Prerequisites:  MB 540, and CH518 or departmental permission.



Description:  Basic biology of pathogenic anaerobic bacteria.  Fundamentals of anaerobic bacteriology and the

development of disease with the avian species used as a model.  Where appropriate pathogenicity in other veterinary

species and humans will be discussed.  Basic clinical methodology, anaerobic bacterial identification,  pathogenesis and

current literature will be dealt in detail.  Current literature will be examined in detail.



I.  Introduction

	A.  Defining an anaerobe.

		1.  How anaerobes differ from aerobic bacteria.

			a.  Basic physiology of anaerobes.

			b.  Substrate-level phosphorylation.

			c.  Electron transport.

			d.  Anaerobic ATP production via respiration.

			e.  Anaerobic ATP production via fermentation.

		2.  Categories of oxygen tolerance.

		3.  Basic redox chemistry.

			a.  Measurement of redox potential.

			b.  Electronegative coupling and electron flow patterns.

			c.  Maintenance of redox equilibrium.

		4.  Oxygen lethality theory.

			a.  Oxygen acting as an oxidant.

			b.  Oxygen acting as a toxic agent.

			c.  Free radical formation and molecular oxygen reduction.

				1.  Superoxide anion formation.

				2.  Hydrogen peroxide formation.

				3.  Hydroxyl radical formation.

				4.  Singlet  oxygen formation.

 				5.  The role of SODs in bacteria and higher organisms.

					a.  Metalloprotein catalyzed dismutation of superoxide 

					     anion.

					b.  Superoxide dismutase theory.

					c.  Two-phase oxygen lethality.



	B.  Anaerobic bacterial ecology.

		1.  Current literature review.

		2.  Alimentary tract function.

		3.  Intestinal contents.

		4.  Mucosal architecture and its affect on colonization.

		5.  Intestinal bacteria.

		6.  Bacterial distribution.

		7.  Bacterial numbers.



	C.  Disease etiology- a conceptual introduction.

		1.  Definition of diseased state.

		2.  Aberration from steady state dynamics.

		3.  Local ecology effects.

		4.  Physiological affects and interaction with local flora.

	D.  Anaerobic bacteriology history.



II.  Disease etiology

	A.  Sporeformers.

		1.  Endospore architecture.

		2.  Endospore formation.

		3.  Endospore germination.

		4.  Current literature review.

		5.  Bacilli

			a.  Clostridium sp.

				1.  Botulinum toxin groups.

				2.  Structural components of pre-toxin and nicked toxin 

				     stereo-chemistry.

				3.  Phage involvement

				4.  Intracorporeal toxin production.

			b.  Bacillus sp.



	B.  Non-sporeformers.

		1.  Gram positive.

			a.  Bacilli.

				1.  Actinomyces sp.

				2.  Arcanobacterium sp.

				3.  Bifidobacterium sp.

				4.  Eubacterium sp.

				5.  Lactobacillus sp.

				6.  Odds and ends.



		2.  Gram negative.

			a.  Bacilli.

				1.  Bacteroides sp.

				2.  Campylobacter sp.

				3.  Porphyromonas sp.

				4.  Prevotella sp.

				5.  Infrequent isolates.

			b.  Spirochetes.

				1.  Treponema sp.

				2.  Borrelia sp.



			c.  Cocci.

				1.  Minor species.

  

		Each of the bacteria will be reviewed in terms of the following criteria:

		1.  Zoonotic potential

		2.  Pathogenesis

		3.  Transmission

		4.  Incubation

		5.  Signs

		6.  Lesions

		7.  Differential diagnosis

		8.   Reported treatments

		9.  Biochemical characteristics useful for identification.



Note:  Some of the bacteria and bacterial diseases discussed in this section will be from other than avian species.  These

have been included for familiarization with the genera and because they are better characterized in other species.





III.  Endogenous flora/Exogenous flora.

	A.  Primary pathogens.

		1.  Etiological development.

	B.  Secondary pathogens.

		1.  Etiological development.

	C.  The "anaerobe scenario".



IV.  Pathogenesis/Virulence Factors.

	A.  Gram positive organisms.

		1.  Toxins- A primer.

			a.  Enterotoxin

				1.  Multi-subunit proteins vs. small heat-stable polypeptides.

				2.  Clostridium perfringens toxins in depth.

					a.  CPE spore coat structure and function.

					b.  Pathophysiological effects of CPE.

					c.  Cytotoxicity expression.

					d.  Neurotoxin mode of action.

						1.  Exocytosis.

						2.  Binding, Internalization and axonal 

						     transport.

						3.  Collagenases.

						4.  Clostridium botulinum toxins.

							a.  Toxin types

							b.  Toxin structure and

							     differentiation.

							c.  Genetics/plasmids in C. 

							     botulinum toxin expression.

