Lecture_30_GAS_infections_2024.pdf

MIIM30011 Medical Microbiology: Bacteriology L30: GAS Infections - Group A Streptococcus Mark Davies Department of Microbiology and Immunology [email protected] SEM Images c/o Dr David Goulding, Sanger Institute ...

MIIM30011 Medical Microbiology: Bacteriology L30: GAS Infections - Group A Streptococcus Mark Davies Department of Microbiology and Immunology [email protected] SEM Images c/o Dr David Goulding, Sanger Institute  Learning objectives Ø Know why Group A Streptococcus is a major human pathogen Ø Describe the key virulence and genome characteristics of S. pyogenes Ø Be able to apply your knowledge of genomic epidemiology to understand the emergence of pathogenic clones of S. pyogenes  Suggested reading Ø Prescott’s Microbiology 9th edition ØChapter 39.1- Airborne human diseases Ø Brouwer et al., Nat Rev Microbiol. 2023 Mar 9:1-17 Ø Barnett et al., Cellular Microbiology. 2015, 17(12), 1721-41 Ø Streptococcus pyogenes: Basic Biology to Clinical Manifestations. 2022. 2nd edition. Ed: Joseph J. Ferretti, Dennis L. Stevens, and Vincent A. Fischetti. open-access book. https://www.ncbi.nlm.nih.gov/books/NBK587111/ Ø Scientific literature cited throughout  Group A Streptococcus- Overview Ø Family Streptococcaceae Ø Genus Streptococcus is made up of medically important pathogens Ø Gram-positive cocci Ø Facultative anaerobes Ø Non-motile Ø Host adapted Ø Commensal of the nasopharyngeal tract and skin ØOpportunistic pathogen ØLargely extracellular living Courtesy of Dr. M. Rhode Ø Transmission by aerosols and direct contact  Group A Streptococcus- Characteristics Ø Classified on the basis of multiple factors ØBeta haemolytic Ø Differentiated on basis of group carbohydrate Ø >12 serological forms Ø Group A carbohydrate ØPolyrhamnose backbone + GlcNAc sidechain Rockefeller University Archive Center Ø Catalase negative ØUnable to convert H2O2 -> H2O + O2  Streptococcus pyogenes – Why worry? Ø Top 10 infectious disease agent Ø Causes ~600,000 deaths/year Ø S. pyogenes infections are endemic in some areas. Ø Antibiotic resistance is a continual emerging problem Ø No vaccine currently available Carapetis et al. Lancet Infectious Diseases 5:685 (2005) www.cdc.gov/drugresistance/biggest_threats.html  Streptococcus pyogenes - Infections Bacterial pharyngitis Puerperal Sepsis Necrotising Fasciitis Strep throat Childbed fever Flesh eating disease Ø 700 million cases superficial disease /year Ø 1.78 million new cases of severe GAS disease/ year Ø > 500,000 deaths p.a. Impetigo Cellulitis Scarlet Fever School sores Streptococcus pyogenes – Post infectious sequelae Ø Repeat GAS infection can lead to post-infectious autoimmune disease ØAcute post-streptococcal glomerulonephritis (Kidney failure) ØAcute Rheumatic Fever/ Rheumatic Heart Disease (Heart failure) Ø Major health concern in regions endemic for streptococcal infection ØContrasting epidemiology ØContrasting dogma of disease ØLow rates of pharyngeal carriage RHD patient heart valve shows thickening and calcification Courtesy of Bart Currie, MSHR Darwin Streptococcus pyogenes – Disease epidemiology > Primarily within developing countries and Indigenous populations of Industrialised regions Carapetis et al. Lancet Infectious Diseases 5:685 (2005)  GAS epidemiology differs globally > GAS epidemiology is based on immunodominant M protein (emm type) > Higher disease burden and strain diversity in developing countries > Presents a challenge for vaccine development > Up to 70% Indigenous school children may have GAS at any one time 100 Cumulative proportion 80 60 40 N.T. Australia Ethiopia 20 Nepal Denmark X U.S.A. 0 0 10 20 30 40 50 60 70 80 Richardson et al. Number of GAS emm types Vaccine 28:5301 (2010)  Streptococcus pyogenes - Pathogenesis ØCauses disease by ability to multiply and spread rapidly in tissues ØLitany of virulence factors drives the spread of disease ØExotoxins ØM protein ØStreptokinase ØHaemolysins ØCysteine and serine proteases ØDNAses ØHyaluronidase ØProtein G ØBacteriocins ØAdhesins ØCapsule ØC5a peptidase ØLeukocydins  GAS – Avoiding the immune system Walker et al. Clin. Microbiol. Rev. 2014;27:264-301  M protein: A key GAS virulence factor > Multi-functional N Factor H Factor H-like protein 1 - Adherence A1 C4b binding protein Hypervariable A2 - Promote internalisation A3 Plasminogen A4 IgA - Anti-phagocytic A5 