VALDOSTA STATE UNIVERSITY

Biology 2900--Summer 2005

PART I. MICROBIAL CELL STRUCTURE AND GROWTH REQUIREMENTS

1. Intro to microbiology in human health
1. What is a disease?
1. process causing damage to tissues or changes physiologic function, typically identified by the signs and symptoms of the damage
2. can be divided into infectious and noninfectious, with infectious diseases divided into communicable and non-communicable
3. this course focuses primarily on the organisms responsible for infectious diseases; note that some microorganisms are also responsible for certain non-infectious diseases (intoxications)
2. discovery of infectious agents one of the huge mile-stones in medicine
1. setting the stage
1. Hook and the discovery of cells (1665)
2. van Leeuwenhoek and the discovery of microscopic organisms (1674)
3. Schleiden and Schwann propose the cell theory of life (1838-39)
4. Pasteur (and Tyndall) refutes the theory of spontaneous generation (1861) -- details
2. the germ theory of disease
1. Henle proposes preliminary germ theory (1840) -- problem with strict germ theory?
2. Pasteur connects wine production and spoilage to activity of microorganisms (1850s and 1860s)
3. Koch connects microorganisms with anthrax and tuberculosis; develops the principle method for determining the cause of an infectious disease (1870s and 1880s)
1. Koch's postulates
1. the purported infectious agent must be present in every case of the disease
2. the purported agent must be grown in pure culture from diseased organisms
3. when healthy hosts are inoculated with pure culture of the purported agent they develop exactly the same signs and symptoms
4. the purported agent must be isolated from the newly infected hosts
3. benefits of the germ theory of disease
1. the development of antiseptic surgery (Semmelweiss (1840s) and Lister (1860s))
2. understanding epidemics (Snow, cholera, and contaminated water--possibly the first epidmiologial study (1850s))
3. development of antibiotics (Ehrlich 1900s) and vaccines (Jenner and cowpox/smallpox, Pasteur and rabies, anthrax, and cholera (1890s))
4. present-day disease surveillance
1. state and county public health agencies
1. monitor local incidence of infectious disease
2. provide health/disease related services
2. National Centers for Disease Control and Prevention (CDC)
1. supports infectious disease research laboratories
2. collects information on diseases of public health importance, including number of new cases of over 50 notifiable diseases (see Table 20.2)
3. publishes Morbidity and Mortality Weekly Report (see http://www.cdc.gov/mmwr)
3. World Health Organization (WHO)
1. provides world-wide guidance in the field of health
2. sets global standards for health
3. works to strengthen national health programs
4. works to transfer develop and transfer appropriate health technology
5. publishes Weekly Epidemiological Record
3. trends and prospects
1. disease eradication and reduction -- targeted vaccination programs
   
1. smallpox almost completely eradication
2. incidence of polio and measles much reduced globally
2. emerging diseases -- new diseases or diseases with marked increases in numbers of cases, usually related to one or more of the following:
   
1. microbial evolution
2. breakdown of the public health network (Russia)
   
3. changes in human behavior (day-care centers)
4. advances in technology (contact lenses)
   
5. larger populations
6. development (schistosomiasis)
7. food distribution networks
8. climate changes
3. nosocomial infections -- hospital-acquired infections
   
1. Major players: Enterococcus spp., Escherichia coli, Pseudomonas spp., Staphylococcus aureus, Staphylococcus spp.
2. Infections often through medical devices, health care personnel, air
3. hospital-based infection control committees
   
