Biology 216 - Lect 20 Campy/Shigella/EHEC Dysentery and invasive - Shigella, Campy, EHEC (VTEC) I. Shigella - Close relative of E. coli; Not normal gut flora; A. Gm- rods, lac-, non-motile, no formate dehydrogenase. 1. 4 species - biochemically and serologically 2. Plasmids - large 220 kbp plasmid - Sereny test; invasive; EIEC; O Ag side chain 37 kb necessary for epithelial cell invasion - escape phagosome (cloned in E.coli) 3. Shiga neurotoxin (S. dysenteriae only): Enterotoxin, neurotoxin, cytotoxin. MOA - Blocks protein synthesis (not ADP-R), alters 60s Role in some inflammation and necrosis (cell death, ulceration) X-some encoded. Evidence for Hemolytic Uremic Syndrome (HUS) - 1. Correlation between HUS and shiga toxin. 2. Shiga-like E. coli toxin acts same 3. Induces cytokines -> damage. Note: Shiga toxin not essential for dysentery symptoms. Mutants still virulent. B. Pathogenesis and disease - appox 20,000/yr in US 1. Transmission - ID50 (10 - 200); person -> person; Survive stomach acid well. Man is only natural host (& S. typhi) Reservoir - patient and carrier (up to 1 month after disease) Fecal -> oral (4 F's - fingers, flies, food, fomites) Poor sanitary practices: Food handlers; day care; mental institution (1350/100K, 6 ave) 2. Multiply as they pass sm intestine - enterotoxin -> diarrhea. 3. Large intestine - 1 - 4 day incubation Plasmid mediated penetration within epithelia to lamina propria Induce phagocytosis by actin/myosin aggregation - kill epithelia inside Genetics of phagocytosis, invasion, cell-cell spread worked out well. Exit phagosome and grow in cytoplasm. Inflammation - multiplication, shallow ulcer, PMN, slough epithlia, bleeding, musus 4. Symptoms: dysentery, fluid loss, abdominal pain, fever, 5. Self limiting (1 - 4 weeks) - mortality in young and old Renal failure from vascular damage, clots.... HUS (only S. dysenteriae) C. Lab diagnosis 1. Clinical - fever, dysentery (suspect Shigella, Campy, EHEC, Giardia, Entamoeba, Yersin) 2. Isolation and ID - Specimen is stool or rectal swab -> Mac, XLD, Biochemicals, serology D. Treat - Fluid if severe dehydration (rare); Antibiotics (though carry R-factor to many) E. Control - Sanitation, hygiene, cohorting. Vaccine - none yet; Exptl live (Mutant penetrate w/o growth; E.coli/Shig hybrid; Cloned O-Ag into plasmid into S. typhi) II. Pathogenic E. coli - Certain O Ag types A. ETEC = cholera-like, LT, ST (STa methanol soluble and STb not) ST activates guanylate cyclase ->, CFA, plasmid mediated. EAggEC - Aggregate in clumps on small intestine, produce ST like toxin. EPEC - Close attachment and effacement at small intestine. Signal transduction and Ca++ uptake -> actin rearrangement -> cupping. Tight adherence -> Loss of water absorption (one explanation for diarrhea) EIEC - large plasmid - disease identical to Shigella, no toxin and no HUS EHEC = VTEC = O157:H7 (90% of cases) (and O111 and others) - Shiga-like toxin(from lysogeny) -> HUS (vascular endothelial cell damage) Now reportable - 1996: 2000 cases (many more) Disease: Carried by 1 % of cattle Hemorrhagic colitis precursor to HUS (D + HUS) HUS - #1 cause of infant renal failure Renal damage, anemia, renal failure, low fever, 3 % mortality Highest mortality less than 5 yr and older than 60 yr B. Lab - Difficult to differentiate - Sorbitol MacConkey for O157, then agglutination by state. Microaerophilic group (Campylobacter and Helicobacter pylori) III. Helicobacter pylori - Correlate with stomach and intestinal ulcers. A. History - Barry Marshall found Campylobacter in old stomach ulcer patients 1986 - Treated 50 % chronic ulcers w/ Tagamet -> 5 % cure Treated 50 % w/antibiotics + bismuth (Peptobismol) -> 80 % cure 1987 - Teaspoon of pure culture - > ulcer in 2 weeks -> treat with antibiotics Current - correlates with gastric carcinoma (2nd leading form of cancer in world) B. Bacteriology Gm- curved and spiral, similar to Campylobacter (DNA different) Urease - neutralize small area, only important in intitial colonization. Ammonium and irritation may cause inflammation. Vacuolating cytotoxin -> tissue damage (speculative) C. Disease - chronic peptic and duodenal ulcers - Good since bacterial diseases treatable. Endogenous or exogenous - unknown Increased carriage with age Urease -> micro habitat neutralized -> initial colonization. Grow in mucin layer close to mucus secreting cells. PMN, lymphocytes -> inflammatory damage. D. Lab - Gastric biopsy from endoscopy -> urease, some culture (like Campy except 37 C) May miss since focal (patchy in non-acid secreting mucosa) Urea orally -> ammonium test Serology - Need for better tests. Anti-urease antibody may be helpful. ELISA and IFA tests getting better. E. Treat - Antibiotics and bismuth effective (or 2 antibiotics). Need for vaccine Old treatment (before known infection) - diet, lifestyle, surgery, IV. Campylobacter (major cause of diarrhea (similar to Shigella) A. Bacteriology - Gm- curved rods, coccobacilli, or gull winged (2 joined), darting motility Obligate microaerophilic - 5% O2, 10 % CO2, 85% N2 (Air is 21 % O2, 78 % N2) C. jejuni grows best at 42 C, fastidious (BAP w/ antibiotics) B. Pathogenesis and disease - C. jejuni (C. fetus systemic disease, but rare) 1. Significance - Before 1972 rare; 1972 media and atmosphere worked out. Today - Estimated 2 million/yr in US (higher than Salmonella and Shigella) 2. Transmission - ID50 low, so foods and person/person transfer. Normal flora of cattle (40 %); Chickens; Humans: Ingestion of food, water, raw milk, day care/ 3. Colonization - Small (primarily small) and large; 1 - 7 day incubation; 4. Invasion - ulceraton, diarrhea (some produce CT related toxin), fever, pain, headache High asymptomatic (1 - 40 %), most get disease. 5. Self limiting in 1 - 2 weeks, antibiotics helpful, 1/4 relapse even with antibiotics. C. Lab - Culture feces and food. 1. Feces - Enriched/Selective (Skirrows, Campy BAP), 42 C, microaerophilic 2. Food - CDHS (Calif Dept Health Serv) - Homogenize; Filter thru 0.65 um; Centrifuge; pellet -> enrichment broth (Thio in Campy atmosph), plate from top. 3. ID - Gm- curved, gull wing, colony (flat, gray, mucoid), Nal, Ceph, Ox, Cat 4. SeroID since invasive - several tests (ELISA) D. Treat/Prevent - self limit, antibiotics, analgesics (pain), no vaccine, pasteurize and cook.