emm1 invasive Group A Streptococcus isolated from Belgian patients during 19942013 J Coppens 1 B B Xavier J Sabirova 1 C Lammens 1 K Loens 2 S MalhotraKumar ID: 1047077
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1. Genome-wide longitudinal analysis of emm1 invasive Group A Streptococcus isolated from Belgian patients during 1994˗2013 J. Coppens1, B. B. Xavier, J. Sabirova1, C. Lammens1, K. Loens2, S. Malhotra-Kumar1, H. Goossens11Laboratory of Medical Microbiology, VAXINFECTIO,University of Antwerp, Antwerp, Belgium2The National Reference Centre for Invasive Group A Streptococci, University Hospital Antwerp, Antwerp, Belgium #O226
2. IntroductionGASGram-positive bacterial pathogenCauses human infections worldwide Diverse clinical manifestationsPharyngitisInvasive infectionsStreptococcal toxic shock syndromeEmm1 geneEncode for M proteinAntiphagocytic cell surface molecule
3. Aim of the studyUsing whole genome mapping and whole genome sequencing to longitudinal study genome wide evolutionary events in emm1 GAS strains collected in Belgium1994-2013invasive strainsnon-invasive strains
4. Whole genome mapping (WGM) WGM is an ordered, high-resolution density whole genome restriction map generated from single DNA molecule Opgen Inc., Gaithersburg, USA DNA extractionDNA molecules immobilized on flow chipRE digestfluorescent stainAnalysisCreating a whole genome map:
5. Longitudinal collection of emm1 GAS
6. Whole genome maps of emm1 invasive strains (1994-2013)ProphageMetabolic genes (LacA, lacB, copA, copC)Mobile elementsMobile elements
7. Whole genome maps of emm1 non-invasive strains (1994-2013)EsteraseMobileelementsMobileelements
8. Whole genome sequencing of invasive emm1 GASMiSeq IlluminaDe novo Genome assembly (Velvet & SPAdes)Annotation (RAST Server http://rast.nmpdr.org/)Comparative genome analysis (MUMmer and mauve)Prophages, crisprAverage genome size (bp): ~1865579Average GC content (%): ~38
9. Non-synonymous SNPs (longitudinaal)Genes with non-synonymous SNPs (Examples)Aminoacid changeRecO Thr > MetLysR (regulator) (region152029-152119)Ile > Asn RpoDAla > ThrSICArg > GlyABC transporterAla > ValEmmAsp > AsnSNP: Single-nucleotide polymorphismShea et al., 2011, Emerg. Infect. Dis.
10. 36 Kb Recombination spot36 kb with high number of mutation (purA ….nadC)Comparison of core genome versus reference SNPsNon-synonymous SNPs1994 versus MGAS500594321994 versus 201382372013 versus MGAS5005112007 invasive versus MGAS5005822008 non-invasive versus MGAS50057037Sumby et al., 2005, J. Infect. Dis. MGAS5005
11. Prophages and other mobile elements in invasive emm1 GAS during 1994-2013ProphagesMobile elements (transposons) http://phast.wishartlab.com/Invasive GAS strains Non-invasive GAS strains
12. Prophages presence in emm1 GASInvasive strains1994 (n=1)1999 (n=2)2001(n=1)2003 (n=1)2004 (n=1)2005 (n=2)2007 (n=1)2008 (n=1)2009 (n=1)2010 (n=1)2012 (n=1)2013 (n=6)PHAGE_Strept_pyogenes_315_522+31(incomplete)112+3332322-4PHAGE_Strept_P911+10111111111-2PHAGE_Entero_G4_sensu_lato00000000001 (S2)0PHAGE_Strept_IC1000000100000PHAGE_Temper_phiNIH1_1 000000000001 (S16)PHAGE_Strept_PH10010000000000Non-invasive strains2007(n=1)2008(n=1)PHAGE_Strept_pyogenes_315_511PHAGE_Strept_P911HNH-Homing endonucleaseshttp://phast.wishartlab.com/
13. Gain of intron containing HNH-Homing endonucleases in phage 315199419992013Belfort et al., 1997, Nucl. Acid. Research; Stoddard et al., 2014, Mobile DNAWithin genome duplication of prophage 315 is probably due to presence of homing endonuclease YearHNHMGAS500511994119991/020010200302004120051200712008120091201012012120131s161Endonucleases encoded by introns promote the homing of their respective genetic elements into allelic intronless site.
14. Superantigens distribution in invasive GASYearSuperantigensSpeGSpeASpeBSpeKSmeZSpeJSpeJ-fragment199411100101999111001120011110110200311101102004111011020051110010200711111102008111111020091110010201011100112012111001020131110010s161111110SpeJ might be phage associated and thus transferable between GAS McCormick et al., 2001 Infect Immun; Berman et al., 2014, BMC Infec. Dis.
15. Correlation of CRISPR and ProphagesStrainCRISPRProphageAccession NumberSF-3702 (3)4NC_002737.1MGAS-50052 (3)3NC_007297.1M1-4762 (3)3NC_020540.2S3-1994 2 (4)3This studyS1-19992 (3)3This studyS8-20072 (3)4This studyS4-20082 (3)4This studyS16-20132 (4)5This studyhttp://crispr.u-psud.fr/Server/; Nozawa et al., 2011CRISPR: Clustered Regularly Interspaced Short Palindromic Repeatsprovide acquired immunity against foreign genetic elements There is no correlation between CRISPR/CAS genes and the gain/ loss of prophages Invasive (Confirmed/ Questionable) Non-invasive strains (Confirmed/ Questionable)
16. ConclusionLongitudinal genome wide analysis showed a rather high conservation of the emm1 GAS strains in Belgium during the 20-year period studiedGain/presence of HNH-Homing Endonuclease in phage 315 linked to duplicationPhage associations of SpeJ No correlation between CRISPRs and prophagesVariations in 2007 and 2008 might be link to specific selection pressureWGM is a high resolution technique for detection of genomic variations, compared to standard molecular typing methods, as MLST, PFGE.
17. AcknowledgementFundingPlatform foR European Preparedness Against (Re-)emerging Epidemics, EU-FP7 (PREPARE; 602525)European public health initiative EUPHi (Opgen Inc., Gaithersburg, USA)The Belgian National Reference Centre for invasive GAS is supported by the Belgian Ministry of Social affairsSabine Chapelle for technical assistance
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