This document provides an overview of microbiology concepts including: 1) It lists common human pathogens and notes that 61% are zoonotic. 2) It discusses the office hours, labs, and lectures for a microbiology course. 3) It summarizes key discoveries and advances in microbiology, infectious disease, and molecular biology from the 18th century to present.

1. MICROBIOLOGY FALL 2011

2. Office Hrs. MW 11-12; STC 239 Tue 10-1; NEWDL 312 Independence Rd. Laboratory M/W 12-3 Lecture MW 3-4:15

3. Syllabus

5. Dasek et al. 2000

6. Human Pathogens 1415 known human pathogens 61% are zoonotic

7. Wild Animals Involved in MPXV Transmission (USA, 2003) Gambian Giant Rat (Crycetomys sp.) Prairie Dog (Cynomis sp.)

8. Ghana TX WI IL * IA 15/4 09/04 Entry and Distribution Route of Crycetomys sp . and Cynomys sp. Involved in MPXV Outbreak (USA, 2003) Source: MMWR 52 (23), CDC, 2003. 15/4

9. Index case in Marshfield, 26 May 2003: Disseminated lesions.

10. Distribution of MPXV Cases in the USA WI IL IN OH KS MO Number of cases = 87 WI = 38 IN = 24 IL = 19 OH = 4 KS = 1 MO = 1 Source: MMWR 52 (23), CDC, 2003.

11. Microbiology Has been defined as the study of organisms and agents too small to be seen clearly by the naked eye – that is, the study of microorganisms

12. Scope and Relevance Microorganisms are exceptionally diverse, are found almost everywhere, and affect human society in countless ways. Modern microbiology is a large discipline with many different specialties: medicine, agricultural & food sciences, ecology, genetics, and biochemistry.

13. Microbiology

14. Microbiology

15. Microbiology Spontaneous Generation Molecular Biology/Genetics Fermentation Infectious Disease

17. Infectious Disease - History 1798 Jenner, smallpox vaccine 1835-1844 Bassi, silkworm disease 1847-1850 Semmelweis 1849 Snow, cholera 1861 Pasteur disproves spontaneous generation 1867 Lister antiseptic surgery

20. Infectious Disease - History 1876-1877 Koch anthrax is caused by Bacillus anthracis 1881 Pasteur develops anthrax vaccine 1884 Koch’s postulates published Autoclave developed Gram stain developed 1885 Pasteur develops rabies vaccine

21. Infectious Disease - History 1887 Petri dish developed 1890 Von Behring prepares antitoxins for diphtheria and tetanus 1899 Ross shows that mosquitoes carry malaria 1910 Ehrlich – magic bullet 1923 – Bergey’s Manual 1 st edition

22. Infectious Disease - History 1929 Fleming discovers penicillin 1935 Domagk discovers sulfa drugs 1975 Lyme Disease 1983 HIV 1986 hepatitis B vaccine – genetically engineered

23. DISEASE Occurrence of Disease

24. Signs & Symptoms of Disease Fever Fatigue White Blood Cell Count Pain Muscle Aches Blood Pressure

25. Molecular Biology 1941 Beadle and Tatum, one gene-one-enzyme hypothesis 1944 Avery shows that DNA carries information during transformation 1952 Hershey and Chase viral infection of bacteria 1953 Watson & Crick

26. Molecular Biology - History 1961 Jacob & Monod propose the operon model of gene regulation 1961-1966 Nirenberg et al. elucidate the genetic code 1970 Arber & Smith – restriction endonucleases 2000 Human genome sequenced

27. Fermentation - History 1857 Pasteur shows that lactic fermentation is due to a microorganism 1897 Buchner prepares yeast extract that ferments

28. Spontaneous Generation 1799 Spallanzani attacks spontaneous generation 1861Pasteur disproves spontaneous generation

29. Kingdoms Animal Plant Fungi Protista Prokaryotic

30. Classification Kingdom Phylum Class Order Family Genus species

32. Infectious Agents / Microrganisms Bacteria Fungi Prions Protozoans Helminths

33. Toxins – Exo and Endotoxins

49. Prokaryotes Greek – before a nucleus

51. Prokaryotes / Eukaryotes There are a number of differences between bacterial cells and plants, animals, fungi and protozoans.

