3/08/2012. EHECO104: Lessons for Australia from the German outbreak. E. coli Pathotypes. EHEC Reservoirs & Transmission. EHEC Virulence Markers

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1 E. coli Pathotypes E. coli O157:H7 EHECO104: Lessons for Australia from the German outbreak Rowland Cobbold Senior Lecturer - Veterinary Public Health School of Veterinary Science University of Queensland E. coli STEC EHEC ETEC EPEC EAEC EIEC Pathogenic Bacteria EHEC Virulence Markers EHEC Reservoirs & Transmission Chromosome Locus for enterocyte effacement (LEE) stx Plasmid ehx eae Enterohaemolysin Attaching & effacing lesion (AE) Shiga toxin The Outbreak The Smoking Cucumber 1 May 23 May 26 May 31 May 2 Jun 10 Jun 13 Jul Suspected outbreak onset Robert Koch Institute issues alert: HC & HUS cases 1 st isolation of STEC O104:H4. WHO confirms novel outbreak strain. RT-PCR developed Whole genome sequencing: BGI, Germany, HPA UK Decline in incidence outbreak peak 3842 cases; 855 HUS; 53 deaths* (ECDC) 1

2 O104 Genome Scaffolded assemblies mapped against reference sequences using BLAST Ring Image Generator (BRIG) Kat Holt Nico Petty Bacpathgenomics. wordpress.com Enteroaggregative E. coli (EAEC) Travellers diarrhoea, chronic diarrhoea in children from developing countries, AIDS patients Human reservoir Defined by phenotypic adherence profile Virulence markers: Stacked brick formation on Hep-2 aggr: aggregation regulator AAF genes: aggregative adherence fimbriae mediator of biofilms and haemagglutination Located on large paa plasmid O104 Outbreak Strain - Novel Not a classical EHEC strain Homology to EC55989 EAEC isolated from Africa 10 y ago Essentially an EAEC that has acquired stx 2a None of the primary EHEC virulence markers: LEE, ehx Presence of classical EAEC virulence markers Entero-aggregative-haemorrhagic E. coli (EAHEC) O104 Outbreak Strain - Novel Not a classical EHEC strain Homology to EC55989 EAEC isolated from Africa 10 y ago Essentially an EAEC that has acquired stx 2a None of the primary EHEC virulence markers: LEE, ehx Presence of classical EAEC virulence markers Entero-aggregative-haemorrhagic E. coli (EAHEC) Sporadic historic clusters/cases of O104 +/- stx reported Multidrug resistance (chromosomal) including ESBL phenotype (TEM, blactx-m-15) (plasmid) What should we take from all this? Surveillance and response Diagnostics, molecular diagnostics Source tracking, control & prevention On-going research ECDC Lessons Learned Considerable delays (21d) from D x to Federal reporting. rationalise coordination and response agencies within Germany: particularly at the Federal and State levels. ECDC should take on role of knowledge broker T x guidance for cases particularly due to high virulence and unusual signalment Need better EU-wide communication, standardisation and networking Still a collection of individual countries Guidance and kit distribution for testing ECDC Director Marc Sprenger: 51st Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC) in Chicago 2

3 D x Recommendations Screening for O104: PCR for wzx O104 K9 live slide agglutination (cross reactivity with O104) Screening for stx: PCR, RT-PCR, commercial EIA kits. Plating of clinical samples: ESBL phenotype: Tryptone Bile X-Glucuronide (TBX) Cefixime tellurite sorbitol MacConkey (CT-SMAC; pink colonies) Confirmation: Standard serotyping, including for H4 PCR of other markers, including absence (e.g. eae) PFGE: non-o157 EHEC CDC Pulsenet protocol, Xba1, Bln1 MLST: ST678 Scheutz et al. Eurosurveillance 16 (24), 16 June 2011 D x Possibilities Specific outbreak strain marker Rapid immuno test (beads, EIA) for O104 Phenotypic marker But what if the next EAHEC (or whatever pathotype) is not the O104 outbreak strain!? WHAT S NEXT? Common virulence markers Stx 2a eae, aggr/aaf, another intestinal adherence analogue (?) Resistance? Virulence markers, pathotype indicators yet to be IDed New Era of Applied Microbial Genomics Rapid generation of de novo sequence data Multiple locations, methods Crowd sourcing approach Technical expertise: do and interpret IT Infrastructure IP and other legal/ethical issues A frontier or a one-off? Source Tracking: Blame & Tame Where: did it come from? Why: Did it emerge? How: Do we stop it from happening again? How is this continued & advanced? Evolutionary model of the current outbreak strain (LB226692) and the historical strain ( ) from 2001 based on whole chromosomal and plasmid data. Source Tracking: Blame & Tame Where did it come from? Not just molecularly Is it born of man or beast? Reservoir carriage: final strain and progenitors Inter-host transmission Conditions for re-combination Mellman et al. PLoS One. 2011; 6(7): e

