Pathogenesis Structure of Shiga toxin

Shiga toxin 1 and shiga toxin 2 o 2 subunits o A enzymatically active A1 and A2 subunit, hairpin disulfide bond between Sensitive to cleavage by trypsin, but is in most cases cleaved by furin Occurs optimally in low pH A1 inhibits protein synthesis, removes one adenenine from 28S RNA of the 60S ribosomal subunit o B binds to cell surface contains 5 identical subunits, binds to glycolipid receptors binds to Gb3 receptor (high expression in kidney)

Entry to cells
via Clathrin-coated pits, mediated by toxin o mechanism behind toxin-induced transport unknown transported to Golgi, required for intoxication o cells that bind but are resistant to toxin lack golgi transport for the molecule o pathway via late endosomes, dependant on Rab9 (GTP-binding protein) responsible for transport of mannose-6-phosphate receptor

Effect of toxin on cells

EAEC is non-invasive o Secretes toxins into gut mucosa o Intestinal epithelial cells able to tranlocate Stx from apical to basolateral side, allows entry of Stx into systemic circulation A subunit contains RNA N-glycosidase o Removes one adenine from adenosine from 28S RNA o General inhibition of protein synthesis Shiga toxin can induce synthesis of (from macrophages) o Interleukins (IL-8, IL-1, IL-6) o TNF Induce apoptosis o Induce DNA degradation, release of cellular content o Facilitate proteolytic attack on neighbouring cells o Associated with enhanced expression of Bax (pro-apoptotic protein)

Transmission Outbreak Monitoring, Some Characteristics and the Mechanism of Spread Outbreak Monitoring What happened? Outbreak of Shiga-toxin producing E. coli enteroaggregative pathotype (1) Second largest outbreak of this pathotype (2) Japan Over 6000 school children Radish sprouts Mainly in Germany (North) Subsequently reported in 15 other countries Most likely travellers Human-to-human transmission (1) Number of HUS cases = 908 Deaths = 34 (1; 2) Number of non-HUS cases = 3167 Deaths = 16 (1; 2) Were the appropriate checks/cautions in place? Not to eat sprouts of any origin Including home-grown raw sprouts and seedling (3) Travellers to follow food safety measures when handling fruits and vegetables (4) Thorough washing of raw vegetables and peeling Avoid consumption of raw vegetables (2) When source discovered, company shut down and all products recalled (5) Concerns there might have been cross contamination of other seed types (3) Who was responsible for monitoring? Federal Institute for Risk Assessment (BfR) (3) European Centre for Disease Control and Prevention (2) What mistakes were made?

 Outbreak initially thought to be from organic cucumbers imported from Spain Authorities warned to avoid eating raw cucumbers, tomatoes and lettuce Other countries banned cucumber imports and removed vegetables from sale to curtail the outbreak (4; 5) Some Characteristics Who was most affected? Usually in children < 5 years In this outbreak, 88% of cases over 20 and around 2/3 women Increase in organic food and sprout consumption (2) Implicated as etiologic agent of diarrhoea in travellers, children and HIV-infected patients as well as several foodborne outbreaks Wide-spread among human populations (1) Mechanism of spread Most likely reservoir was sprouts from farm in Northern Germany Lower Sacony, Bienenbuettel (5) Not unusual source of outbreak (2) Originated from Fenugreek Seed shipment from Egypt (3) Human-to-human transmission from bad hygiene practices Faecal-oral Especially in food industry (2) Symptoms: Characteristics of the disease Who was affected most children, adults elderly? Development of disease, symptoms, complications, comparison with enterohemorrhagic E. coli May 2011: unusual strain characterised by high incidense and severity of diarrhea caused by Shiga-toxin producing E. coli. 908 cases (34 deaths) with hemolytic-uremic syndrome,yea 3,167 cases (16 deaths) without Definition of STEC (without HUS) requires presence of (as well as lab confirmation: o At least on of diarrhea (3+ loose stools in 24 hours), abdominal cramps or vomiting. Physicians were required to report clinical symptoms that appeared compatible with HUS t health department, fowarded to state level. All suspected cases sent through based on physician diagnoses. Disease onset defined as onset of diarrhea regardless of HUS development. Definition of HUS Syndrome thrombo cyto penia (platelet count <150,000/cubic cm), hemolytic anemia, and acute renal dysfunction. Acute renal dysfunction only met if at least one of the follow is present: o Increase in serum creatinine level (non-specific?), oliguria (small amounts of urine production), anuria (no urine production), proteinuria (abnormal protein content in urine), or hematuria (bloody urine) o abnormal urine production (2) In patients presenting at Hamburg Univeristy Medical Centre positive for Shiga-toxin-

producing E. coli between May 19 and June 1 2011 at first presentation (N= 141): o 91% bloody diarrhea, 89% Abdominal pain, 34% nausea (surprising?), 26% vomiting. o However when comparing those without HUS to those with HUS in adults: Bloody diarrhea 95: 100% with, Nausea 30:50% with, Vomiting 16:56 with. o In children appeared to be opposite? Nausea 75:40% without! o Only symptom that was more severe without was abdominal pain 90:82% without. However abdominal pain not necessarily assoc with mortality (fluid loss, organ failure). o 25% of patients in outbreak developed HUS despite public knowledge to seek medical attention if see bloody diarrhea developed world problem? Author predicts led to more cases. Prospectively: o Only 22% total had previous contact with another STEC patient

Diagnosis - Methods (traditional vs current), limitations; genome sequencing how was it used in this outbreak? Traditional methods: refer to old categorical diagnoses by clinicians Enterohemorrhagic E. coli - such strains contain enterocyte effacement pathogenicity island facilitating colonisation of large intestine. (2) STEC diagnosed by screening for Stx using enzyme immunoassays or PCR. Immunoassay time consuming vs. PCR faster and more specific. PCR also used to detect virulence factors National Reference Centre for Salmonella and Other Bacterial Enteric Pathogens confirms, culturally isolates and characterises from samples positive for Stx. Biochemical characterisation minimal detail. PCR assays (1-3) showed German outbreak steain lacked pathogenicity island, rapidly released genomic sequencing results (7) and cell adherence assays (1) confirmed that the O104:H4 strain was enterrohemorrhagic. similar to strain isolated from Central African Republic patient suffering HIV whos strain did not produce Shiga toxin. Previously, only 3 enteroaggregative E. coli genomes had been sequenced genome-wide understanding was lacking. Used 3rd gen or next gen DNA sequencing technology to determine genome sequwnces of O104:H4 stain, 7 other enteroaggregative strains, and 4 reference strains. Three sequencing instruments were used in parallel fast. Mean read length 2067 bases in 5 hours per isolate. Found 99.97% of bacterial chromosome at 99.97% accuracy.