52. Prokaryotes / Eukaryotes Size of cell 1-10 u m diameter 10-100 u m diameter Nucleus No nuclear membrane True nucleus Membrane bound organelles absent present Flagella 2 protein building blocks complex

53. Prokaryotes / Eukaryotes Glycocalyx Slime layer Absent Cell Wall Usually present Complex When present, simple Plasma membrane No CHO and lack sterols Sterols and CHO present Cytoplasm No cytoskeleton cytoskeleton

54. Prokaryotes / Eukaryotes Ribosomes Small size 70s Large 80s; small 70s Chromosome Circular, lacks histones Linear, with histones Cell division Binary fission mitosis Sexual reproduction No meiosis meiosis

55. Bacterial Classification Cellular Characteristics Morphology – cell shape, cell size, arrangement of cells, arrangement of flagella, capsule, endospores

59. Cellular Characteristics Staining Reactions – Gram stain, acid-fast stain

74. Cellular Characteristics Growth and nutritional characteristics – appearance in liquid culture

75. Cellular Characteristics Growth and nutritional characteristics–colonial morphology

77. Cellular Characteristics Growth and nutritional characteristics pigmentation

79. Cellular Characteristics Growth and nutritional characteristics energy sources, C, N sources,

80. Cellular Characteristics Growth and nutritional characteristics fermentation products

81. Cellular Characteristics Growth and nutritional characteristics modes of metabolism

83. Cellular Characteristics Biochemical Characteristics – cell wall constituents, pigment biochemicals, storage inclusions, antigens, RNA molecules

84. Cellular Characteristics Physiological and Ecological Characteristics – temperature range and optimum

85. Cellular Characteristics Physiological and Ecological Characteristics oxygen relationships

86. Cellular Characteristics Physiological and Ecological Characteristics – pH tolerance range

87. Cellular Characteristics Physiological and Ecological Characteristics –salt requirement and tolerance

89. Cellular Characteristics Genetic Characteristics- DNA G + C DNA hybridization

90. Fimbriae & Pili G- bacteria have short, fine, hairlike appendages that are thinner than flagella and not involved in motility

91. Fimbriae & Pili slender tubes composed of helically arranged protein subunits and are about 3 to 10 nm in diameter and up to several u m long

92. FIMBRIA (s) FIMBRIAE (pl) Composition varies, contain protein Tendency to stick to each other and surfaces Bacterial attachment in aqueous environments Role in colonization  infection

94. PILUS (s) PILI (pl) Hollow, non-helical (9-10nm dia) Filamentous appendages Thinner than flagella, more numerous Example F-pilus ( SEX PILUS ) entry of genetic material during conjugation GRAM -VE BACTERIA ONLY

98. E.M Pili on E. coli – N. gonorrhea

99. Pili & Fimbriae Some types of fimbriae attach bacteria to solid surfaces such as rocks in streams and host tissues Pili – about 1 to 10 per cell, differ from fimbriae: are larger (9 to 10 nm in diameter), they are genetically determined by sex factors or conjugative plasmids and are required for bacterial mating

100. Pili & Fimbriae Some bacterial viruses attach specifically to receptors on sex pili at the start of their reproductive cycle

101. Flagella & Motility Most motile bacteria move by use of flagella, threadlike, locomotor appendages extending outward from the plasma membrane and cell wall. Slender, rigid structures, about 20 nm across and up to 15 to 20 u m long

102. Flagella - Arrangements Monotrichous Amphitrichous Lophotrichous Peritrichous

103. Flagellar Ultrastructure Filament Hook Basal body

106. ARRANGEMENT OF FLAGELLA POLAR - at one or both ends MONTRICHOUS - single e.g., Vibrio sp. LOPHOTRICHOUS - small tufts at same site e.g., Pseudomonas sp.

107. AMPHITRICHOUS - at both poles, e.g., Spirillum sp. LATERAL PERITRICHOUS - surrounding entire cell, e.g., Proteus sp.