	B.  Gram negative organisms.

		1.  Capsules.

		2.  Adherence.

		3.  Other virulence factors.



V.  Anaerobic etiology signs.

	A.  The "magnificent seven".



VI.  Selection and collection of anaerobic samples.

	A.  Defining acceptable specimens.

	B.  Defining unacceptable specimens.

	C.  Site selection and preparation.



VII.  Transport of anaerobic specimens.

	A.  Aspirates.

	B.  Swabs.

	C.  Tissue.

	D.  Blood and other fluids.



VIII.  Anaerobic specimen processing.

	A.  Macroscopic examination.

		1.  Colony morphology.

	B.  Microscopic examination.

		1.  Modified gram stain techniques.

		2.  Cellular morphology.

	C.  Media selection.

		1.  Selective media.

		2.  Non-selective media.

		3.  The use of PRAS media for strict anaerobes.

		4.  Specimen techniques.

			a.  Aspirates.

			b.  Swabs. 

			c.  Tissue.

			d.  Blood and other fluids.



IX.  Media preparation.

	A.  PRAS media.

	B.  Reducing agents and indicators.

	C.  Media containing "Oxyrase".



X.  Primary isolation setup.

	A.  Selective media selection.

	B.  Non-selective media selection.

	C.  Solid vs. liquid media.

	D.  Aerotolerance testing setup.



XI.  Principles of anaerobiosis.

	A.  Anaerobic induction.

	B.  Anaerobic incubation systems.

		1.  Anaerobic chambers.

		2.  Anaerobic jars.

		3.  Anaerobic bags.



XII.  Processing suspect anaerobes.

	A.  Clues to the presence of anaerobes.

	B.  Colony descriptions.

	C.  Colony characteristics.

	D.  Confirmation of gram stain results.

		1.  Special potency disks.

	E.  Aerotolerance testing.



XIII.  Presumptive identification of anaerobes.

	A.  Quick tests.

		1.  Fluorescence.

		2.  Disk tests.

		3.  Spot tests.

		4.  Motility tests.

		5.  Lecithinase/Lipase reactions.

		6.  Urease test.

		7.  Oxidase tests.



XIV.  Presumptive identification flow charts.

	A.  Anaerobic, gram-positive cocci.

	B.  Anaerobic, gram-positive bacilli/coccobacilli.

	C.  Anaerobic, "Clostridium-like", gram-positive bacilli.

	D.  Anaerobic, gram-positive diphtheroids.

	E.  Anaerobic, gram-negative bacilli/coccobacilli.

	F.  Anaerobic, Vancomycin-resistant, gram-negative bacilli/coccobacilli.

	G.  Anaerobic, gram-negative, bacilli/coccobacilli resistant to Vancomycin, Colistin and

   	      Kanamycin.

	H.  Anaerobic, gram-negative bacilli/coccobacilli with pitting colonies.

	I.  Fusobacterium sp.

	J.  Anaerobic, gram-negative bacilli/coccobacilli resistant to Vancomycin and Kanamycin,   	 	                          susceptible to Colistin.



Note:  Some of the bacteria discussed in this section will be from other than avian species but are included for

familiarization with the use of flow charts in identification.



XV.  CDC differential media-"Presumpto Plates".

	A.  Plate 1 reactions.

	B.  Plate 2 reactions.

	C.  Plate 3 reactions.



XVI.  Definitive identification of anaerobes.

	A.  Conventional methods.

		1.  Hungate technique.

		2.  Tube techniques.

	B.  Minisystems

		1.  API 20A system.

		2.  Minitek system.

		3.  Rapid ID Ana II panel.

		4.  Crystal ID system.

	C.  Metabolic end-product analysis- gas liquid chromatography.

	D.  Cellular fatty acid analysis- high resolution GLC.



XVII.  Susceptibility testing of anaerobes.

	A.  Antibiotic resistance.

	B.  Criteria for susceptibility testing.

	C.  Testing methods.

		1.  Agar dilution.

		2.  Disk diffusion.

		3.  Broth dilution.

		4.  Broth microdilution.

		5.  Epsilometer techniques.

		6.  Beta-lactamase testing.



XVIII.  Antimicrobial  agents.

	A.  Veterinary medications.

	B.  Human medications.



XIX.  Future directions in anaerobic bacteriology.







Requirements:





Tests:

	Test 1:  Covers sections I-II. (100 points).

	Test 2:  Covers sections III-VIII.  (100 points).

	Test 3:  Covers sections IX-XIV.  (100 points).

	Test 4:  Covers Sections XIV-XIX (100 points).

	Final:   Comprehensive Sections I-XIX.  (200 points).



Missed tests require an official excuse.  Test must be made up by the next class date upon return.

Exams will consist of multiple choice questions, and essays.



Current literature topic presentation:  Subject must be approved by end of first week of class.  Presentations will 

consist of a 30 minute talk (with slides), on a subject approved by the instructor,  presented to the class.  Presentations 

will be made starting on Tuesday of the fourth week of class at 6 PM.  Two presentations will be made each week 

until all are complete.  Grade will be based on: a) Thoroughness of literature review (100 pts), b) Organization and 

presentation of the material (50 points), c) Ability to discuss material and answer questions from audience (50 points).  

Total 200 points.  





