Genomic comparative analysis and gene function prediction in infectious diseases: application to the investigation of a meningitis outbreak

BMC Infectious Diseases

Luisa Barzon³  Giorgio Palù³  Riccardo Manganelli³  Marco Falda¹  Francesca Finotello²  Barbara Di Camillo²  Elisa Franchin³  Stefano Toppo¹  Enrico Lavezzo¹ 

[1] Department of Molecular Medicine, University of Padova, Padova, Italy;Department of Information Engineering, University of Padova, Padova, Italy;Regional Reference Laboratory for Infectious Diseases, Microbiology and Virology Unit, Padova University Hospital, Padova, Italy

关键词: Gene function prediction;    Molecular epidemiology;    Meningitis outbreak;    454 pyrosequencing;    Comparative genomics;    capsule locus;    Next generation sequencing;    Whole genome sequencing;    Neisseria meningitidis;   

Others  :  1145365
DOI  :  10.1186/1471-2334-13-554

 received in 2013-05-03, accepted in 2013-11-13,  发布年份 2013

【 摘 要 】

Background

Next generation sequencing (NGS) is being increasingly used for the detection and characterization of pathogens during outbreaks. This technology allows rapid sequencing of pathogen full genomes, useful not only for accurate genotyping and molecular epidemiology, but also for identification of drug resistance and virulence traits.

Methods

In this study, an approach based on whole genome sequencing by NGS, comparative genomics, and gene function prediction was set up and retrospectively applied for the investigation of two N. meningitidis serogroup C isolates collected from a cluster of meningococcal disease, characterized by a high fatality rate.

Results

According to conventional molecular typing methods, all the isolates had the same typing results and were classified as outbreak isolates within the same N. meningitidis sequence type ST-11, while full genome sequencing demonstrated subtle genetic differences between the isolates. Looking for these specific regions by means of 9 PCR and cycle sequencing assays in other 7 isolates allowed distinguishing outbreak cases from unrelated cases. Comparative genomics and gene function prediction analyses between outbreak isolates and a set of reference N. meningitidis genomes led to the identification of differences in gene content that could be relevant for pathogenesis. Most genetic changes occurred in the capsule locus and were consistent with recombination and horizontal acquisition of a set of genes involved in capsule biosynthesis.

Conclusions

This study showed the added value given by whole genome sequencing by NGS over conventional sequence-based typing methods in the investigation of an outbreak. Routine application of this technology in clinical microbiology will significantly improve methods for molecular epidemiology and surveillance of infectious disease and provide a bulk of data useful to improve our understanding of pathogens biology.