4 Source Tracking: Blame & Tame Why did it emerge? Drivers for virulence gene exchange? Why the unusual signalment in HUS patients? Why the change in EHEC host paradigm? Modelling to predict future emergence? Role of resistance?: Driver for selection and dissemination: especially ESBL? In animals: if EHEC model. In humans: if EAEC model. Other?: alternative host, environmental residues? Molecular mechanisms: plasmid exchange, role in stx transfer? What is an E. coli pathotype? How do we stop it from happening again? Questions Comments Phylogentic placement of German EAHEC O104:H4 outbreak strain. Minimum-spanning tree based on allelic profiles of E. coli (n=1,144) and an in silico generated hypothetical O104:H4 progenitor. Each dot represents an allelic profile, the number on connecting lines represent the number of alleles that differ between two profiles. Mellman et al. PLoS One. 2011; 6(7): e Food source: Sprouts: farm in Lower Saxony What s Next Source? Definitive source and means of sprout contamination? Localised to seed stock Fenugreek imported from Egypt Ultimate source: bovine vs human vs? Contaminating seed Mixing vessel Itoh et al., 1998 AEM 64:4 4

5 Stx2 phage in O104 most closely related to that of an O111 strain. Stx phage phylogeny usually related to that of the host genome: not so for O104. Indications of recent acquisition of phage Laing 2012 ECDC Director Marc Sprenger at the 51st Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC) in Chicago The current isolate diverges from common enteroaggregative E. coli isolates in the number and nature of its SPATE proteases. Most diarrheagenic E. coli strains encode a single SPATE, and enteroaggregative E. coli strains ordinarily encode two SPATEs 37 ; the presence of three SPATE-encoding genes in the outbreak strain is unusual. One of these SPATEs, Pic, is common among enteroaggregative E. coli strains and cleaves host intestinal epithelial-cell mucins, thereby promoting intestinal colonization, 43 and mucin-related glycoproteins associated with leukocyte immune functions. 44 The outbreak strain also encodes SigA, a SPATE that cleaves the cytoskeletal protein spectrin, inducing rounding and exfoliation of enterocytes. 45 The third SPATE, SepA, is associated with increased enteroaggregative E. coli virulence, but its function is unknown (unpublished observations). We speculate that the combined activity of these SPATEs, together with other enteroaggregative E. coli virulence factors, accounts for the increased uptake of Shiga toxin into the circulation, resulting in the high rates of the hemolytic uremic syndrome. Rasko et al N Engl J Med August 25; 365(8): LOTHAR BEUTIN AND ANNETT MARTIN Journal of Food Protection, Vol. 75, No. 2, 2012, Pages EHEC Pathogenicity The stx2a gene was located on a lambdoid bacteriophage, which was integrated into the genome of EAHEC O104:H4 strains. The Stx2a type belongs to a group of Shiga toxin genes in the Stx2acd group (stx2a, stx2c, and stx2d), which are genetically closely related (84). The presence of these Stx2acd types in classical EHEC strains has been reported to be associated with HUS in infected patients aggr could be used as a candidate marker in addition to stx and eae for future risk assessment of STEC strains from all kinds of samples to detect EAHEC strains. Int. Attaching & effacing lesion Shigatoxin target organs 5

6 EHEC Clinical Manifestations Enteric Asymptomatic Non-bloody diarrhoea Haemorrhagic colitis (HC) Extra-intestinal Haemolytic uremic syndrome (HUS) Thrombotic thrombocytopaenic purpura (TTP) Relatively rare, but severe clinical outcomes 5y av. Of 116 cases/year HUS + EHEC (NNDSS) E. coli O157:H7 Most important serotype Higher virulence: eae, stx 2 Predominant: USA, UK, Japan, [Australia] Economic / trade / regulatory issues Zero tolerance policy for meat stringent testing requirements Massive recalls Produce associated outbreaks Other serotypes: O26, O111, O113, O103, O45, O121 Hungry niche theory USDA big six, EU gang of five Public / Media Perception E. coli O157 - D x Sorbitol MacConkey agar (SMAC) O157 sorbitol-negative colourless colonies Rapid methods =!!!! = $$$ - but what of non- O157 EHEC and other pathotypes? 6