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1. Introduction and History (Chapter 1)a. Why study microbiology

i. the most important biological science(1) ubiquitous, wherever life = microbes

(a) 1E8/g soil, 200 - 500 lb/acre fertile soil(b) only microbe-free live environment is inside healthy tissues

(2) more biomass than plants or animals(3) role in human health

(a) all over skin surfaces (1013 body cells vs 1014 microbes)(b) present on all food not sterilized or freshly cooked(c) present in drinks, soil, dust, air, etc(d) majority are harmless, many beneficial, pathogens in the minority, except where

sickness is prevalent(4) life as we know it could not exist without microbes

(a) evolution(b) photosynthesis, oxygen production (c) elemental cycles (C, N, O, S, metals)(d) nutrition(e) waste degradation

ii. scientific knowledge(1) basic biological science

(a) accessible research tools for investigating life processes(b) deepest understanding of living processes due to studies with microbes

(i) molecular biology(ii) metabolism

(2) applied biological science(a) impact on medicine, agriculture, industry(b) many diseases (especially communicable)

(i) disease major cause of death at turn of century, better sanitation and health practiceshave lowered risk tremendously

(ii) microbial disease still a major cause of death in many developing countries(c) soil fertility and animal production

(i) N2 fixation(ii) rumen microbiology(iii) nutrient cycling

(d) food microbiology(i) preservation (antimicrobial)(ii) fermented foodstuffs (dairy, grain, vegetables)(iii) chemical production (fructose from corn syrup, amino acids for aspartame, citric

acid for soft drinks)(e) biotechnology (especially pharmaceuticals, stock chemicals, genetic engineering)(f) sanitation (food treatment, landfills, sewage treatment)

(i) wastes carried away with flowing water (pipelines) over 4000 years ago(ii) today, 15,000,000 children die each year from poor sanitation and lack of

immunization and simple medical treatmentb. Overview of Microbiology and Cell Biology

i. Classes of microorganisms(1) bacteria

(a) prokaryotes = cells that lack a true, membrane-enclosed nucleus, having genetic material

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in a nucleoid instead(b) Archaea = archaebacteria(c) Bacteria = eubacteria

(2) algae(a) eucaryotic = cells that have a membrane-delimited nucleus(b) photosynthetic = convert light to energy (via chloroplasts)(c) lack roots, stems, and leaves, but have chlorophyll, like other plants(d) microscopic (e.g. Euglena) to macroscopic (e.g. brown kelp)(e) phycology or algology is the study of algae

(3) fungi(a) "eucaryotic, spore-bearing organism that has absorptive nutrition and lacks chlorophyll;

that reproduces asexually, sexually, or by both methods; and that normally hasfilamentous hyphae surrounded by cell walls, which usually contain chitin" (Prescott)

(b) important as decomposers(c) molds, yeasts, mushrooms (toadstools, truffles)

(4) protozoa(a) eucaryotic, animal-like, usually motile, unicellular(b) important in food chains; many are animal parasites; some have become useful in

molecular biology(5) viruses

(a) simple, acellular entities consisting of DNA or RNA enclosed in a protein coat(b) obligate intracellular parasites(c) specific viruses infect bacterial, plant, animal cells

ii. Microorganisms as Cells(1) the cell is the fundamental unit of living matter

(a) membrane = barrier(b) nucleus or nucleoid(c) cytoplasm = contains machinery for cell growth and function

(2) cell components = proteins, nucleic acids, lipids, polysaccharidesiii. Cells are nonrandom entities that transform energy to maintain order

(1) energy generation is an important component of metabolism (catabolism)(2) chemical reactions catalyzed by enzymes encoded by DNA

c. History of early microbiology (Chapter 1)i. Francesco Redi demonstrated that maggots do not arise from spontaneous generation

(1) spontaneous generation = living material arising from non-living material(2) biogenesis = living material arising from living material(3) experimental procedure:

(a) meat was left uncovered or covered with gauze or paper(b) maggots developed in uncovered meat(c) maggots did not develop in meat covered with paper(d) flies laid eggs on gauze where they developed into maggots, meat was not infested(e) important toward development of scientific method (reproducible, controls)

ii. microorganisms first seen and described by Antony van Leeuwenhoek (1676)(1) simple, single lens microscopes (50 - 300x magnification)(2) huge curiosity, observed anything he could

(a) animalcules (intestinal organisms, including Giardia)(b) bacteria from overnight pepper infusions(c) seeds and plant embryos(d) small invertebrates

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(e) spermatozoa(f) red blood cells(g) essentially all main unicellular organisms we know today

(i) yeasts(ii) protozoa(iii) algae(iv) bacteria

(3) "I have had several gentlewomen in my house, who were keen on seeing the little eels invinegar; but some of them were so disgusted at the spectacle, that they vowed they'd neveruse vinegar again. But what if one should tell such people in the future that there are moreanimals living in the scum on the teeth in a man's mouth, than there are in a wholekingdom?"

(4) superior observation skills but did not allow others to copy his techniques and verify hisresults

(5) compound microscopes, at that time, provided higher magnification, but quality of duallenses was lower than Leeuwenhoek's single lens

(6) renewed argument for spontaneous generation(a) animimalcules formed spontaneously(b) arose from "seeds" or "germs" of animalcules always present in air(c) difficult to prove a negative

iii. Louis Pasteur (1) filtered air through guncotton

(a) dissolved cotton in alcohol/ether and found trapped objects resembling plant spores,protozoan cysts, and other microbial cells

(b) placing the cotton in sterile medium resulted in growth(2) sterilized nutrient broth in open flasks with swan necks

(a) no growth occurred(b) if necks were broken (shortened), growth occurred immediately

(3) By 1861 Pasteur had almost completely resolved the controversy and demonstrated how tokeep solutions sterile

iv. Pasteur's studies led him to discover the existence of life forms that can live only in the absenceof free oxygen(1) microscopic observation of butyric acid bacteria showed loss of motility at margin of

flattened drop(2) cells in center (away from air) remained motile(3) introduced the terms aerobic and anaerobic (4) "fermentation is life without air"

v. Sterilization is killing all microorganisms in or on objects(1) sterile = devoid of life(2) sterilization procedures were essential to development of microbiology as a science(3) basis of the canning preservation of many foods

vi. Aseptic technique = handling of objects so as to prevent contaminationvii. Pasteur termed the microbial spoilage of wine and beer "diseases"

(1) considered that microorganisms could act as agents of infectious disease(a) already known that fungi could cause disease in wheat and rye (b) a fungus was responsible for the great Potato Blight of Ireland

(2) introduction of anesthesia (1840) allowed longer, more complicated surgeries(a) surgical sepsis increased(b) bacteria in air (demonstrated by Pasteur) considered as possible cause

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viii. Ignaz Semmelweiss improved hospital sanitary practices(1) observed mortality rates lower with midwives than doctors

(a) strep infection = child bed fever or puerperal sepsis (P.S.)(b) got fired

(2) friend died of P.S. from cut during autopsy of P.S. victim(3) hypothesized an invisible agent responsible for disease(4) sanitized hospitals by required handwashing and changing lab coats (fired again)(5) died in insane asylum of P.S.

ix. John Lister sterilized instruments with heat and used phenol on dressings and sometimes onwounds

