Enterics Flashcards
Strain vs serotype
Strain - genetically identical, single precursor
- via mutation or horizontal acquisition
- more specific than species, subspecies
Serotype - same antigenetic determinants
- only surface proteins - may include different strains
- O antigen = LPS (repeating oligosacch + side chains)
- H antigen = flagellin
- K antigen = polysacch capsule
Enteric bacteria
Enterobacteriaciae
All Gram (-) that live in gut
(other Gram + are NOT “enterics”)
Salmonella
Shigella
E coli
Typhoid epidemiology
22 million cases, 200,000 deaths
Mostly developing world, travellers
Fecol oral transmission (contaminated food, water)
Only humans (no reservoir)
Incubation 7-14 days
Salmonella clinical syndromes
S typhi -> typhoid/enteric fever
S cholerasuis -> septicemia
S enteriditis, typhimurium -> acute gastroenteritis
Typhoid presentation
Distinct phases of disease, NOT diarrhea
week 1 - fever, pain, constipation
week 1-2 - bacteremia, Rose spots (bacterial emboli), hepatosplenomegaly, leukopenia, fever
week 2-3 - bowel hemorrhage, rare perforation
can become chronic
Typhoid pathogenesis
Requires high dose (10’3-5 in immunocompetent)
Resistant to stomach pH
Adhesins -> endothelium -> induces endocytosis (via T3SS)
Survive and divide within macrophages (resistant to lysosomes)
Lysis/release -> bacteremia -> spleen, liver, gall bladder
(also endotoxic shock)
Reinfect GI -> bleed
Salmonella virulence factors
Pathogenecity islands - horizontal transposon sequences (aka SPI)
SPI encode for Type 3 Secretion Systems (T3SS)
- “needle” that injects effector protein across bacterial and host membranes
SPI1 -> invasion and endocytosis (“membrane ruffling”)
SPI2 -> protection in endosome
Shigella presentation
Fever (LPS) Bloody diarrhea with mucous (T3SS), cramps Usu self-limiting - bacteremia rare - hemolytic uremic syndrome possible Shed for 1-4 weeks post-sx
Shigella epidemiology
14,000 cases confirmed (likely 20x higher)
Spread: Feces -> Food, Fingers, Flies
No animal reservoir
Children more susceptible
Four species - similar presentation
- dysenteriae - developing world
- flexneri -
- sonnei - most common US
- boydii - India
Shigella pathogenesis
Low inoculum (100), acid tolerant
Specific phagocytosis into epithelium (T3SS, fimbrae)
Escape phagosome -> direct cell-cell spread (polymerize actin)
Apoptosis of macrophages
TNF, IL-1 -> systemic sx (fever)
Shiga toxi -> diarrhea
Shigella tx
Fluids, electrolytes
Antibiotics if severe
- Ciprofloxacin, Bactrim (trimethoprim-sulfonamide)
- resistance increasing
Shiga toxin
Only produced by S. dysenteriae
Exotoxin (released vs endotoxin)
Subunit B binds to intestinal receptor
Subunit A -> inhibits 60S ribosome -> inhibits protein synthesis
- > fluid malabsorption -> diarrhea
- > apoptosis or necrosis -> ulceration
Detect via immunochromatographic after growth in broth
(rare to have toxin +, culture -…wtf?)
Shigella prevention
Sanitation (only humans)
- pools, food, daycare, nursing homes
No effective vaccine (live attenuated not effective)
- possible O-antigen + inactivated Shiga toxin
Overview of enteric identification
Stool sample (blood or gall bladder for Salmonella)
Enrichment broth - bile suppresses non-enterics
Selective and differential media
- bile to suppress others, pH or metabolic indicators
Specific tests
- MacConkey, Hektoen agar
- Kligler agar
- oxidase
- motility
- urease
- Shiga toxin
All grow on glucose, oxidase negative, reduce nitrate
Hektoen agar
Bile + dyes - inhibit non-enteric
Lactose, sucrose, salicin + pH (acid = yellow)
Na2S2O3, Fe
E coli - ferments lactose -> yellow/salmon + bile precipitate (dt acid)
Shigella - non-lactose fermenter = green
Salmonella - non-fermenter, produces H2S -> black (with Fe)
MacConkey agar
Bile + dyes - inhibit Gram +
Lactose + pH (acid = red)
E coli - lactose fermenter -> pink with bile precipitate (dt acid)
Salmonella, Shigella - non-fermenters -> colorless colonies
Treatment for enterics
Treat with antibiotics if bacteremia common!