Laboratory:  (200 points total including 100 points for laboratory exercises and attendance, 50 points for laboratory

notebook, 50 points for identification of 5 unknown using presumptive methods ).

Laboratory notebook due at completion of final laboratory session.  Attendance and laboratory exercises can not 

be made up.  Because of equipment availability, students may have to work after class hours to complete some 

exercises.  



Schedule:  3 one hour lectures and 2 three hour labs per week. 





Required Textbooks:  Principles and  Practice of Clinical Anaerobic Bacteriology

			P.G. Engelkirk, J.D. Engelkirk, V.R. Dowell

			Star Publishing Company, 1992.





Supplementary readings:  	Free Radicals in Biology and Medicine, 2nd ed.

			B. Halliwell, J.M.C. Gutteridge

			Clarendon Press, 1993.

			

			Intestinal Microbiology

			B.S. Draser, P.A. Barrow

			American Society of Microbiologists, 1985.



			Current literature handouts.



Laboratory Exercises:



Lab 1.

	1.  Safety Briefing.  At the end of this session students will sign a safety SOP.  Violation of this SOP may

	     threaten the health and welfare of the student or instructors.  It is critical to recognize the seriousness

	     of working with pathogenic anaerobic bacteria and anaerobic bacteriological equipment.  Violation of the         	     safety SOP may lead to immediate suspension from this class.

	2.  Introduction to anaerobic bacteriology equipment.  Training in anaerobic chamber use. Emergency

	     procedures.  Standards of good laboratory practice. 



Lab 2.  

 	1.  Oxygen tolerance exercise.  Set up: Clostridium perfringens, Bacteroides fragilis, Escherichia coli.  Incubate

	     anaerobically in chamber and in anaerobic jar, aerobic, CO2, gassed tube incubated in aerobic chamber.

	2.  Gram Stain exercise with same.  Read and record results.



Lab 3.  

	1.  Read  and record results from lab 2.  Record colony morphology. 

	2.  Re-gram stain new growth.  Record results.

	3.  Identify bacteria grown from Lab 2.  Use the Ana system, OneE.

	4.  Subculture Clostridium perfringens, Bacteroides fragilis.



Lab 4.  

	1.  Set up primary plates with Clostridium perfringens, Bacteroides fragilis.

		a.  Brucella agar

		b.  BBE

		c.  KVLB

		d.  PEA

		e.  Thioglycolate backup tube.

	2.  Receive mixed culture

 		Aerobic

			a.  BA

			b.  CA

			c.  MAC

			d.  PEA

        		Anaerobic

			a.  PRAS BRU

			b.  PRAS PEA

			c.  PRAS BBE

			d.  PRAS KVLB



Lab 5.

	1.  Record Results from Lab 4.

	2.  Gram stain.

	3.  Set up EYA with Clostridium perfringens.

	4.  Set up EYA with Fusobacterium necrophorum.

	5.  Subculture Clostridium perfringens, Fusobacterium necrophorum, purified mixed culture bacteria on 	                         BRU/BA.



Lab 6.

	1.    Record results from Lab 5.

	2.  Set up CCFA with Clostridium difficile.

	3.  Identify bacteria growth from Lab 5 BRU/BA using ANA, OneE.

	4.  Subculture the Bacteroides fragilis and Clostridium perfringens.



Lab 7.

 	1.  Set up Porphyromonas spp., Prevotella spp., Clostridium difficile, Veillonella spp. on BRU.

	2.  Set up Bacteroides fragilis and Clostridium perfringens on quad plate.



Lab 8.

	1.  Read results from quad plates.

	2.  Receive chicken ceca and process on:

		 Aerobic

			a.  BA

			b.  CA

			c.  MAC

			d.  PEA

		Anaerobic

			a.  PRAS BRU

			b.  PRAS PEA

			c.  PRAS BBE

			d.  PRAS KVLB



Lab 9.

	1.  Subculture bacteria from Lab 8.

	2.  Perform Gram stain, read and record results.



Lab 10.  

	1.  Set up Clostridium ramosum,  Clostridium clostridioforme, Fusobacterium spp. with special potency disks                        on BRU.

	2.  Set Staphylococcus aureus, Peptostreptococcus anaerobius with SPS disks.

 	3.  Set up Bacteroides fragilis with disk.  



Lab 11.

	1.  Read results from lab 10.

	2.  Set up Clostridium perfringens, Bacteroides fragilis on BRU for E test. 

	3.  Sample recovery and transport exercise with chicken.



Lab 12.

	1.  Record results from E test.

	2.  Discussion of anaerobic susceptibility testing.



Lab 13.

	1.  State Diagnostic Laboratory Visit 

  

Lab 14.

	1.  Computer exercise.	



Lab 15.  

	1.  Computer exercise.



Lab 16.  

	1.  Receive unknown bacterial mixture.

	2.  Work on unknown.



Lab 17.

	1.  Work on unknown.



Lab 18.

	1.  Work on unknown.



Lab 19.  

	1.  Verify ID with biochemical tests.



Lab 20.

	1.  Turn in notebook.

	2.  Group discussion.