(a) phenol kills bacteria(b) less wounds became infected(c) indirect evidence for role of bacteria in infection

x. Direct evidence for role of bacteria in disease was by Robert Koch, working with anthrax(a) injected a series of 20 healthy mice with anthrax bacilli(b) inoculated broth with spleen from infected mouse(c) isolated anthrax bacilli spores(d) injection of spores into mice resulted in anthrax

xi. Koch's Postulates(1) The organism should be present in every case of the disease, but absent in healthy

individuals(2) The suspected microorganism must be isolated and grown in pure culture(3) The disease must result when the isolated microorganism is inoculated into a healthy host(4) The same microorganism must be isolated again from the diseased host

xii. Vaccination developed by Pasteur (1) against cholera and rabies(2) named in honor of Edward Jenner (vacca is Latin for cow)

(a) Jenner saw that milkmaids who had cowpox were immune from smallpox(b) started intentional inoculation with cowpox to prevent smallpox

xiii. The germ theory of disease (from Pasteur’s and Koch’s work) describes the demonstrationthat microbes can be the agents of disease

(1) general belief was that epidemics were penalties of God(2) greatest impetus for development of microbiology(3) contagious diseases spread through populations by contagions(4) after discovery of microbes, contagions = microorganisms

xiv. Koch developed spread plate techniques on solid nutrient surfaces(1) started with potato slices(2) added gelatin to nutrient fluid (e.g. blood serum)

(a) digested by many bacteria(b) melted above 28o C

(3) agar suggested by Fanny Angelina Eilshemius the wife of Walter Hesse, one of Koch'sassistants(a) not attacked by most bacteria(b) did not melt below 100o C

xv. biochemistry born when Buchner discovered that sugar added to disrupted yeast (physicalgrinding) was fermented

(1) key to chemical analysis of energy-yielding metabolic processes(2) similarities later observed between glycolysis by muscle and alcohol fermentation by yeast(3) vitamins required by animals identical to growth factors required by some bacteria and yeast

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d. Microbes and human welfarei. microbes are critical for some of the steps in nature’s mineral cyclesii. natural abilities of microorganisms are exploited to treat solid wastes and recycle wateriii. bacteria and fungi are used to clean up environmental pollutants (bioremediation)iv. experiments conducted this century with microbes helped establish the science of genetics and

molecular biology(1) mechanisms of genetic transfer(2) establishment of DNA as hereditary material(3) biotechnology (recombinant genetics)(4) gene therapy

e. Microbes and human diseasei. normal flora are the microorganisms that co-exist on us

(1) perform functions that help us(2) protects us from invasion by pathogenic bacteria

ii. microbes (bacteria, protozoa, fungi, viruses) can cause diseaseiii. emerging diseases are those that have come to our notice within the last two decades

(1) AIDS (HIV)(2) mad cow disease = Creutzfeldt-Jakob in humans (spongiform encephalopathy)(3) Ebola(4) hantavirus

2. Chemistry (Chapter 2)a. The structure of atoms and how they form bonds

i. components of the atom(1) electrons(2) protons(3) neutrons

ii. atomic number = number of protonsiii. atomic mass or weight = number of protons plus number of neutronsiv. isotope = elements whose atoms have different numbers of neutrons (some may be radioactive)v. chemical bonds are the result of interactions involving electronsvi. ions are atoms that have gained or lost an electron

(1) no longer a balance between positive and negative charges(2) negative ions are attracted to positive ions and form an ionic bond(3) negative ions = anions, positive ions = cations(4) ionic bonds can dissociate in water

vii. covalent bonds form when two atoms share electrons (1) orbit both nuclei(2) very strong bond

viii. hydrogen bonds are an attraction between hydrogen covalently bound to one molecule withoxygen or nitrogen bound to another molecule

(1) hydrogen is weakly positive(2) oxygen or nitrogen is weakly positive(3) overall bond is weak

b. Chemical reactionsi. reactions progress from more energy to less energy (energy given off)ii. activation energy is the energy required for a reaction to get started (Figure 2.5)

(1) a catalyst lowers the activation energy and speeds up the reaction(2) enzymes are biological catalysts made of protein(3) catalysts are not permanently altered by the reaction

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c. Organic Compoundsi. Four important classes of macromolecules

(1) carbohydrates(2) lipids(3) nucleic acids (4) proteins

ii. carbohydrates contain carbon, hydrogen, and oxygen(1) polymers (polysaccharides) made up of sugar monomers (saccharides)(2) the same monomers can make up different polymers, depending on the types of bonds

(a) cellulose is made of glucose with $-1,4 linkage(b) starch is made of glucose with "-1,4 linkage

(3) sugars are components of many key cell structures(a) nucleic acids (ribose or deoxyribose)(b) lipopolysaccharide

iii. lipids contain carbon, hydrogen, and oxygen(1) a typical lipid has a backbone of glycerol linked to fatty acids (Figure 2.10)

(a) glycerol part is usually hydrophilic (water-loving)(b) fatty acid region is hydrophobic (water-hating)

(2) important components of membranes (Figure 2.11)(a) form bilayers(b) glycerol (with phosphate attached) on the outsides(c) fatty acid chains on the inside

iv. nucleic acids contain carbon, hydrogen, oxygen, nitrogen, phosphorus(1) composed of nucleotides (Figure 2.17)

(a) nitrogen base (adenine, guanine, cytosine, thymine, uracil)(b) sugar (ribose or deoxyribose)(c) phosphate

(2) DNA occurs as a two-strand helix, held together by hydrogen bonds between the bases(a) backbone is made of phosphate (negative charge) and deoxyribose(b) center contains bases in complementary pair between strands

(i) adenine - thymine (2 hydrogen bonds)(ii) guanine - cytosine (3 hydrogen bonds)

(c) RNA contains ribose instead of deoxyribose(i) occurs as a single strand(ii) uracil replaces thymine(iii) can form secondary structures (hydrogen bonds between complementary bases)

v. proteins contain carbon, hydrogen, oxygen, nitrogen, and may contain sulfur(1) made up of chain of amino acids connected by peptide bonds (Figure 2.15)

(a) chain of amino acids = polypeptide(b) functional polypeptide = protein

(2) proteins can have several levels of structure (conformation) (Figure 2.16)(3) primary structure = sequence of amino acids (peptide bond is a strong covalent bond)(4) secondary structure occurs through hydrogen bonding between amino acids

(a) two primary structures(i) "-helix(ii) $-sheet or pleated sheet

(b) easily denatured(5) tertiary structure is additional folding of secondary structure

(a) may involve disulfide bridges (between cysteines), hydrogen bonding, other interactions

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(b) more difficult to denature than secondary structure(6) quarternary structure is when the mature protein is made up of subunits

(a) not all proteins have subunits(b) subunits held together by hydrogen bonds

(7) denaturation is when bonds are broken, altering the conformation, and affecting the activityof the protein (a) harsh denaturation by heat is almost always irreversible(b) gentle denaturation (urea) can often be reversed, with activity restored

vi. isomer = chemical compounds with the same formula but different structures vii. stereoisomer = isomers that exist as mirror images

(1) L- and D- forms(2) enzymes are so specific they can only react with a specific stereoisomer

(a) L-amino acids are used by biochemical systems(b) D-sugars are used by biochemical systems