IgG - Proinflammatory B1 B2 B3 > Conveys GAS type specific Variable B4 Fibrinogen immunity B5 - epidemiological molecular C1 - Leading vaccine target Factor H C2 Human serum albumin > Implicated in development of C3 D1 RHD Conserved D2 D3 - Share structural moieties with D4 Pro/Gly heart myosin Hydrophobic C  Streptococcal toxins - superantigens > GAS possess over 20 exotoxins that are variably distributed between strains > Bind different Vb chains of T cell receptors > Potent immunostimulators that contributes to severity of infection Conventional Superantigen Barnett et al. Cellular Microbiology 17(12) 1721-41 (2015)  Group A Streptococcal toxins - STSS > Streptolysin O is a cholesterol dependent cytolysin that disrupts cell membranes Barnett et al. Cellular Microbiology 17(12) 1721-41 (2015)  Streptococcus pyogenes: genomics Key Features Core genome ~ 70% Accessory ~ 30% - Single chromosome - Central metabolism - Bacteriophage - ~ 1,800,000 bp (~1800 genes) - House keeping - Genomic islands - Low GC - Carbohydrate - Transposons - Polylysogenic - M protein - Antibiotic resistance - Bacteriophage carry - SpeB virulence genes - Adhesins - Plasmids are not common - Antiphagocytic proteins - Sortase Sumby et al. JID, 2005;192(5):771-782 Davies et al. Nat Genet. 2019 Jun;51(6):1035-1043.  Genomic plasticity > Dynamic nature of prokaryotic genomes > Enable rapid adaptation to changing environments > Bacteriophage, Transposons, Plasmids, DNA rearrangement etc  GAS Population Genetics Example: A Genomic Approach to Investigate the Re-emergence of Scarlet Fever Illumina Hi-seq PacBio  Genomic epidemiology: A re-cap > Linking genomics with epidemiology Klemm and Dougan. 2016. Cell Host Microbe. 19(5), 599–610 Genomic epidemiology: Application to disease outbreaks 1. Clinical observation 2. Epidemiological Investigation 3. Isolate causative agent eg. Bacterial pathogen (variety of sources) 4. Extract DNA 5. Genome Sequence 6. Map sequence reads to a reference genome - Generate a sequence alignment and call SNPs 7. Reconstruct phylogeny - Incorporate temporal/geographical information - Identify genetic signature(s) 8. Inform disease control - Public health (contact tracing) - Develop diagnostic tools +  GAS causes scarlet fever Ø Scarlet fever is a toxin-mediated disease primarily of young children Ø GAS produces a variety of distinct toxins (superantigens such as SpeA, SpeC etc.) ØSuperantigen expression leads to hyperstimulation of the host immune system ØWell documented scarlet fever pandemics throughout the 15th – early 20th centuries Case Fatality 30% 20% Katz and Morens, CID, 14:298-307 (1992) 10% 1863 1870 1878  2011 Hong Kong scarlet fever outbreak Total cases reported Total cases reported Tse et al. Journal of Infectious Diseases 206: 341 (2012) You* Davies* et al. EBioMedicine 28: 128 (2018)  GAS epidemiology at start of the 2011 outbreak 2 predominant GAS emm types were circulating within the population Mainland China and Hong Kong scarlet fever outbreak isolates: emm12 >80% emm1 >10% macrolide >95% resistant tetracycline >80% resistant 2016 incidence/100,000 You* Davies* et al. EBioMedicine 28: 128 (2018) Chen et al. Pediatric Infectious Diseases 31: E158 (2012) Tse et al. Journal of Infectious Diseases 206: 341 (2012) Liu et al. Lancet Infectious Diseases 18: 30321 (2018)  Learning from a single genome Complete genome sequence of an emm12 GAS from a scarlet fever patient ϕ1 46.4kb HKU16 1,908,100bp 1942 CDS ϕ2 45.1kb ϕ3 45kb ICE 64.9kb PacBio  Learning from comparative genomics HKU16 (emm12) versus published non-scarlet fever emm12 genomes MGAS9429 (M12) MGAS2096 (M12) ϕHKU.vir (NEW) HKU16 1,908,100bp 1942 CDS ϕ9429.3 ϕ9429.2 MDR ICE-HKU.emm12 (NEW) Tse et al. Journal of Infectious Diseases 206: 341 (2012)  Characterisation of ‘new’ elements 64 kb Integrative conjugative element (ICE HKU.emm12) Tn916 0 kb 20 kb 40 kb 60 kb tetM ermB 46.4 kb prophage (ϕHKU.vir) spd1 speC 0 kb 20 kb 40 kb ssa Tse et al. Journal of Infectious Diseases 206: 341 (2012)  Transmission (Source) Outbreak possible sources ‘Other’ diseases Hospital Community International Evolution or acquisition New Clone - Fitness advantage of new traits or  Learning from population genomics A total of 137 emm12 GAS from Hong Kong were subjected to whole genome sequencing using the Illumina Hi-seq platform (3 x PacBio). Ø 52 strains from 2011 outbreak