Core Genome Multilocus Sequence Typing and Single Nucleotide Polymorphism Analysis in the Epidemiology of Brucella melitensis Infections

Janowicz, Anna; Massis, Fabrizio De; Ancora, Massimo; Cammà, Cesare; Patavino, Claudio; Battisti, Antonio; Prior, Karola; Harmsen, Dag; Scholz, Holger C.; Zilli, Katiuscia; Sacchini, Lorena; Giannatale, Elisabetta Di; Garofolo, Giuliano

doi:10.1128/jcm.00517-18

Cited by 58 publications

(82 citation statements)

References 37 publications

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“…The number of SNPs encountered between the ten isolates ranged from zero to five, in the same range as the wgMLST variation and insufficient to decipher transmission of strains between patients (data not shown). Of note, a recent cgMLST study for Brucella melitensis revealed similar findings: epidemiological cutoff values for nonvariance were defined as Ͻ6 loci for wgMLST and Ͻ7 loci for wgSNP analyses, similar to what we document here for S. marcescens (19).…”

Section: Resultssupporting

confidence: 71%

Epidemiological Typing of Serratia marcescens Isolates by Whole-Genome Multilocus Sequence Typing

et al. 2019

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Serratia marcescens is an opportunistic bacterial pathogen. It is notorious for its increasing antimicrobial resistance and its potential to cause outbreaks of colonization and infections, predominantly in neonatal intensive care units (NICUs). There, its spread requires rapid infection control response. To understand its spread, detailed molecular typing is key. We present a whole-genome multilocus sequence typing (wgMLST) method for S. marcescens. Using a set of 299 publicly available whole-genome sequences (WGS), we developed an initial wgMLST system consisting of 9,377 gene loci. This included 1,455 loci occurring in all reference genomes and 7,922 accessory loci. This closed system was validated using three geographically diverse collections of S. marcescens consisting of 111 clinical isolates implicated in nosocomial dissemination events in three hospitals. The validation procedure showed a full match between epidemiological data and the wgMLST analyses. We set the cutoff value for epidemiological (non)relatedness at 20 different alleles, though for the majority of outbreak-clustered isolates, this difference was limited to 4 alleles. This shows that the wgMLST system for S. marcescens provides prospects for successful future monitoring for the epidemiological containment of this opportunistic pathogen.

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Section: Resultssupporting

confidence: 71%

Epidemiological Typing of Serratia marcescens Isolates by Whole-Genome Multilocus Sequence Typing

et al. 2019

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“…Phylogenetic analysis of Brucella melitensis loci [16], and the other runs on a pay-per-use platform (RIDOM SeqSphere) despite involving a wider core gene set of 2704 targets [17].…”

Section: Plos Onementioning

confidence: 99%

“…With the technological advances and decreased cost of whole genome sequencing, new methods of pathogen typing, including gene-by-gene comparison using core genome multilocus sequence typing (cgMLST), as well as single-nucleotide polymorphism (SNP) calling based on a reference sequence analysis, are considered to be a suitable and more informative replacement of the gold standard typing schemes [16]. Although the SNP-based analysis may constitute a better option for phylogenetic analyses of conserved genomes (because this approach covers the entire genome, including the intergenic regions) [17], very recently, efforts to develop cgMLST schemes for Brucella have been done [16,17]. One of these schemes involves 2704 genes and is based on a pay-per-use platform [17], whereas the other involves a strikingly low number of genes (n = 164) for differentiating purposes [16].…”

Section: Introductionmentioning

confidence: 99%

“…Although the SNP-based analysis may constitute a better option for phylogenetic analyses of conserved genomes (because this approach covers the entire genome, including the intergenic regions) [17], very recently, efforts to develop cgMLST schemes for Brucella have been done [16,17]. One of these schemes involves 2704 genes and is based on a pay-per-use platform [17], whereas the other involves a strikingly low number of genes (n = 164) for differentiating purposes [16].…”