3. Cell Structure and Function (Chapter 4)a. Prokaryotic Cell Structure

i. Overview of prokaryotic cell structure(1) size, shape, arrangement

(a) small, 0.1 - 0.2 :m diameter to several hundred :m long(b) largest about the size of a hyphen (Epulopisicum fishelsoni)(c) E. coli 1.1 - 1.5 :m x 2.0 - 6.0 :m(d) coccus = roughly spherical

(i) diplococci (Neisseria), 1 plane(ii) streptococci (Streptococcus), 1 plane(iii) grapelike clusters (Staphylococcus), random planes(iv) tetrads (Micrococcus), 2 planes(v) sarcina (Sarcina), 3 planes

(e) bacillus = rod(i) coccobacillus = short rod (Corynebacterium)(ii) vibrio = curved (iii) ends can be flat, rounded, cigar-shaped, or bifurcated

(f) long rods twisted into spirals or helices(i) spirilla if rigid(ii) spirochete if flexible

(2) cell organization(a) almost all cells are bounded by cell walls(b) plasma (cell, cytoplasmic) membrane inside wall(c) genetic material is contained in the nucleoid(d) ribosomes and inclusion bodies are scattered throughout the cytoplasm

ii. Cell membranes(1) in all organisms(2) barrier that contains the cytoplasm and inclusions, separating it from the surroundings(3) selectively permeable (allows specific atoms and molecules to diffuse freely, while

preventing passage of others)(a) prevents loss of essential components(b) requires transport systems for non-diffusible

(4) site of many critical processes(a) respiration(b) photosynthesis

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(c) lipid and cell wall synthesis(5) contain receptors that help detect and respond to chemicals in surrounding environment(6) composed of lipids and proteins

(a) amphipathic = structurally asymmetric (polar and non-polar ends)(i) polar ends = hydrophilic(ii) non-polar ends = hydrophobic

(b) lipids form a bilayer, with hydrophobic portion inside(c) proteins are embedded within the bilayer

(7) very thin, visible with EM (5 - 10 nm)(8) fluid-mosaic model

(a) 20 - 30% protein is membrane bound (peripheral or extrinsic) and easily washed off withaqueous solutions

(b) 70 - 80% protein are integral (intrinsic); insoluble in aqueous solutions (amphipathic)(c) membrane is highly organized and asymmetric, but also flexible and dynamic

(9) nutrient uptake(a) membrane-bound transport mechanisms required for uptake of nutrients by microbial cell

(i) mechanism must be specific (no use to transport unusable compound)(ii) membrane is selectively permeable(iii) transport of dilute solutes against a gradient

(b) passive diffusion = molecules move from a region of higher concentration to one oflower concentration as a result of random thermal agitation(i) rate is dependent on size of concentration gradient(ii) requires relatively high external concentration(iii) inefficient, not extensively employed

(c) facilitated diffusion = diffusion process aided by a carrier(i) permeases embedded in membrane function as carrier proteins(ii) more efficient than passive diffusion

1) more rapid at lower concentrations2) saturatable (levels off, compared to linear increase with passive diffusion)3) no energy requirement, still gradient driven (still diffusion)

(iii) permeases may function by changing conformation, allowing buildup of internalconcentration

1) allows lipid-insoluble molecules to enter cell2) can work in reverse if internal concentration is high enough

(iv) does not appear to be very important in procaryotes (used with glycerol); largerrole in eucaryotes (for sugar and amino acids)

(d) active transport = energy-linked transport of solute molecules(i) works against a concentration gradient(ii) employs carrier proteins

1) high specificity2) saturatable

(iii) can use ATP, other high energy phosphate compounds, or proton motive force(iv) many compounds have multiple transport systems (may have selective advantage

in different environments)(v) typically used with sugars and amino acids(vi) symport = linked transport of two substances in the same direction (H+ - linked

uptake of amino and organic acids)(vii) antiport = linked transport of two substances in opposite directions (Na+/H+)(viii) occurs in G- and G+

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(e) group translocation = molecule is transported and chemically altered at the same time(i) phophenolpyruvate:sugar phosphotransferase system (PTS) best studied

1) transports sugars into procaryotes2) phosphorylates sugar using phosphoenol pyruvate (PEP) as phosphate donor

(ii) systems are fairly complex, with cytoplasmic and membrane enzymes involved(iii) present in facultative anaerobes and anaerobes (rare in aerobes, except for some

Bacillus species)(f) siderophores = low molecular weight molecules that complex with ferric iron

(i) hydroxamates or phenolates-catecholates(ii) complex binds to cell surface receptor protein and iron or complex is transported

inside(iii) iron reduced to ferrous form inside cell

iii. Cytoplasmic components(1) cytoplasm (cytoplasmic matrix) = cellular contents inside the plasma membrane, except for

the nucleoid(2) protoplast = everything inside the plasma membrane(3) inclusion bodies = organic or inorganic materials, often visible with light microscopy

(a) glycogen (i) starch-like glucose polymer ("-1,4 and "-1,6)(ii) used for carbon and energy

(b) poly-$-hydroxybutyric acid (PHB) (i) lipid-like linear polymer of $-hydroxybutyrate(ii) used for carbon and energy

(c) volutin (metachromatic granules) = polyphosphate(d) sulfur granules

(4) ribosomes(a) complex structures made of protein and RNA(b) site of protein synthesis(c) located throughout the cytoplasm or loosely attached to the membrane

iv. Nucleoid(1) nucleoid, nuclear body, chromatin body, nuclear region(2) region containing the prokaryotic chromosome(3) 60% DNA, some RNA, some protein(4) chromosome is a single circle of double stranded DNA

(a) tightly packaged(b) E. coli, 2 - 6 :m long, DNA = 1400 :m

(5) plasmids = circular, double stranded DNA molecules existing extrachromasomally(a) generally not required for cell growth and replication, but may confer selective

advantages(b) commonly contain genes for antibiotic resistance mechanisms

v. Cell wall(1) general features

(a) very important for several reasons (present in all but mycoplasmas and a few Archae)(i) provide shape and prevent lysis(ii) many pathogens have cell wall components that contribute to pathogenicity(iii) protect cells from toxic substances(iv) site of activity of many antibiotics

(b) Gram stain(i) differential stain developed by Christian Gram

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(ii) mechanism unknown, but appears related to cell wall structure(iii) G+ (purple) have thick cell walls, G- (pink) have thin cell walls

(c) G+ cells have a thick (20 - 80 nm) homogeneous layer of peptidoglygan or mureinoutside the plasma membrane

(d) G- cells have a more complex cell wall structure(i) 1-3 nm peptidoglycan layer(ii) outer membrane 7 - 8 nm thick(iii) periplasm or periplasmic space = space between cell wall and plasma

membrane (present but smaller in G+)(iv) periplasmic space contains many hydrolytic enzymes

(e) archaebacteria lack peptidoglycan and have cell walls composed of proteins,glycoproteins, or polysaccharides

(2) peptidoglycan(a) enormous polymer containing two sugar derivatives and several amino acids

(i) N-acetylglucosamine (NAG)(ii) N-acetylmuramic acid (NAM)(iii) of amino acids, D-glutamic acid, D-alanine, meso-diaminopimelic acid (DAP) are

not found in proteins(b) in a typical molecule, the backbone is composed of alternating NAG and NAM residues