4. major groups of infectious agents
1. cellular forms are divided into three domains
1. Eubacteria -- mostly small, single-celled organisms with a rigid wall with peptidoglycan and no nucleus; reproduce by binary fission; responsible for most of the "bacterial diseases"
2. Archaea -- similar in size and shape to Eubacteria; also without a nucleus; wall does not contain peptidoglycan
3. Eucarya -- more structurally complex than Eubacteria and Archaea; cells with a nucleus, cytoskeleton, and other complex structures (mitochondria) not found in the other domain; many also form complex multicellular associations (Fungi, Animalia, Plantae)
2. non-cellular forms (are they alive?)
1. viruses -- obligate intracellular parasites (no metabolic machinery); consist of proteins and nucleic acid, sometimes covered in an envelop derived from the hosts membrane
2. viroids -- naked bits of RNA
3. prions -- infectious proteins (reproduce?)
5. final note -- not all microorganisms are harmful
1. geochemical cycling -- carbon, nitrogen, etc.
2. food production -- beer, bread, yogurt, cheese, tofu, etc.
3. industrial chemicals
4. antibiotics, dietary supplements, anti-cancer agents, vaccines
2. Quick review of some basic chemistry (review on your own, be sure you know and understand these concepts from your chemistry course)
   
1. atoms, molecules, chemical bonds (ionic, covalent, hydrogen)
2. CHNOPS
3. water and water solutions (water is about 70% of mass of the cell)
1. properties of water (polar)
2. concentrations of solutions (molarity vs percent composition)
3. acids, bases, buffers
4. electrolytes (about 1% of the dry weight of a cell)
5. macromolecules -- four major classes, all built of smaller subunits by dehydration reactions
1. proteins
1. built from amino acids (amino group, acid group, side chain)
2. joined by peptide bonds
3. four levels of structure
1. primary -- sequence of amino acids
2. secondary -- twisting around the peptide bond
3. tertiary -- three dimensional shape
4. quaternary -- more than one chain held together by weak bonds
4. substituted or conjugated proteins have other molecules attached to the side chain
2. carbohydrates -- sugars and starches; (CH₂O)_n
1. monosaccharides can be ring or linear; rings either alpha or beta depending on OH at 1 carbon
2. disaccharides -- two monosaccharides joined
3. polysaccharides -- multiple monosaccharides joined; linkage is important
1. starch -- primarily alpha 1, 4 linked glucose
2. cellulose -- primarily beta 1, 4 linked glucose
3. dextran -- primarily alpha 1, 6 linked glucose
3. lipids
1. simple lipids
1. mono-, di-, and triglycerides
2. cholesterol
2. compound lipids -- contain other types of molecules (phospholipids, lipopolysaccharides, lipoproteins)
4. nucleic acids -- chains of nucleotides
1. nucleotide structure; RNA vs DNA
2. complementary base pairing
3. single-stranded molecules
4. double-stranded molecules (Watson-Crick model)
3. Structure of microorganisms
1. tools
1. microscopy
1. general concerns:  resolution, magnification, contrast
2. visible light microscopy
1. resolution at best 0.2 micrometers (200 nanometers)
2. contrast increased either optically (phase, interference) or by stains
1. stains usually target particular molecules or classes of molecules
2. staining procedures often involve use of a primary stain (binds to the target chemical), a mordant, a decolorizing agent, and a secondary (counter) stain
1. example: in Gram stains, the primary is crystal violet, the mordant is iodine, the decolorizing agent is alcohol, and the counterstain is safranin
2. other important techniques: acid-fast stain, endospore stain, capsule stain, flagellar stain,
3. possible to link fluorescent dyes to antibodies to target particular molecules not easily stained or not easily visible with normal stains (immunofluorescence)
3. confocal microscopes - three dimensional imaging
3. electron microscopy (scanning and transmission)
1. resolution at best 0.1 nanometers
2. contrast increased with stains
4. scanning probe microscopy
1. uses new technology to look at surfaces
2. resolution can be greater than that of scanning electron microscopes
2. cell fractionation -- basically, we break the cells open, then separate the pieces
1. breaking techniques include: chemical digestion, grinding and smashing, pressure
2. separation techniques include: differential centrifugation, electrophoresis, chromatography
3. basic results -- cells divided into three domains: Bacteria, Archaea, Eucarya (see table 1.2)
