Biology 216 - Lect 18
Introduction to Enterics
Enteric = intestine, but ubiquitous, so 
	opportunists in other sites
Entero as a prefix: enterotoxin, enteropathogenic, 
	enterohemorrhagic....
3 families we will discuss (all Gm- rods):
   1. Spirillaceae (microaerophilic, curved/spiral) - 
	Helicobacter, Campylobacter (5% O2,10% CO2)
	Air is:21% O2, 2% CO2
   2. Vibrionaceae (Ox+) - Vibrio, Aeromonas, Plesiomonas
   3. Enterobacteriaceae (Glu+,Ox-,NO3+) - 
      Escherichia, Salmonella, Shigella, Yersinia, 
      Klebsiella, Enterobacter,Proteus
I. GI tract locations for infection.
	A. Mouth
	B. Esophagus
	C. Stomach (Helicobacter - pyloric ulcer)
	D. Small intestine (duodenum, jejunum, ileum) - 
		Vibrio, ETEC, Yersinia,
		Salmonella, Campylobacter transient.
	E. Large intestine (colon) - Shigella, VTEC (EHEC), 
		Campylobacter, EPEC 
		Clostridium difficile, infant botulism.
	F. General symptoms helpful
		1. Watery - cholera
		2. Dysentery (bloody, mucus, PMN) - 
			Shigella, VTEC, Campylobacter
II. ID50 - dictates transmission - 
	(Vibrio vs Shigella)
	Also note reservoir (human only or 
		non-human carrier)
III. Intoxications - absorbed in small intestine
	A. Staphylococcus aureus
	B. Adult botulism
	C. Diagnosis - history, organisms in 
		food, toxin in feces.
IV. Isolation (primary media depends on specimen)
	Fecal - Highly selective; Selective, 
		Enrichment, Campy 42C
	Non-fecal - selective/BAP
	Principles - inhibit Gm+, Gm- fastidious, 
		allow enterics (bile salts)
	A. Highly selective/diff - fecal only; 
			very high bile; 
		Discourage swarming, some 
			non-pathogenic enterics
		Some so selective, no need to autoclave.
		Ex. XLD (xylose lysine desox); HE (Hektoen Enteric); 
			Salmonella/Shigella
	B. Selective/dif - for both fecal and non-fecal
		Ex. MacConkey (1905) - first used since lac- 
			were pathogens (Salm/Shig)
		Others: MAC, EMB, Endo, Desoxycholate citrate; 
			Sorbitol Mac if suspect O157
	C. Enrichment broth - For Salmonella, Shig, 
			Yersinia in low #
		Principle - pathogen often low #; 
			Place in environ which encourages path
		Ex. 10(2) Salmonella with 10(9) E. coli
			Enrich 6 hours then plate on Mac.
		Ex. Selenite, GNB....
	D. Campy-BAP(Ex Skirows) - BAP with antibiotics
		Incubate 42 C, microaerophilic atmosphere
	E. Others if suspect non-conventional pathogen - 
		Ex. TCBS for Vibrio
V. Tests for ID - principles of majors 
	(variation in results within species)
	A. Triple sugar iron (TSI) and Kligler (no sucrose) 
		- stab and streak
		0.1 % glu; 1 % lac; 1 % sucr - 24 hr 
		Sodium thiosulfate to detect H2S -> 
			ferrous sulfate black
		Reactions: Slant/butt? H2S? Gas? Ex. K/A H2S
		All Enterobacteriaceae are Glu+; K/K = 
			non fermenters (other family)
	B. Sugar fermentation - trypticase; pH indicator; 
		1 % sugar; Durham tube (extra)
	C. ONPG - tests for B-galactosidase ->
			 galactose + ONP (yellow)
		Principle: Need permease and B-gal
		Slow fermenters show up negative 
			since poor transport
	D. IMViC - Originally to differentiate 
		E. coli/Enterobacter (++-- vs --++)
		Indole test: Trp -> indole, filter strip test
		Methyl red - mixed acid ferm (pH< 4.4)
		Voges Proskauer - acetoin by 2,3-butanediol
		Citrate - utilization (Simmon citrate -> 
			basic; other look for cloudy media)
	E. Decarboxylases (Lys, Orn, Arg) - enzyme 
			which specifically removes COO-
		Decarboxylase activated by acid environment; 
			Performed anaerobically.
	F. Phenylalanine deaminase -> 
		phenylpyruvate (green with FeCl3)
		Only tribe + is Proteae (Proteus, Providencia) 
			and some Yersinia
	G. Urease -> 2 NH3 + CO2 (basic magenta color)
	H. Rapid tests - show kit tests
	I. Serotyping - used for Salmonella and 
		to speciate Shigella
VI. Enterobacteriaceae ID schemes - SLIDES
	A. Grouping by Tribes - Edwards/Ewing divided 
		into 7 tribes by stable characters.
	B. Checkerboard matrix - tedious; Confidence 
		levels per test >90 %; Cumulates error
	C. Flow diagram - Groups of tests to increase confidence
 
Biochemical Tests
1. B-galactosidase
	
	ONPG	---------> galactose + ONP (yellow)
 
2. Tryptophanase
 
	Tryptophan------------> indole + ammonium + pyruvate
 
3. Decarboxylases (Lys, Orn, Arg) - 
	Enzyme which specifically removes COO-
	Activated by acid environment
 
	Ex. Lysine ----------------> Alkaline amine + CO2 
 
4. Phenylalanine deaminase
 
	Phenylalanine ------> phenylpyruvate (green w/ FeCl3)
	Helpful with Proteae (Proteus, Providencia) 
 
5. Urease 
 
	Urea------------------>CO2 + 2NH3 (basic magenta color)