(i) peptide chain of 4 alternating L- and D-amino acids connected to the carboxyl groupof NAM

(ii) peptidoglycan subunits joined by cross-links between the peptides(c) isolated peptidoglycan sacs are strong enough to retain their shape and integrity, yet are

also elastic and somewhat stretchable (unlike cellulose)(3) gram positive cell walls

(a) besides peptidoglycan, also contain teichoic acids (polymers of glycerol or ribitol joinedby phosphate groups)

(b) teichoic acids extend to surface of peptidoglycan, giving the cell wall a negative charge(c) functions are unclear(d) teichoic acids are not present in G-

(4) gram negative cell walls(a) made of peptidoglycan, but in a much thinner layer(b) the outer membrane has typical membrane structure(c) OM contains lipopolysaccharides (LPS)

(i) lipid A (major component of OM)(ii) core polysaccharide(iii) O side chain or O antigen

(d) LPS important for several reasons(i) the core polysaccharide contains charged sugars and phosphates, contributing to

overall negative charge of cell surface(ii) lipid A helps stabilize the outer membrane (iii) lipid A is often toxic, so LPS can act as an endotoxin (responsible for many

symptoms in G- bacterial infections(e) OM acts as a barrier, but is more permeable than plasma membrane

(i) blocks bile salts, antibiotics, other toxic compounds(ii) allows passage of small molecules (monosaccharides)

1) porin proteins form channels spanning the OM2) porins pass molecules # 600 - 700 daltons3) larger molecules require transport mechanisms with specific carriers

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(iii) prevents loss of constituents like periplasmic enzymes(5) gram reaction

(a) appears due to physical nature of cell walls(i) G+ with cell wall removed stain G-(ii) peptidoglycan itself is not stained

(b) thought that EtOH shrinks pores in thick peptidoglycan(i) G- peptidoglycan is thin, not as highly cross-linked, and has larger pores(ii) may also extract lipids from G- cell wall and increase porosity

(6) role of cell wall, osmotic protection(a) usually required to protect bacteria against destruction by osmotic pressure

(i) cytoplasm has higher solute concentration than environment (hypotonic medium)(ii) osmosis moves water into the cell, increasing osmotic pressure to as high as 20

atmospheres(iii) cell membrane would burst (lysis) without the CW(iv) in hypertonic habitats (external solute concentration higher than cells), water

flows outward, cytoplasm shrivels and pulls away from cell walls1) plasmolysis2) useful for food preservation

(b) Removal of cell wall removes osmotic protection(i) lysozyme hydrolyzes NAM-NAG bond of peptidoglycan(ii) penicillin inhibits peptidoglycan synthesis(iii) cells grow normally in isotonic solutions

1) G+ converted to protoplasts2) G- retain OM and are called spheroplasts (some CW remains)3) if either transferred to dilute solution, lysis

vi. External components(1) capsules, slime layers, RS layers

(a) capsule = layer of material external to the cell, well organized, not easily removed(b) slime layer = zone of diffuse, unorganized material that's easily removed(c) glycocalyx = network of polysaccharides extending from cell surface (could be capsule or

slime layer)(i) usually composed of polysaccharides, but could also have protein(ii) capsules protect cells from phagocytosis(iii) Streptococcus pneumoniae non-pathogenic without a capsule(iv) unencapsulated strains easily destroyed (v) capsules contain a lot of water and can protect against desiccation(vi) exclude phages and hydrophobic toxic agents, like detergents(vii) aids attachment to surfaces or tissues

(d) RS (regularly structured) layer(i) protein or glycoprotein(ii) pattern similar to floor tiles(iii) may act as protection

1) ion and pH fluctuations2) osmotic stress3) enzymes4) predacious bacteria

(2) pili and fimbriae(a) fimbriae = thin, hairlike appendages often called pili

(i) G- cells

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(ii) helically arranged protein subunits (3 - 10 nm x several :m)(iii) some types used to attach cells to surfaces

(b) sex pili = similar to fimbriae, but larger (9 - 10 nm diameter) and fewer (1 - 10/cell)(i) genetically determined by sex factors(ii) required for mating(iii) some phages bind to receptors on sex pili

(3) flagella and motility(a) flagella = threadlike locomotor appendages

(i) mechanism for majority of motile bacteria(ii) inserted through plasma membrane(iii) 20 nm diam. x 15 - 20 :m long

(b) distribution patterns(i) monotrichous = one flagellum (polar = at one end)(ii) amphitrichous = single flagellum at each pole(iii) lophotrichous = cluster of flagella at one or both ends(iv) peritrichous = flagella spread evenly over whole surface

(c) structure(i) filament, made of single protein flagellin(ii) basal body embedded in cell(iii) hook, links filament to basal body

(d) synthesis(i) flagellin subunits transported through core to outer end(ii) subunits spontaneously aggregate (self-assembly)

(e) mechanism of movement(i) filaments are in a rigid helix(ii) when filament rotates, it acts as a propeller and bacterium moves(iii) rotation rates as fast as 40 - 60 rpm(iv) flagella rotate counterclockwise during swimming, clockwise during random

tumbling1) monotrichous cells turn clockwise (when flagella turn counterclockwise)2) peritrichous arrangements do not appear to spin the cell

(v) mechanism of "motor" still unclear, but proton flow appears required (ATP has nodirect role)

(vi) bacterial movement is relatively rapid (2 - 100 body lengths/sec) through a fairlyviscous environment

(f) spirochetes move by flexing and spinning caused by an axial filament(g) some bacteria use gliding motility

vii. Bacterial taxis(1) net movement to or away from an attractant or repellent

(a) run = travel in straight or slightly curved line for a few seconds(b) tumble = stop in linear movement(c) tumble followed by run in another direction(d) in direction of attractant, tumbling less frequent, runs longer, so net movement towards

attractant(2) receptors may influence the direction of flagellar rotation(3) different types of taxis

(a) chemotaxis = response to chemical stimulus(b) aerotaxis = response to oxygen concentration(c) phototaxis = response to light energy

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(d) magnetotaxis = positive tactic response to magnetic field lines(i) large amounts of iron as magnetite located near location of flagellar insertion(ii) isolates from northern hemisphere seek the north

1) magnetic lines are about 70o

2) tactic response bring cells downward into more anoxic zones (cells are anaerobesor microaerophiles)

3) northern isolates die in southern hemisphere (mutants can reverse fields andsurvive)

viii. Endospores(1) endospore = specialized dormant structure resistant to adverse conditions

(a) endo because they form inside vegetative cells(b) resist boiling (remember Tyndall), desiccation, and disinfection(c) can remain viable for hundreds of years

(2) formed by several genera, but Clostridium and Bacillus most common(a) Sporosarcina(b) Desulfotomaculum(c) Oscillospira

(3) exosporium = thin, delicate, outer covering(4) spore coat = several protein layers

(a) beneath exosporium(b) can be fairly thick(c) impermeable(d) primarily responsible for spore's resistance to chemicals

(5) cortex = peptidoglycan layer(a) beneath spore coat(b) can fill half spore volume(c) less cross-linked than in vegetative cells

(6) spore cell wall (core wall) = inside cortex, surrounding protoplast or core(7) protoplast = normal cell structures, including ribosomes and nucleoid

(a) largely dehydrated(b) important to heat resistance

(8) dipicolinic acid = up to 15% of spore weight(a) complexed with calcium(b) role unknown