Section: Introductionmentioning

confidence: 99%

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Genome–scale approach to study the genetic relatedness among Brucella melitensis strains

et al. 2020

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Brucellosis is an important zoonotic disease that affects both humans and animals. To date, laboratory surveillance is still essentially based on the traditional MLVA-16 methodology and the associated epidemiological information is frequently scarce. Our goal was to contribute to the improvement of Brucella spp. surveillance through the implementation of a whole genome sequencing (WGS) approach. We created a curated ready-to-use speciesspecific wgMLST scheme enrolling a panel of 2656 targets (http://doi.org/10.5281/zenodo. 3575026) and used this schema to perform a retrospective analysis of the genetic relatedness among B. melitensis strains causing human infection in Portugal (a country where brucellosis is an endemic disease) from 2010 to 2018. The strains showed a phylogenetic clustering within genotype II (25 out of 36) and IV (4 out of 36), and shared clades with strains isolated from countries with which Portugal has intense food trading, tourism and similar eating habits, such as Spain, Italy and Greece. In addition, our results point to the identification of strong associations between B. melitensis strains, likely underlying missed "outbreaks" as 22 out of the 36 strains showed genetic linkage with others. In fact, the applied gene-by-gene approach grouped these strains into six genetic clusters each one containing putative epidemiological links. Nevertheless, more studies will be needed in order to define the appropriate range of cut-offs (probable non-static cut-offs) that best illustrate the association between genetic linkage and epidemiological information and may serve as alerts for the health authorities. The release of this freely available and scalable schema contributes to the required technological transition for laboratorial surveillance of brucellosis and will facilitate the assessment of ongoing and future outbreaks in order to prevent the transmission spread.

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“…Despite the growing amount of comparative studies (de Been et al, 2015;Li et al, 2017;Ghanem and El-Gazzar, 2018;Halbedel et al, 2018;Janowicz et al, 2018;Meehan et al, 2018), it is currently often unknown which approach is the most suitable for a given organism and in a given epidemiological situation, and the dissimilarities in the output obtained using alternative approaches have been poorly characterized. This is a second problem that slows down the adoption of WGS in routine, i.e., the absence of an established methodology for the evaluation and comparison of WGS data analysis schemes.…”

Section: Introductionmentioning

confidence: 99%

Detailed Evaluation of Data Analysis Tools for Subtyping of Bacterial Isolates Based on Whole Genome Sequencing: Neisseria meningitidis as a Proof of Concept

et al. 2019

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Whole genome sequencing is increasingly recognized as the most informative approach for characterization of bacterial isolates. Success of the routine use of this technology in public health laboratories depends on the availability of well-characterized and verified data analysis methods. However, multiple subtyping workflows are now often being used for a single organism, and differences between them are not always well described. Moreover, methodologies for comparison of subtyping workflows, and assessment of their performance are only beginning to emerge. Current work focuses on the detailed comparison of WGS-based subtyping workflows and evaluation of their suitability for the organism and the research context in question. We evaluated the performance of pipelines used for subtyping of Neisseria meningitidis, including the currently widely applied cgMLST approach and different SNP-based methods. In addition, the impact of the use of different tools for detection and filtering of recombinant regions and of different reference genomes were tested. Our benchmarking analysis included both assessment of technical performance of the pipelines and functional comparison of the generated genetic distance matrices and phylogenetic trees. It was carried out using replicate sequencing datasets of high-and low-coverage, consisting mainly of isolates belonging to the clonal complex 269. We demonstrated that cgMLST and some of the SNP-based subtyping workflows showed very good performance characteristics and highly similar genetic distance matrices and phylogenetic trees with isolates belonging to the same clonal complex. However, only two of the tested workflows demonstrated reproducible results for a group of more closely related isolates. Additionally, results of the SNPbased subtyping workflows were to some level dependent on the reference genome used. Interestingly, the use of recombination-filtering software generally reduced the similarity between the gene-by-gene and SNP-based methodologies for subtyping of Frontiers in Microbiology | www.frontiersin.org 1 December 2019 | Volume 10 | Article 2897 Saltykova et al. WGS Subtyping of Neisseria: BenchmarkingN. meningitidis. Our study, where N. meningitidis was taken as an example, clearly highlights the need for more benchmarking comparative studies to eventually contribute to a justified use of a specific WGS data analysis workflow within an international public health laboratory context.

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Core Genome Multilocus Sequence Typing and Single Nucleotide Polymorphism Analysis in the Epidemiology of Brucella melitensis Infections

Cited by 58 publications

References 37 publications

Epidemiological Typing of Serratia marcescens Isolates by Whole-Genome Multilocus Sequence Typing

Epidemiological Typing of Serratia marcescens Isolates by Whole-Genome Multilocus Sequence Typing

Genome–scale approach to study the genetic relatedness among Brucella melitensis strains

Detailed Evaluation of Data Analysis Tools for Subtyping of Bacterial Isolates Based on Whole Genome Sequencing: Neisseria meningitidis as a Proof of Concept

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