Biology 216 - Lect 28 - Obligate Anaerobes
Obligate anaerobes - Grow only in absence of O2 and 
	killed by exposure to O2
Disease from non-sporeforming anaerobes 
	(non-Clostridia) is endogenous.
A. General bacteriology - probably left over from pre-plant era
	Habitat - soil, water, animals
	Often associated with facultative (reduce O2)
	Mouth 50 % anaerobes; Colon 1000:1 anaerobes; 
		Sebacious glands
	Why restricted to anaerobic environment?
		1. O2 toxicity - no SOD, catalase or peroxidase 
		(some have these)
		2. Redox potential too low for proper e- transport
		 (oxidation of enzymes and carriers)
B. Organisms (Sporeformers Clostridium 
	discussed next period in detail)
	1. Gm negative rods - 2 groups of major medical importance
		a. Bacteroides - short, vacuolated.
		     B. fragilis group of 5 species - colon, abdominal infection,
			 virulence capsule, Pen resist (B-lactamase)
		     Prevotella melaninogenicus and Porphyromonas - 
			black pigmented, red fluorescing, oral, lung
		b. Fusobacterium - long thin tapering (fusing) 
			- normal flora in GI, oral
		     Lung and brain, ulcerative gingivitis
	2. Gm negative cocci - rare in disease - omit
	3. Gm positive rods - few in disease - Actinomyces, 
		Bifidobacterium, Propionibacterium
	4. Gm positive cocci - (anaerobic strept) -  
		Peptostreptococcus, 
		oral cavity, lung/brain
C. Pathogenesis - little is known
	1. Endogenous (except Clostridium) - 
		Normal flora dictates disease site.
	2. Colonization - areas with low O2
		Trauma - devitilization of tissue
		Ischemia - diabetes, necrotic areas, inflammation necrosis
		 (decreased O2 killing by PMN)
		Facultative coinfection - polymicrobic
	3. Lesion - abscess, pus, putrification (proteolysis)
	4. Diseases - based on location
		Lung - anaerobes 50 % of lung abscesses; 
			Aspiration or orals (alcoholic, viral, anaesthesia, ...)
		CNS - hematogenous spread from lung (Bacteroides, 
			Fuso, Peptostrep, Actino)
		Intraabdominal - large inoculum spills into peritoneum 
			(Trauma, appendicitis, Carcinoma, Surgery)
			Inflammation walls off low inoculum; Large inoculum -> 
				abscess, necrosis, debride.
D. Diagnosis - Becoming more aware of anaerobe disease 
		with better isolation techniques.
	1. Clinical - abscess, gas in lesion, odor, history, near mucosal site
	2. Specimen collection - beware normal flora 
		contamination and O2 exposure
		Ex. Sputum will have normal flora anaerobes; 
			blood, CSF.... any anaerobe is significant.
		Collect anaerobically (aspirate wound)
		Process immediately (15 min) or hold in transport medium 
			(reduced) - inject specimen into capped.
	3. Direct exam - Gm stain (stain poorly, pleomorphic, fusiform), 
		Fl-Ab for Bacteroides. 
	4. Inoculation of media -care and expediency - 
		examine blood culture and blind subculture.
		Minimal O2 exposure, pretreat media to reduce, 
			PRAS (Pre-reduced anaerobic sterilized),
			boil broth, Store plates in N2 (fish tank), thioglycolate, 
				enriched (Vit K), antibiotics to select.
	5. Anaerobic growth systems
		a. Glove box - 90 % N2, 10 % CO2 (flush 20 times); 
			Entry chamber, media, incubator, electric loop.
		b. Anaerobe jar - Evacuate with non-O2 gas
			Gas Pak - H2 generator, palladium -> H2O; Small versions; 
			Redox indicator (LMB or reazurin)
	6. ID - Biochems, kits (API-20 A), MiniteK
		Aerotolerance test; Presumpto (esculin, casein, starch...)
		Antibiotic sensitivity is species indicator - Kn, Colistin, Vanco...
		GLC - acid end products; Ether extract, 
			derivatize (methylation), compare retention w/ standards
E. Treatment - Surgical debridement; Combination antibiotics 
	(Pen effective except B. fragilis - clinda, metronidazole)
	Metronidazole (flagyl) - reduced inside anaerobes 
		& necrotic area -> active DNA damage