(9) spore formation usually triggered by lack of nutrients, but not by lack of energy(10) activation prepares spores for germination and usually results from treatments like

heating(11) germination = breaking spore's dormant state

(a) spore swells, spore coat cracks(i) loss of resistance to heat and other stresses(ii) loss of refractivity(iii) release of spore components(iv) increase in metabolic activity

(b) can be triggered by normal metabolites or nutrients(i) amino acids(ii) sugars

(12) outgrowth = protoplast makes new components, emerges from remains of spore coat,develops into active bacterium

b. Eukaryotic Cell Structure

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i. Overview(1) unit of structure in plants, animals, protozoa, fungi, alga (except cyanobacteria)(2) differ from procaryote cells most obviously by having variety of membrane-bound organelles

in cytoplasm and membrane-bound nucleus(a) organelle = intracellular structures that perform specific functions in the cell(b) partitioning by membranes allow simultaneous biochemical and physiological activities

under independent control and proper coordination (necessary due to greater size andcomplexity)(i) greater membrane surface area increases membrane bound activities

1) respiration2) photosynthesis

(ii) internal membrane system serves as transport system(3) cytoskeleton instead of cell wall

(a) made up of small organelles microtubules, microfilaments, and other filaments(b) involved in cell movements

(4) cell walls not always present(a) constructed from polysaccharides (e.g. cellulose)(b) structurally simpler than prokaryotic cell walls

(5) Motile through several mechanisms, but flagella and cilia most common(a) structure very different than prokaryotic flagella(b) cilia and flagella similar structure

ii. Cytoplasmic matrix and organelles(1) endoplasmic reticulum (ER) involved in protein synthesis(2) Golgi apparatus (composed of one or more stacks of cisternae)

(a) prepares and packages products for secretion(b) form lysosomes (intracellular digestion, including materials taken up by endocytosis)

(3) lysosomes(4) ribosomes (free or bound to ER)(5) mitochondria

(a) bounded by two membranes(b) inner membrane folded into cristae(c) energy generation

(i) TCA cycle(ii) electron transport(iii) oxidative phosphorylation

(6) chloroplasts(a) site of photosynthesis(b) thylakoid membranes = site of reaction centers(c) stroma = site of CO2 incorporation

iii. The nucleus and cell division(1) nuclear structure

(a) contains cell's chromosomes(b) double membrane envelope perforated by pores]

(2) nucleolus(a) within the nucleus(b) synthesis of ribosomal RNA and ribosomal subunits

(3) mitosis and meiosis(a) chromosomes distributed to daughter cells by mitosis(b) meiosis is a reductive division for sexual reproduction

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c. Endosymbiote theoryi. hypothesis that some organelles evolved from bacteria

(1) mitochondria and chloroplasts are bacterial in size(2) both contain closed, circular DNA and reproduce semiautonomously(3) ribosomes similar to bacterial ribosomes; similar sequences in rRNA and tRNA

ii. Protozoan flagellate Cyanophora paradoxa has photosynthetic organelle (cyanellae) withstructures similar to cyanobacteria(1) some peptidoglycan in cell walls(2) DNA < cyanobacteria, about same as chloroplasts(3) lacks OM that cyanobacteria have

4. Nutrition and Growth (Chapter 5)a. General nutrition

i. general requirements(1) nutrients = substances required for microbial growth

(a) biosynthesis(b) energy production

(2) analysis of cells shows that 95% of dry weight is made up of carbon, oxygen, hydrogen,nitrogen, sulfur, phosphorus, potassium, calcium, magnesium, and iron(a) termed the macroelements or macronutrients(b) CHONPS are major components of carbohydrates, lipids, proteins, and nucleic acids(c) cations have several roles, many of them enzyme related

(3) micronutrients = microelements = trace elements(a) manganese, zinc, cobalt, molybdenum, nickel, copper(b) small amounts required (contaminants in water, glassware, media components often

enough for growth)(c) usually part of enzymes and cofactors

ii. growth factors(1) organic compounds that are essential cell components that cannot be synthesized by the cell(2) three major classes

(a) amino acids (required for protein synthesis)(b) purines and pyrimidines (required for nucleic acid synthesis)(c) vitamins

(i) small organic molecules(ii) all or part of enzyme cofactors

iii. media composition (enrichment, pure culture)(1) culture medium = nutrient mixture prepared for the growth of microorganisms

(a) culture = microbes that grow in or on a culture medium(b) many different types of media, depending on the organism of interest or the application

(i) no single medium to grow all organisms(ii) some organisms have specific requirements(iii) estimated only 10% of bacteria are culturable(iv) different media used to test anti-microbials, water qualities, vitamin

concentrations, etc.(v) enrichment media

(2) defined medium = synthetic medium = a medium in which all components are known(a) allows accurate assessment of nutrient utilization(b) can limit contamination(c) "clean" medium often useful in physiology/ecology studies

(3) complex medium = medium that contains some ingredients of unknown chemical

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composition(a) extracts from meat, yeasts, plants, soil, water, rumen, etc.(b) digests of proteins (c) very rich(d) good for growth of a wide variety of bacteria

(4) solid medium(a) useful for isolation, enumeration(b) agar (gelling agent made from seaweed derivative) most common agent

(i) generally non-bacterially degraded(ii) solid below 100o C(iii) suggested for use by Fanny Angelina Hesse

(c) gelatin can be used but is degraded by many bacteria and liquifies above 28o C(d) gel-rite, a gellan gum, also in use

(5) general purpose media support a wide range of organisms(6) selective media favor the growth of particular organisms, often inhibiting the growth of

others(a) addition of basic fuschin or crystal violet inhibits G+(b) used to enrich for bacteria with specific traits

(7) differential media distinguish between different groups based on identifiable reactions(8) selective and differential media can be combined

(a) MacConkey agar contains bile salts and crystal violet that inhibit G+, and lactose identifylactose fermentors

(b) Mannitol salts agar contains 7.5% NaCl to select for Staphylococcus and a pH indicatorto differentiate between mannitol fermentors and non-fermentors

b. General growthi. growth = increase in cell number or cell mass (usually refers to number in case of bacteria)ii. most bacteria reproduce by binary fission

(1) cell numbers increase by powers of 2(2) generation time = amount of time required for population to double (reproduce)(3) Let No = initial cell number

Nt = cell number at time tn = number of generations in time tNt = No x 2n

log Nt = log No + n(log 2)n = (log Nt - log No)/log 2 = (log Nt - log No)/0.301

(4) cell numbers increase logarithmically, so numbers are usually plotted on a semi-log scaleiii. bacterial growth curve = plot of cell growth over time, usually in a batch culture or closed

system(1) lag phase = period of little or no cell division

(a) "gearing up" phase(b) intense metabolic activity, particularly DNA and enzyme synthesis

(2) log or exponential phase = period of most rapid growth, where cells are in continual state ofcell division(a) generation time reaches a constant minimum based on genetic potential and medium

limitations(b) cells most active metabolically(c) microorganisms particularly sensitive to adverse conditions(d) single bacterium weighing 9.5 x 10-13 g with doubling time of 20 minutes could,

theoretically, result in 80,000 tons of cell material

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(3) stationary phase = period of equilibrium between growth and death of cells(a) depletion of nutrients and accumulation of waste products(b) changes in pH