Ø 78 other clinical cases Ø 7 from other geographical regions and ‘old’ isolates PacBio Illumina Hi-seq  Mapping sequence reads to a reference HKU16 1,908,100bp Reference AGATTCTTCGAGAGTTCTGAGATTAGGATATTTTATTATTTACTCTCTGGG................................................... AGATGC TCGAGA TTCTGAGA TCGGATATT TATTATTT CTCTCTG Reads GATGCTTCG AGTTCTGAGAT GGATATTTTATTA TTTCTCTCT AGATGCTT GAGAGTTCTGAGATTCGG TATTTTATTA CTCTCTGGG AGATGCTTCG GAGTTCTGAGAT CGGATA TTTATTA TTTCTCTCTGGG ATGCTTCGAGAG GAGAT CGGATA TTA TTTCTCTCTG GATGCTTC GTTCTGAGAT CGGATA TTTATTA TTTCT * * SNPs * Sidetrack: An example of a ‘clonal’ outbreak… Outbreak GAS Residents isolates Staff Conclusion: All outbreak isolates came from a single ‘clone’ Non-outbreak GAS isolates Worthing et al 2020. EID May;26(5):841-848.  Genomic analysis of Hong Kong emm12 GAS CLADE IV 1991 1935 CLADE III 1983 Ø 4 predominant clades 1955 Ø Major clades diverged from a common ancestor during the 1950s and expanded during the 1980s CLADE I 1982 CLADE II 1927 1954 1982 2009 Davies et al. Nature genetics. 2015. Jan;47(1):84-7 2011 emm12 scarlet fever outbreak is multiclonal Red = scarlet fever CLADE IV 1991 Yellow = NOT scarlet fever 1935 CLADE III 1983 Ø 4 predominant clades 1955 Ø Major clades diverged from a common ancestor during the 1950s and expanded during the 1980s CLADE I 1982 Ø 3 of 4 clades contain scarlet fever isolates CLADE II 1927 1954 1982 2009 Davies et al. Nature genetics. 2015. Jan;47(1):84-7 SSA confined to ‘scarlet fever-associated’ clades a ss CLADE IV CLADE III Ø Of the known toxins, only ssa was variably distributed in the scarlet fever- associated clades (I, II and III) but absent from clade IV. Ø Date of ssa phage acquisition CLADE I correlates to the predicted date of expansion of the major scarlet fever clades CLADE II 1927 1954 1982 2009 Davies et al. Nature genetics. 2015. Jan;47(1):84-7 GAS ‘ssa’ pathogenesis model Brouwer et al. Nat Commun 2020 Oct 6;11(1):5018.  Scarlet fever outbreak - conclusions Ø The 2011 Hong Kong outbreak was multiclonal involving emm12 GAS. ØAcquisition of macrolide resistance genes reduces effectiveness of front-line treatment (azithromycin and clindamycin), yet remain sensitive to penicillin. Ø Acquisition of the SSA and SpeC encoding phage appear to act in synergy to enhance colonization, leading to enhanced virulence, triggering scarlet fever GAS emm12 outbreaks. ØWhy 2011? Host/environmental factors driving upsurge? - Host immune status? - Unknown environmental factors? - Co-infection?  The story continues…. SF iGAS https://www.who.int/emergencies/disease-outbreak- news/item/2022-DON429 What would be your next steps?  Scarlet fever – M12 Scarlet fever / iGAS – M1 Frequency of M1 clones in QLD 100 Percentage of isolates 75 M1UK 50 M1T1 25 0 2006 2008 2010 2012 2014 2016 2018 n = 4, 2, 5, 5, 5, 10, 4, 9, 10, 10, 10, 22, 55, 65, 17 Frequency of M1 clones in VIC 100 Percentage of isolates 80 60 M1uk 40 WT 20 0 2014 2015 2016 2017 2018 2019 n = 3 n=11 n=5 n=24 n=34 -> polyclonal -> M1UK in Australia -> difference sub-lineage in UK/China -> iGAS rates increasing in Aus -> importation into Aus -> presence of ssa phage in Aus M1uk sub- -> ssa ‘globally’ representative lineages -> MDR low in UK  Why do we sequence genomes? Ø Understand bacterial evolution (eg. outbreaks) Ø Identify virulence factors/mechanisms of pathogenesis Ø Design new vaccines / improve vaccine design Ø Develop new antibiotics Ø Develop new molecular diagnostics Ø Design public health interventions Ø monitor how pathogen populations respond How can genomics be used to improve vaccine design Ø Population genomics allows you to assess ‘naturally’ occurring genetic variation. Ø GAS isolates > Sequence > Assemble > Blast > Output Davies et al. Nat Genet. 2019 Jun;51(6):1035-1043.  Learning objectives Ø Know why Group A Streptococcus is a major human pathogen ØKey epidemiological differences Ø Describe the key virulence and genome characteristics of S. pyogenes ØMajor contributors to genetic differences between strains ØFunctional attributes of major GAS virulence factors Ø Be able to apply your knowledge of genomic epidemiology to understand the emergence of pathogenic clones of S. pyogenes ØWhat are some of the molecular markers? ØSteps in using genomics to characterise bacterial outbreak(s) ØCould you use similar approaches to study other pathogens?