108. MOTILITY CHEMOTAXIS: movement towards/away from chemicals MAGNOTAXIS: orientation of movement in magnetic field Aquaspirillium magnetotacticum - Magnetosomes (Fe 3 O 4 crystalline magnetic iron oxide) PHOTOTAXIS: Differences in light intensity THERMOTAXIS: heat

109. Rotation of flagellar motor: reversible Clockwise (CW) or Counterclockwise (CCW) Smooth swimming/running motion Motor rotates CCW direction Flagella sweep around cell (in common axis)

110. Tumbling motion Motor reverse (CW direction) Flagella disperse

112. Flagellar Synthesis Complex process – involving at least 20 – 30 genes Flagellin subunits are transported through the filament’s hollow internal core. When they reach the tip, the subunits spontaneously aggregate so that the filament grows at its tip rather than at the base Self - assembly

113. The Mechanism of Flagellar Movement The filament is in the shape of a rigid helix, and the bacterium moves when this helix rotates

114. The Mechanism of Flagellar Movement Act like propellers on a boat

115. The Mechanism of Flagellar Movement E. coli rotates 270 r.p.s., Vibrio alginolyticus averages 1,100 r.p.s.

116. Distance and speed: 20-90  m/sec Equivalent to: 6ft human running 5 body lengths/second

117. AXIAL FILAMENTS Modified flagellum Long thin microfibril, inserted into a hook, entire structure enclosed in periplasmic space ENDOFLAGELLUM

119. CELL SURFACE 3 Basic Layers - GLYCOCALYX CELL WALL CELL MEMBRANE collectively termed CELL ENVELOPE

120. Glycocalyx Is a network of polysaccharides extending from the surface of bacteria and other cells Aids in bacterial attachment to surfaces of solid objects in aquatic environments or to tissue surfaces in plant and animal hosts

121. GLYCOCALYX External mucilaginous layer Surrounds cell Shows organisation SLIME LAYER - abundant, easily washed off (poorly organised) CAPSULE - abundant, not easily washed off

122. Capsule Well organized and not easily washed off Composed of polysaccharides, but may be constructed of other materials i.e. Bacillus anthracis has a capsule of poly-D-glutamic acid Visible with the light microscope Resist phagocytosis

126. Capsules Contain a great deal of water – protect cell from desiccation Exclude viruses Exclude most hydrophobic toxic substances

127. FUNCTIONS Provide protection (drying) Block attachment of bacteriophages Antipathogenic (inhibit engulfment of pathogenic bacteria by WBC’s) Contributes to VIRULENCE or INFECTIVE ABILITY Promote attachment to surfaces Streptococcus mutans : adheres to teeth ( GLUCAN ), DENTAL CARIES

128. Complement Activation Some capsules prevent formation of C3 convertase on the bacterial surface

129. Host response – Antibody Subvert this type of protective host response by having capsules that resemble host polysaccharides.

130. CELL WALL Important in bacterial characteristics Determines shape Provides support/rigidity

131. STRUCTURE Composed of PEPTIDOGLYCAN (MUREIN) Insoluble, porous Cross-linked polymer (glycan), provides strength and rigidity N -acetyl Muramic Acid (NAM) N -acetyl Glucosamine (NAG)  1-4 glycosodic bonds

132. Differences in structure - basic principles of GRAM STAIN REACTION Christian Gram 1884 Differential stain : ability of eubacterial cells to retain dye (crystal violet) after discolouration with 95% ethanol Cells retain stain: Gram +ve (thick cell walls) PURPLE Cells lose stain: Gram -ve (thin cell walls) RED

137. Peptidoglycan Structure Peptidoglycan or murein is an enormous polymer composed of many identical subunits.

138. Peptidoglycan Structure Most G- cell wall peptidoglycan lacks the peptide bridge.

139. Gram Negative Cell Walls More complex than the G+ cell walls. Peptidoglycan 5-10% of the wall weight. Braun’s lipoprotein – a small lipoprotein covalently joined to the underlying peptidoglycan and embedded in the outer membrane by its hydrophobic end. Lipopolysaccharides (LPSs)

141. Lipopolysaccharide Structure Known as ENDOTOXIN Complex molecule: Inner most LIPID (Lipid A), achors LPS to outer membrane Polysaccharide portion (external to Lipid A) known as O-antigen O-polysaccharide long repeating sequence of sugars

142. LPSs Contain both lipid and carbohydrate Consist of three parts: lipid, the core polysaccharide and the O side chain

143. LPSs The lipid A region contains two glucosamine sugar derivatives, each with three fatty acids and phosphate or pyrophosphate attached.