(4) death phase = period when more cells are dying than dividing(a) logarithmic decline phase (constant proportion dies with time)(b) population can diminish or become extinct

iv. measurement of growth(1) growth can be measured in terms of cell numbers, cell components (e.g., protein or DNA

concentration), or cell mass(2) direct, viable counts

(a) can take some time for results(b) pour plates and spread plates

(i) assumes that inoculum is homogeneous and cells are not clumped(ii) culture is usually serially diluted to provide inoculum with 30 - 300 cells

(c) most probable number(i) statistical estimation based on number of cultures showing growth in serially diluted

series(ii) useful with cells that won't grow on solid media

(3) direct microscopic counts(a) counting chamber(b) counts viable and nonviable cells

(4) indirect methods(a) turbidity

(i) absorbance is proportional to cell density(ii) requires cell densities above 106 cells/ml(iii) can be standardized with viable counts or total counts

(b) metabolic activity(i) accumulation of product(ii) depletion of substrate(iii) useful for ecological studies

(c) dry weight(i) can be nearly direct, in the case of unicellular organisms (standardized with direct

method)(ii) especially useful for filamentous fungi(iii) can be extended to cellular components

1) protein2) DNA3) membrane lipids

(iv) cell yields can be determined for a limiting nutrientY = (mass of cells)/(mass of substrate consumed)1) basis of microbial assays for vitamins and other growth factors2) can be extended to YATP if pathways known

v. batch vs. continuous culture(1) continuous culture = open system (continual introduction of nutrients and removal of wastes)

(a) bacteria maintained in exponential phase(b) growth rate dependent on dilution rate (rate of introduction of fresh medium)(c) cell density dependent on concentration of limiting nutrient(d) if dilution rate is greater than the growth rate, cells are washed out

vi. influence of environmental factors (role of physical parameters on bacterial growth)

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(1) water activity(a) describes the availability of water to the organism(b) aw = ratio of vapor pressure of air over a substance or solution divided by vapor pressure

of water at the same temperature(c) high water activity = dilute environment(d) low water activity inhibits bacterial growth

(i) high solute concentration(ii) method of preservation

(e) halotolerant = able to survive with salt, but less than optimal growth(f) halophiles = require salt for growth(g) osmophiles = live in environments with low water activity (could be due to non-salt

solutes)(h) xerophiles = live in dry environments

(2) pH(a) each organism has a pH range where growth is possible, along with an optimum pH(b) most life occurs around neutrality, but ranges from 2 - 10(c) acidophiles live at low pH(d) alkalinophiles live at high pH(e) neutraphiles live around pH 7

(3) temperature(a) every organism has a minimum temperature, a maximum temperature, and an optimum

temperature(b) psychrophile opt. # 15o C

(i) oceans (avg temp 5o C)(ii) organisms growing under ice in polar regions (snow alga)(iii) membranes rich in unsaturated fatty acids

(c) mesophile opt about 35o C(i) best studied group(ii) corresponds to pathogens

(d) thermophile opt about 60o C(i) hot springs(ii) water heaters(iii) compost heaps and digestors(iv) membranes rich in saturated fatty acids(v) changes in enzyme structures

(e) extreme thermophile opt$ 85o C(i) thermal vents(ii) steam vents

(4) oxygen concentration(a) bacteria vary in their need for and tolerance(b) obligate anaerobes are killed by oxygen(c) aerotolerant anaerobes can grow in the presence of oxygen but cannot use it as an

electron acceptor(d) facultative anaerobes can use oxygen, but can also grow without it(e) microaerophiles require oxygen, but at lower levels than in air (2 - 10%)(f) aerobes require oxygen for growth(g) toxic forms of oxygen

(i) superoxide anion, O2-

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1) occurs during reduction of O2 to H2O2) highly reactive3) can cause oxidative destruction of lipids and other biochemical components

(ii) peroxide, O22-

1) formed during respiratory processes2) often used as a disinfectant

(iii) hydroxyl free radical, OH.

1) formed by ionizing radiation 2) formed by reaction of superoxide anion with peroxide

(h) several enzymes to protect against oxygen toxicity(i) catalase

1) H2O2 + H2O2 6 2H2O + O2

2) present in aerobes and facultative anaerobes(ii) peroxidase

1) H2O2 + NADH + H+ 6 2H2O + NAD+

2) no oxygen produced(iii) superoxide dismutase

1) O2- + O2

- 6 H2O2 + O2

2) present in aerobes, facultative anaerobes, and aerotolerant anaerobes(5) pressure

(a) most cells are barotolerant(b) barophilic organisms grow more rapidly at high pressures

(i) isolated from guts of deep-sea invertebrates(ii) hard to study (cultivate)

(6) radiation5. Control of Growth (inanimate objects) (Chapter 7)

a. Terminologyi. sterilization = destruction of living cells, viable spores, viruses, viroidsii. disinfection = killing, inhibition, or removal of organismsiii. sanitization = reduction of microorganisms to safe health levelsiv. antiseptic = chemical agents applied to tissues to kill or inhibit pathogensv. -cide = kills organismsvi. -lytic = lyses organismsvii. -static = inhibits growth of organisms

b. Effectiveness of antimicrobialsi. population death is generally exponential or logarithmicii. efficiency of antimicrobials influenced by at least 6 factors

(1) population size - larger population requires more time to die(2) population composition - different degrees of resistance between different organisms or

structures(a) spores more resistant than vegetative cells(b) Mycobacterium tuberculosis (acid-fast) more resistant than most bacteria(c) young cells more readily destroyed than older cells

(3) concentration of antimicrobial(a) usually greater concentration = greater effectiveness(b) 70% ethanol more effective than 95% ethanol

(4) exposure duration

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(a) longer exposure = greater death(b) sterilization = reduction of survival probability to #10-6

(5) temperature; increase usually increases effectiveness of chemical(6) local environment

(a) heat kills better at acid pH(b) efficiency higher with lower organic matter

c. Heati. moist heat

(1) boiling kills viruses, bacteria, fungi(2) 10 minutes boiling kills vegetative cells, not endospores(3) pressurized steam (autoclave)

(a) autoclave = fancy pressure cooker(b) combines wet heat with pressure; allows temperatures above 100o C(c) temperatures above 100o C required to destroy endospores(d) chamber filled with saturated steam for 121o C, 15 psi(e) materials exposed for $15 minutes

(4) pasteurization (a) reduces microorganism numbers but retains flavor of foods, especially dairy, beer, and

other beverages(b) brief heating, followed by rapid cooling(c) 63o - 66o C for 30 minutes (batch or older method)(d) flash pasteurization = 72o C for 15 seconds(e) ultrahigh temperature (UHT) = 134o C for 1-2 seconds

(5) tyndallization = discontinuous boiling or fractional steam sterilization(a) heat material to 90o-100o C for 30 minutes on 3 consecutive days, incubated at 37o C(b) 1st heating destroys cells but leaves endospores(c) 2nd and 3rd heating destroys germinating endospores

ii. Dry heat sterilization(1) 160o-170o C for 2-3 hours(2) cell constituents oxidize(3) less effective than moist heat

iii. Filtration(1) physical removal of microorganisms

(a) excellent for heat sensitive materials(b) can be used with gases

(2) depth filters = thick layers of fibrous or granular material(a) twisting channels of small diameter(b) microbes removed by physical entrapment and adsorption to filter material

