciało (3,6,23,24). Dotychczas opracowano wiele technik wykrywania i analizy materiału genetycznego bakterii i wirusów, z których najbardziej istotnymi wydają się: łańcuchowa reakcja polimerazy (polymerase chain reaction -PCR) (25,26,36) wraz z jej pochodnymi modyfikacjami oraz techniki amplifikacji DNA w warunkach stałej temperatury (29,40).…”