144. LPSs Lipid A is buried in the outer membrane and the remainder of the LPS projects from the surface.

145. LPS The core polysaccharide is joined to lipid A. In Salmonella it is constructed of 10 sugars, many of them unusual in structure.

146. LPS The O side chain or O antigen is a short polysaccharide chain extending outward from the core. It has several peculiar sugars and varies in composition between bacterial strains.

147. LPS G- bacteria can rapidly change the nature of their O side chains to avoid detection. Contributes to the negative charge of the bacterial surface LPS helps stabilize membrane structure Lipid A is toxic – endotoxin Serves as a protective barrier

148. LPS – protective barrier Prevents or slows entry of bile salts, antibiotics, and other toxic substances.

149. Porin Proteins Cluster together and span the outer membrane to form a narrow channel through which molecules smaller than about 600 to 700 daltons can pass. .

150. Porin Proteins Larger molecules such as vitamin B 12 must be transported across the outer membrane by specific carriers. The outer membrane also prevents the loss of constituents like periplasmic enzymes.

151. COMPARISON OF GRM+VE /GRM-VE CELL WALLS

154. Periplasmic Space Contains enzymes involved in peptidoglycan synthesis and the modification of toxic compounds that could harm the cell.

155. Periplasmic Space Gram – contains many proteins that participate in nutrient acquisition. Ex. hydrolytic enzymes attacking nucleic acids and phosphorylated molecules, and binding proteins involved in transport of materials into the cell. Denitrifying and chemolithoautotrophic bacteria – electron transport proteins in periplasm

159. CELL/CYTOPLASMIC MEMBRANE 4-5nm thick composed 1 o phospholipids 30-40% and protein 60-70% Phospholipid bilayer: Polar heads (outwards into aqueous phase - membrane surface) Fatty acyl tails (inwards - semi/liquid phase at interior)

160. FLUID MOSAIC MEMBRANE Davison & Danelli

161. ALSO CONTAINS: PROTEINS INTEGRAL - removed by destruction i.e., Detergents PERIPHERAL - loosely attached, easily removed i.e., Osmotic shock

163. FUNCTION Transport - control nutrients Oxidative phosphorylation (Respiration) Secretion - discharge of metabolic products Anchoring DNA (during cell division) Metabolism - enzyme sites

164. INTERNAL CONTENTS Cell Material divided into PROTOPLASM Granular appearance Site of biochemical activity Water 70-80% acts as solvent for nutrients, sugars, Aa’s & salts

165. CHROMATIN AREA no distinct membrane enclosed nucleus no mitotic apparatus BACTERIAL CHROMOSOME Typically single circular strand of DNA ( CHROMATIN BODY ) Exception Streptomyces & Borrelia sp (Linear) Rhodobacter sphaeroides (2 separate chromosomes) all genes are linked Aggregated in one area ( NUCLEOID )

166. Bacterial Chromosome

167. PLASMIDS Additional to chromosome 1 or more, small circular macromolecules of DNA Capable of self-replication

168. Types: Fertility (F-plasmid): genes for mating in conjugation Resistance (R-plasmids): antibiotics, metals

169. Virulence factor: enterotoxin, fimbriae, antibiotic production Colicinogenic (col-plasmids): gene for protein ( COLICINS ) toxic to closely related bacteria (eliminates competitors) Transformation (Ti-plasmids): plant microbiology (formation of crown gall tumors) Agrobacterium Metabolic: utilization of camphor, toluene

170. OTHER FEATURES RIBOSOMES Located in Protoplasm RNA/PROTEIN bodies Composed of 2 sub units (70S) Svedberg Units Sites of Protein Synthesis

171. MESOSOMES: Extensive invaginations (infoldings) of cyto membrane Continuous with membrane Function NOT KNOWN Corynebacterium parvum

172. INCLUSIONS/VACUOLES compensate for poor availability of nutrients present in Protoplast VOLUTIN GRANULES/METACHROMATIC ( coloured ) composed of POLYPHOSPHATE energy rich storage structures

173. VOLUTIN i.e., POLY-  -HYDROXYBUTYRATE serve as carbon and energy source METACHROMATIC i.e., Aquatic bacteria - colored crystals ( Blue or Red dyes) e.g., Corynebacterium

174. Cyanobacterium: Microcystis (12,600x) Gas vacuoles - blue Storage granules - red