(3) membrane filters = thin (0.1 mm) membranes (a) made of cellulose acetate, cellulose nitrate, polycarbonate, polyvinylidene chloride, or

other synthetic materials(b) vegetative cells removed with 0.2:m pore size

iv. Radiation(1) alters DNA, causing lethal mutations(2) UV= ultraviolet light (260 nm)

(a) doesn't penetrate glass, dirt films, water, many plastics(b) often used to sterilize cabinets or entire rooms

(3) ionizing (e.g., gamma)(a) penetrates objects

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(b) also called cold sterilization(c) widely used with food

d. Chemical methodsi. phenolics

(1) early use by Lister(2) Lysol contains a mixture of phenolics(3) denature proteins and disrupt cell membranes(4) excellent for surfaces, but can cause skin irritation

ii. alcohols(1) bactericidal and fungicidal, but not sporicidal(2) denature proteins and dissolve membrane lipids

iii. halogens(1) iodine most common, followed by chlorine(2) tincture of iodine = $2% iodine in water/ethanol solution of potassium iodide

(a) effective antiseptic(b) stains and may damage skin(c) iodophor = complex of iodine + organic carrier

(i) water soluble, stable, nonstaining(ii) slow release to prevent skin burns and irritation

(3) chlorine disinfectant of choice for municipal water supplies and swimming pools(a) added in many forms; forms hypochlorous acid(b) oxidizes several materials, destroying cells but not endospores(c) Halzone tablets used for personal drinking water(d) excellent household disinfectant

(i) 1:100 dilution of household bleach (1.3 oz/gal) + 0.7% nonionic detergent (1 oz/gal)(ii) cleans and kills bacteria

iv. heavy metals(1) Hg, Ag, As, Zn, Cu used to be common germicides

(a) most heavy metals are bacteriostatic, not bactericidal(b) currently using less toxic, more effective germicides(c) 1% silver nitrate added to eyes of infants to prevent ophthalmic gonorrhea; being replaced

by erythromycin, which is also effective against Chlamydia and Neisseria(d) silver sulfadiazine used on burns(e) copper sulfate used as algicide in lakes and swimming pools

(2) combine with proteins (sulfhydryl groups), inactivating themv. quaternary ammonium compounds

(1) detergents = organic molecules (non-soaps) that serve as wetting agents and emulsifiers(a) amphipathic molecules(b) effective cleansing agents

(2) cationic detergents more antimicrobial than anionic detergents(a) quaternary ammonium compounds most popular(b) positively charged quaternary nitrogen with long hydrophobic aliphatic chain

(3) disrupt membranes and may denature proteins(4) kill most cells, but not endospores or M. tuberculosis(5) often used as disinfectants for food utensils, small instruments, and skin antiseptics

vi. aldehydes(1) formaldehyde and gluteraldyde most common(2) combine with and deactivate proteins(3) 2% glutaraldehyde commonly used to disinfect hospital equipment

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vii. gases(1) ethylene oxide is both microbicidal and sporicidal

(a) combines with cell proteins(b) penetrates packing materials, even plastic wraps

(2) explosive, usually done in special sterilizer6. Antimicrobial chemotherapy (living systems) (Chapter 20)

a. Historyi. chemotherapeutic agents = chemical agents used to treat disease

(1) destroy or inhibit growth of pathogens(2) concentrations low enough not to damage host(3) include antibiotics = microbial products or their derivatives that can kill or inhibit growth of

microorganismsii. Paul Erlich began the modern age of chemotherapy

(1) magic bullet = chemical (toxic dye) that would specifically bind to and destroy pathogens (a) particularly interested in syphilis and African sleeping sickness(b) normal syphilis treatment was ingestion of toxic mercury compounds (often patients died)(c) trypanosomiasis treated similarly with arsenic compounds(d) deliberately tried preparing organic material containing arsenic which would be less lethal

to humans(2) arsphenamine (Erlich 606) was effective against syphilis (trade name Salversan)

(3) noted that dyes were strongly taken up by bacteria, so studied those for specific toxicity(a) trypan red was effective against the trypanosome that causes African sleeping sickness(b) acriflavine (yellow dye still in use)

(i) good for superficial wounds and skin infections(ii) too toxic for internal use

(4) established the concept of selective toxicity; led to testing of hundreds of compounds(a) Gerhard Domagk found that Prontosil Red killed pathogenic staphylococci and

streptococci, but didn't harm the animal(b) Jacques and Therese Trefouel discovered that Prontosil Red was converted to

sulfanilamide, the true active factor

iii. led to development of sulfa drugs (sulphonamides)(1) specifically designed go stay in gut or be absorbed in bloodstream(2) often more active against microbes and less toxic to humans than sulfanilamide(3) tremendously effective against pneumonia, puerperal fever (systemic infection due to

Streptococcus pyogenes)(4) general structure similar to PABA (required for folic acid synthesis)

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(5) some microbes require PABA for growth(6) sulfa drugs are competitive inhibitor; prevent growth and allow body's defense mechanisms

to deal with microbes(7) during 40's and 50's, many vitamins discovered and inhibitory analogs made

(a) worked in test tubes(b) too toxic in humans, eliminated too well by kidneys, vitamin concentration in tissues too

high, or infecting bacteria did not require vitamin(c) not one drug was of practical therapeutic importance

iv. penicillin = first antibiotic to be used therapeutically(1) originally observed by 21-year old French medical student Ernest Duchesne, rediscovered by

Alexander Fleming(2) Fleming observed a contaminant mold which seemed to be dissolving Staphylococcus

colonies(a) found that broth from a Penicillium culture could destroy a number of pathogens(b) unable to purify the active compound very well(c) published some papers and abandoned the area

(3) Howard Florey and Ernst Chain (1939) obtained Penicillium notatum from Fleming(a) purified penicillin(b) destroyed staph and strep infections in mice(c) Fleming, Florey, and Chain shared 1945 Nobel

v. Selman Waksman (1944) discovered streptomycin, an antibiotic produced by the actinomyceteStreptomyces griseus (Nobel 1952)(1) led to increased search for other antibiotic producers(2) by 1953, microbes producing chloramphenicol, neomycin, terramycin, and tetracycline were

isolatedb. Characteristics of antimicrobials

i. selective toxicity = kill or inhibit pathogen without damaging host(1) therapeutic dose = drug level required for clinical treatment of a particular infection(2) toxic dose = drug level at which the agent becomes too toxic for host(3) therapeutic index = ratio of toxic dose to therapeutic dose (larger number = better)(4) drugs specific for microbial function that doesn't occur in host(e.g., cell wall synthesis) have

highest therapeutic indexii. range of effectiveness varies

(1) narrow-spectrum = effective against a limited number(2) broad-spectrum = effective against many types of pathogens(3) can be classified based on targeted group

(a) antibacterial(b) antifungal(c) antiprotozoan(d) antiviral

(4) some agents are effective against more than one group (e.g., sulfa drugs work against bacteriaand some protozoa)

iii. chemotherapeutic agents can be natural, synthetic, or semi-synthetic

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(1) synthetics are(a) sulfa drugs(b) trimethoprim(c) chloramphenicol(d) isoniazid(e) dapson(f) many antiviral and antiprotozoan drugs