- S typhi
- S cholerasuis
- NOT S typhimurium, Shigella
Differential diagnosis of diarrhea
Bacteria
- Shigella
- Salmonella
- E coli
- Campylobacter
- C difficile
- rare: Yersinia enterocolitica, Vibrio cholerae, Vibrio parahemolytica
Toxin: S aureus, Bacillus cereus
Viral: Rotavirus, Norwalk
Protozoa: Giardia
Helminths: Strongyloides
Kligler Iron Agar
Tube = anaerobic conditions
Some glucose (all grow)
Lots of lactose (-> more fermentation)
Na2S2O3, Fe
E coli - ferments lactose -> completely yellow + gas
Shigella - only glucose -> some yellow
Salmonella - only glucose, produces H2S, gas -> black (with Fe), gas
Cytochrome oxidase
Final enzyme of electron transport chain
(for aerobic metabolism)
e- + O2 -> H2O (also pumps H+)
Redox dye (TMPD) -> purple if present
All enterics are negative (E coli, Salmonella, Shigella)
Positive for other Gram (-) (Pseudomonas, Neisseria)
Sulfide identification
Specific sulfur metabolism enzyme
S2O3 + H20 -> H2S + SO4
Detect with ferric citrate
Fe + H2S -> Fe2S3 (black) + H+
Salmonella - positive
E coli and Shigella - negative
Salmonella identification
Fecal (or blood for S typhi)
Lactose non-fermenter (black MacConkey, colorless Hektoen)
Motile (vs Shigella)
H2S producer (vs Shigella - black MacConkey and Kligler)
Gas producer from glucose (vs Shigella)
Urease negative (vs Proteus)
Indole negative (vs E coli)
Can serotype O and H antigens, PCR for epidemiology
Shigella identification
Fecal sample
- PMNs in stool = invasive disease
Lactose non-fermenter (green MacConkey, colorless Hektoen)
No H2S (vs Salmonella)
Non-motile (vs Salmonella) - no H antigen (flagellin)
No gas production
Indole negative (vs E coli)
Urease negative
Shiga toxin can be tested via immuno-assay
Overview of Salmonella vs Shigella
Antigens:
O (aka LPS) - both
H (aka flagellin) - Salmonella
Vi - S typhi only
Infectability
100,000 for Salmonella vs 100 Shigella
Reservoir
Salmonella poultry, reptiles, Shigella none
Bacteremia - common in S typhi, S choleraesuis
Vi antigen
Capsule polysaccharide
Specific to S typhi
May promote survival in endosome
Target of ViCPS vaccine (injection)
Endotoxin
= lipid A component of LPS (lipopolysaccharide)
Gram negative outer membrane
Strong innate immune response even to low concentrations
- macrophages -> TNFa -> phagocytes, permeability, PLT adhesion -> micro-emboli
Gram (-) bacteremia -> endotoxic shock
- 20-80% mortality dt fever, inflammation, hypotension, DIC
Typhoid diagnosis
Week 1 = subclinical -> + stool culture
Week 2-3 = symptomatic -> + blood culture
Week 3 = gal bladder colonization -> + stool culture again
Characterize as Salmonella
Typhoid treatment
Generally treated due to bacteremia Acute: Ciprofloxacin Cephalosporin (Ceftriaxone) Chronic: Ampicillin Ciprofloxacin Cholecystectomy
Typhoid prevention
Epidemiology -> control food, water
No animal reservoir -> identify carriers
- may be chronic, assymptomatic gall bladder secretion - ex Typhoid Mary
Vaccines
- Ty21a = live attenuated (oral)
- ViCPS (targets capsule polysacch) = injection
S cholerasuis
Rare - only source is contaminated pork
Ingest 1000 organisms -> short incubation 6-72 hrs ->
Bacteremia, fever, gastroenteritis, microabcesses ->
High mortality
Similar to typhoid (SPI1, SPI2 -> T3SS, endotoxin)
Susceptibility if young, malaria, sickle cell, immunocompromised
E coli overview
Enteric
Normal microflora -> Vitamin K, protective vs pathogens
Pathogenic if acquired virulence genes via phage, plasmid, transposon
- diarrheal - often endemic (25-50% of diarrheal mortality)
- Shiga toxin - epidemic, high mortality
- sometimes also need to lose commensal genes
Enterohemorrhagic E coli
Acquired Shiga toxins (STEC)
(EHEC is specific type of STEC)