【 授权许可】

   
2013 Lavezzo et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Barzon L, Lavezzo E, Costanzi G, Franchin E, Toppo S, Palù G: Next-generation sequencing technologies in diagnostic virology. J Clin Virol 2013, 13:00086-3. doi: S1386-6532
• [2]Barzon L, Lavezzo E, Militello V, Toppo S, Palù G: Applications of next-generation sequencing technologies to diagnostic virology. Int J Mol Sci 2011, 12:7861-7884.
• [3]Didelot X, Bowden R, Wilson DJ, Peto TE, Crook DW: Transforming clinical microbiology with bacterial genome sequencing. Nat Rev Genet 2012, 13:601-612.
• [4]Kahvejian A, Quackenbush J, Thompson JF: What would you do if you could sequence everything? Nat Biotechnol 2008, 26:1125-1133.
• [5]Kostic AD, Ojesina AI, Pedamallu CS, Jung J, Verhaak RG, Getz G, Meyerson M: PathSeq: software to identify or discover microbes by deep sequencing of human tissue. Nat Biotechnol 2011, 29:393-396.
• [6]Frasson I, Lavezzo E, Franchin E, Toppo S, Barzon L, Cavallaro A, Richter SN, Palù G: Antimicrobial treatment and containment measures for an extremely drug-resistant Klebsiella pneumoniae ST101 isolate carrying pKPN101-IT, a novel fully sequenced bla(KPC-2) plasmid. J Clin Microbiol 2012, 50:3768-3772.
• [7]Ferro A, Baldo V, Cinquetti S, Corziali P, Gallo G, Lustro G, Paludetti P, Menegon T, Baldovin T, Palù G, Trivello R: Outbreak of serogroup C meningococcal disease in Veneto region, Italy. Euro Surveill 2008, 13(2):8008.
• [8]Fazio C, Neri A, Tonino S, Carannante A, Caporali MG, Salmaso S, Mastrantonio P, Stefanelli P: Characterisation of Neisseria meningitidis C strains causing two clusters in the north of Italy in 2007 and 2008. Euro Surveill 2009, 14(16):19179.
• [9]Maiden MC, Bygraves JA, Feil E, Morelli G, Russell JE, Urwin R, Zhang Q, Zhou J, Zurth K, Caugant DA, Feavers IM, Achtman M, Spratt BG: Multilocus sequence typing: a portable approach to the identification of clones within populations of pathogenic microorganisms. Proc Natl Acad Sci USA 1998, 95:3140-3145.
• [10]Suker J, Feavers IM, Achtman M, Morelli G, Wang JF, Maiden MC: The porA gene in serogroup A meningococci: evolutionary stability and mechanism of genetic variation. Mol Microbiol 1994, 12:253-265.
• [11]Lavezzo E, Toppo S, Barzon L, Cobelli C, Di Camillo B, Finotello F, Franchin E, Peruzzo D, Toffolo GM, Trevisan M, Palù G: Draft genome sequences of two Neisseria meningitidis serogroup C clinical isolates. J Bacteriol 2010, 192:5270-5271.
• [12]Finotello F, Lavezzo E, Fontana P, Peruzzo D, Albiero A, Barzon L, Falda M, Di Camillo B, Toppo S: Comparative analysis of algorithms for whole-genome assembly of pyrosequencing data. Brief Bioinform 2012, 13:269-280.
• [13]Li W, Jaroszewski L, Godzik A: Clustering of highly homologous sequences to reduce the size of large protein database. Bioinformatics 2001, 17:282-283.
• [14]Li W, Jaroszewski L, Godzik A: Tolerating some redundancy significantly speeds up clustering of large protein databases. Bioinformatics 2002, 18:77-82.
• [15]Delcher AL, Bratke KA, Powers EC, Salzberg SL: Identifying bacterial genes and endosymbiont DNA with Glimmer. Bioinformatics 2007, 23:673-679.
• [16]Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ: Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 1997, 25:3389-3402.
• [17]Fontana P, Cestaro A, Velasco R, Formentin E, Toppo S: Rapid annotation of anonymous sequences from genome projects using semantic similarities and a weighting scheme in gene ontology. PLoS One 2009, 4:e4619.
• [18]Falda M, Toppo S, Pescarolo A, Lavezzo E, Di Camillo B, Facchinetti A, Cilia E, Velasco R, Fontana P: Argot2: a large scale function prediction tool relying on semantic similarity of weighted Gene Ontology terms. BMC Bioinformatics 2012, 13(Suppl 4):S14. BioMed Central Full Text
• [19]Radivojac P, Clark WT, Oron TR, Schnoes AM, Wittkop T, Sokolov A, Graim K, Funk C, Verspoor K, Ben-Hur A, Pandey G, Yunes JM, Talwalkar AS, Repo S, Souza ML, Piovesan D, Casadio R, Wang Z, Cheng J, Fang H, Gough J, Koskinen P, Törönen P, Nokso-Koivisto J, Holm L, Cozzetto D, Buchan DW, Bryson K, Jones DT, Limaye B, et al.: A large-scale evaluation of computational protein function prediction. Nat Methods 2013, 10:221-227.
• [20]Elias J, Vogel U: IS1301 fingerprint analysis of Neisseria meningitidis strains belonging to the ET-15 clone. J Clin Microbiol 2007, 45:159-167.
• [21]Vogel U, Claus H, Frosch M, Caugant DA: Molecular basis for distinction of the ET-15 clone within the ET-37 complex of Neisseria meningitidis. J Clin Microbiol 2000, 38:941.
• [22]Jolley KA, Wilson DJ, Kriz P, McVean G, Maiden MC: The influence of mutation, recombination, population history, and selection on patterns of genetic diversity in Neisseria meningitidis. Mol Biol Evol 2005, 22:562-569.
• [23]Schoen C, Tettelin H, Parkhill J, Frosch M: Genome flexibility in Neisseria meningitidis. Vaccine 2009, 27(Suppl 2):B103-B111.
• [24]Swartley JS, Marfin AA, Edupugnanti S, Liu LJ, Cieslak P, Perkins B, Wenger JD, Stephens DS: Capsule switching of Neisseria meningitidis. Proc Natl Acad Sci USA 1997, 94:271-276.
• [25]Hao W, Ma JH, Warren K, Tsang RS, Low DE, Jamieson FB, Alexander DC: Extensive genomic variation within clonal complexes of Neisseria meningitidis. Genome Biol Evol 2011, 3:1406-1418.
• [26]Didelot X: Genomic analysis to improve the management of outbreaks of bacterial infections. Expert Rev Anti Infect Ther 2013, 11:335-337.
• [27]Vogel U, Szczepanowski R, Claus H, Jünemann S, Prior K, Harmsen D: Ion Torrent Personal Genome Machine sequencing for genomic typing of Neisseria meningitidis for rapid determination of multiple layers of typing information. J Clin Microbiol 2012, 50:1889-1894.
• [28]Jolley KA, Maiden MC: Automated extraction of typing information for bacterial pathogens from whole genome sequence data: Neisseria meningitidis as an exemplar. Euro Surveill 2013, 18:20379.
• [29]Jolley KA, Hill DM, Bratcher HB, Harrison OB, Feavers IM, Parkhill J, Maiden MC: Resolution of a meningococcal disease outbreak from whole-genome sequence data with rapid Web-based analysis methods. J Clin Microbiol 2012, 50:3046-3453.
• [30]A.r.g.o.t.2, Functional annotation of proteins using the semantic similarity in the Gene Ontology. http://www.medcomp.medicina.unipd.it/Argot2 webcite