(2) semisynthetic are natural antibiotics that have been chemically modified to make them lesssusceptible to inactivation(a) ampicillin(b) carbenicillin(c) methicillin

iv. chemotherapeutic agents can be cidal or static(1) can be concentration dependent(2) effect varies with species(3) static effect relies on host's defense mechanism for elimination of infection

c. Determining activity levelsi. dilution susceptibility tests

(1) a series of broth tubes containing a range of antibiotic concentrations inoculated with testorganism

(2) minimal inhibitory concentration (MIC) = lowest concentration that prevents growth (nogrowth after 16-20 hr)

(3) minimal lethal concentration (MLC) = lowest concentration that kills the organism (nogrowth in subculture)

(4) cidal drugs usually kills at 2-4x MIC, static drugs kill at much higher concentrations (if at all)ii. disk diffusion tests

(1) antibiotic impregnated disks are placed on agar previously inoculated with the test bacterium(a) antibiotic diffuses, forming a gradient(b) resistant organisms grow up to the disk(c) susceptible organisms grow some distance from the disk, displaying a clear zone around

the disk(i) wider the clear zone = more susceptible(ii) zone width is a function of initial concentration, solubility, diffusion rate,

susceptibility of organism(iii) zone width cannot be used to compare 2 antibiotics

(2) Kirby-Bauer most used disk diffusion test(a) Mueller-Hinton agar inoculated with lawn of bacteria(b) disks placed on surface(c) incubation at 35o C for 16-20 hr(d) diameters of zones measured and compared to tabulated values to determine degree of

microbial resistance(i) plot MIC vs zone diameters for different strains(ii) determine from plot if treatment dosage would result in MIC

d. General mechanisms of activityi. pathogen damage can occur through several mechanisms

(1) most selective antibiotics interfere with cell wall synthesis(2) high therapeutic index since cell walls not sound in eucaryotes

ii. Cell wall synthesis inhibition(1) penicillin, ampicillin, carbenicillin, methicillin, cephalosporins

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(2) inhibit enzymes for peptidoglycan cross-linking; activate cell wall lytic enzymes(3) bacitracin inhibits CW synthesis by interfering with lipid carrier that transports precursors

across the plasma membraneiii. protein synthesis inhibition

(1) streptomycin, gentamicin; bind to 30S ribosome subunit and causes misreading of mRNA(2) chloramphenicol binds to 50S ribosomal subunit, inhibits peptidyl transferase, blocking

peptide fond formation(3) tetracyclines bind to 30S, interfere with aminoacyl-tRNA binding(4) erythromycin binds to 50S, inhibits peptide chain elongation(5) high therapeutic index because drugs differentiate between procaryotic and eucaryotic

ribosomesiv. nucleic acid synthesis inhibition

(1) rifampicin(2) inhibits DNA-dependent RNA polymerase, blocking RNA synthesis(3) often toxic to eucaryotic systems also

v. Cell membrane disruption(1) polymyxin B(2) binds to cell membrane, disrupts structure and permeability

vi. metabolic antagonism (antimetabolites)(1) sulfa drugs compete with PABA, inhibits folic acid synthesis(2) trimethoprim inhibits dihydrofolate reductase, blocking tetrahydrofolate synthesis(3) dapsone interferes with folic acid synthesis(4) isoniazid may disrupt pyridoxal or NAD metabolism and functioning; inhibits synthesis of

mycolic acid "cord factor"vii. Several factors determine effectiveness of antimicrobial drugs

(1) drug must reach site of infection, so delivery system important(a) penicillin G unstable in stomach acid(b) gentamicin (aminoglycosides) not well absorbed through gut and must be injected

intramuscularly(c) parenteral routes = non-oral administration

(2) concentration must exceed MIC(a) dependent on amount administered,(b) speed of uptake,(c) rate of elimination from body(d) best if drug is absorbed slowly over a long period and excreted slowly

(3) infecting organism(a) dormant bugs less susceptible(b) pathogen must have proper target site

(4) many agents less effective due to resistance mechanisms, spread quickly via plasmidse. Classes of antibiotics

i. sulfa drugs(1) structural analog (similar to metabolic intermediate)(2) similar to PABA, necessary for synthesis of folic acid

ii. quinolones(1) synthetic drug, broad spectrum, bactericidal(2) inhibits DNA replication and repair, transcription(3) nalidixic acid, fluoroquinolones (ciprofloxacin, norfoxacin, ofloxacin)

iii. penicillins(1) $-lactam ring is common feature (side chains vary)

1113- p.26

(2) penicillinase destroys ring(3) block peptidoglycan cross-linking, leading to lysis(4) many people are allergic

iv. cephalosporins(1) originally isolated from Cephalosporium (fungus)(2) $-lactam ring, like penicillins(3) useful for people allergic to penicillin(4) broad spectrum

v. tetracyclines(1) naturally produced by Streptomyces or semi-synthetic(2) bind to 30S ribosomal subunit, inhibiting protein synthesis(3) bacteriostatic(4) broad spectrum

vi. aminoglycoside antibiotics(1) Streptomyces make streptomycin, kanamycin, tobramycin(2) Micromonospora purpurea synthesizes gentamicin(3) bind to small ribosomal subunit, inhibiting protein synthesis(4) bactericidal, most effective against gram negatives(5) quite toxic to humans(6) widespread resistance

vii. erythromycin(1) macrolide, synthesized by Streptomyces erythraeus(2) broad spectrum, bacteriostatic, most effective against G+(3) bind to 23S rRNA of 50S ribosomal subunit, inhibiting protein elongation(4) macrolides have 12- to 22-carbon lactone rings

viii. chloramphenicol(1) synthetic, but originally from Streptomyces venezuelae(2) acts like erythromycin(3) broad spectrum, bacteriostatic(4) quite toxic to humans

f. Mechanisms of drug resistancei. drug cannot enter cell

(1) G- unaffected by penicillin G because it can't penetrate the outer membrane(2) changes in binding proteins render cells resistant

ii. chemical modification(1) penicillinase hydrolyzes the $-lactam ring(2) groups can be added which inactivate drugs

iii. modification of target(1) changes in 23S rRNA protects against chloramphenicol or erythromycin(2) change binding site for sulfanilamide

iv. genes for drug resistance can be chromosomal or on plasmids(1) spontaneous mutations in chromosome are rare(2) chromosomal changes usually result in changes in drug receptors, preventing binding(3) R plasmids (resistance plasmids) often code for enzymes that destroy or modify drugs

(a) implicated in resistance to aminoglycosides, penicillins, cephalosporans, erythromycin,tetracyclines, sulfonamides, chloramphenicol, and others

(b) plasmids transferred rapidly through populations(c) single plasmid can carry resistance to many drugs

v. Overuse of antibiotics has led to many resistant strains

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(1) increase drug concentrations to destroy susceptible and spontaneous mutants(2) use two drugs together(3) limit use, especially broad-spectrum antibiotics

g. Antifungal drugsi. eukaryotic, so drugs often toxic to humansii. most fungi have efficient detoxification mechanismsiii. often target membrane sterols or cell walls

h. Antiviral drugsi. most drugs disrupt critical stages in virus life cycle or synthesis of viral-specific nucleic acidsii. difficult to use drug therapy, since viruses use host's cell machinery