Labeled by O and H antigens - O = LPS polysaccharide - H = flagellin Most common is O157:H7 Also O111, O26, O157:H-, O104:H4
vs Commensal (ex K12)
STEC presentation
Very low toxic dose ( endothelial damage)
-> acute renal failure, anemia, thrombocytopenia
E coli is extracellular, localized to GI
Toxin is released -> kidney, etc
STEC pathogenesis
E coli common pilus -> weak attachment (similar to commensals)
-> trigger A/E lesions -> effacement = loss of microvilli
T3SS -> stronger attachment (Tir, Intimin)
-> actin polymerization -> “pedestal”
Hemolysin
Shiga-like toxin
Others
- capsule (K antigen)
- LPS -> inflammation -> sx
- nutrient acquisition pathways
LEE pathogenicity island
Virulence genes + IS sequences -> E coli
“Locus of Enterocyte Effacement”
Type 3 Secretion system (T3SS) - “needle” and pore induced by contact
- Tir -> host cell surface
- Intimin -> bacterial surface
- both on eae gene -> bind together for strong adhesion
EHEC Shiga-like toxin
aka verotoxin
Acquired via phage (more than bacteria)
Multiple different sequences
Bloody (hemorrhagic) diarrhea
Cytotoxic -> endothelium -> HUS, kidney, CNS
Subunit B -> binding and uptake
(cows don’t have host target receptor CD77 -> immune to E Coli)
Subunit A -> ribosome binding and RNA cleavage
Hemolysin
aka RTX toxin (“repeats in toxin”)
Plasmid
Pore-forming protein -> lysis
Common in other Gram (-), meningitis
Enteropathogenic E coli (EPEC)
Diarrheagenic E coli
Usu person-person vs food
Leading cause of childhood diarrhea in developing
Pathogenesis:
- Bundle forming pili (Bfp) -> adherence
- A/E effacement lesions -> T3SS -> Tir, Intimin
- No toxin in stool (diarrhea is direct effect of bacteria)
“Moderately invasive”
Diarrheagenic E coli
Enteropathogenic (EPEC) - childhood in developing world
Enterotoxigenic (ETEC) - travellers, infants
Enteroaggregative (EAEC) - persistent -> weight loss
Enteroinvasive (EIEC)
Enterotoxigenic E coli (ETEC)
Traveller’s diarrhea and infants in developing
Virulence factors
- fimbriae - adherence to small intestine (NOT pili and T3SS)
- heat labile toxin (LT) - similar to cholera toxin
- > adenylate cyclase -> cAMP -> CFTR (transmembrane channel) -> Cl- secretion, less Na uptake -> watery diarrhea
- heat stable toxin (ST) -> guanylate cyclase -> cGMP -> ion movement -> watery diarrhea
Not invasive
Enteroaggregative E coli (EAEC)
Childhood -> persistent diarrhea -> weight loss
Similar to EPEC with more aggressive epithelial adhesion (biofilm)
No A/E effacement lesions
Not invasive
Virulence factors:
Enteroaggregative stable toxin (EAST) - similar to heat stable
Pet = plasmid-encoded toxin
Hemolysin
ex 2011 Germany outbreak = EAEC + Shiga-toxin phage
Enteroinvasive E coli (EIEC)
Mostly developing countries
Non-fimbrial adhesions
Invades cells -> multiplication -> spread to neighboring
Watery diarrhea + blood/mucous (similar to Shigella)
Not systemic
Does not produce familiar toxins (ST, LT, etc)
Diffuse adhering E coli (DAEC)
Older children, developing
Individual (diffuse) attachment
Poorly characterized pathogenesis and toxins
E coli diagnostics
Stool enriched in Gram (-) bile broth
Lactose fermenter -> yellow on MacConkey or EMB
Sorbitol - O157:H7 negative vs commensals positive
Confirmation: O157 antigen -> latex agglutination H7 serology Shiga-toxin immunoassay PCR, DNA probe (vs stx1, stx2, eae, hlyA)
Epidemics (spinach outbreak):
pulsed field gel electrophoresis ->
E coli treatment
Supportive fluid management (prevent dehydration, HUS pathology)
STEC:
Do NOT give antibiotics (more toxin released) or anti-motility
ETEC:
Loperamide, Fluoroquinolone (Cipro), Azithromycin, rifaximin
EPEC: susceptible antibiotics if severe
EAEC: fluoroquinolones for travellers, HIV