1. note that not all infectious agents are cellular or even alive: viruses, viroids, and prions
2. what are cells
1. functionally: boundary, metabolic machinery, genetic material
2. structurally: cell membrane, cytoplasm, DNA
3. connections: boundary = cell membrane; metabolic machinery = cell membrane + cytoplasm; genetic material = DNA
3. structure of eubacteria
1. size and shape
2. associations: diplococci and diplobacilli; chains, packets, and clusters
3. subcellular (chemical) structures: cell membrane, cell wall, cell envelope, cytoplasm, nucleoid
1. cell membrane
1. phospholipid bilayer with associated proteins (fluid mosaic model)
2. function as a barrier
1. phospholipids are semipermeable
2. some associated proteins function as transporters and permeases
1. facilitated transport
2. active transport via proton motive force
3. active transport via ATP breakdown (ABC = ATP binding cassette)
4. group translocation involves an alteration of the molecule during transport
3. other functions (binding) also use (different) associated proteins
2. cell wall
1. in most eubacteria
2. functions primarily as protection against osmotic shock and osmotic-induced rupture
3. basis is a chemical called peptidoglycan (murein in older texts) present only in eubacteria
1. consists of alternating units of N-acetyl muramic acid and N-acetylglucosamine
2. the NAM-NAG chains are linked by 4 amino acids attached to NAM units: L-alanine, D-glutamic acid, diaminopimelic acid, and D-alanine; in some bacteria (Gram negatives) the chains are directly linked, in others (Gram positives) they are joined by small linking proteins
3. some bacteria (Gram positives) have teichoic acid mixed in with and attached to the peptidoglycan; teichoic acid is a mix of ribitol-phosphate or glycerol-phosphate and other molecules that attach directly to NAM subunits; they stick out of the peptidoglycan giving the cell a negative charge
4. Gram negative vs Gram positive eubacterial walls
1. cells stain differently in the Gram staining procedure
2. differences associated with extra layer in Gram negative bacteria--the outer membrane
1. special type of bilayer with phospholipids on one side and lipopolysaccharides on the other
1. lipopolysaccharide contains two parts
1. Lipid A--nonpolar part in the membrane; causes immune response (endotoxin)
2. O-specific polysaccharide--strain specific
2. outer membrane contains porins--protein channels with a degree of specificity regarding what can pass through, can block some toxins
3. space between outer membrane and cell membrane is the periplasm
3. differences in the peptidoglycan layer
1. Gram positive bacteria contain teichoic acid (attached to NAM unit) and lipoteichoic acid (linked to the cell membrane)
2. in Gram positive bacteria the 4-amino-acid-long chains in peptidoglycan are linked through small linker-proteins; in Gram negatives they are directly linked
4. note that Mycoplasmas do not have a cell wall
3. glycocalyx (capsule, slime layer) composed of polysaccharides or proteins (proteins sometimes with abundance of D-amino acids); aids in attachment and/or in defense
4. appendages
1. bacterial flagellum
1. cork-screw type propellor that pushes cell through surroundings; aids in taxis
2. uses proton motive force for energy
3. composed principally of protein called flagellin
4. number and position of flagella is sometimes used for identification
2. pili or fimbriae
1. hollow protein rods
2. type and function depends on the type of protein
1. some used for attachment--have adhesin or glue at end
2. twitching motion on agar
3. conjugation (bacterial sex)
5. internal structures
1. the nucleoid (bacterial chromosome)
2. plasmids (extraneous circles of DNA)
3. ribosomes--protein + RNA, used to make proteins, smaller than in eukaryotes
4. storage granules--glycogen, poly-beta-hydroxybutyrate, volutin (metachromatic granules, poly-phosphate granules)
5. gas vesicles
6. endospores--protected, dormant cells produced within a cell; many can withstand hours of boiling water, radiation, desiccation, etc.; have a wall containing peptidoglycan covered in a special structure called the spore coat; core is rich in dipicolinic acid which binds calcium ions and may help resist environmental conditions; special DNA-binding proteins are also found which might protect and repair the DNA
7. more advanced environmental bacteria have internal membranes
4. structure of archaean cells
1. size and shape similar to eubacteria (only recently separated)
2. subcellular structures
1. cell membrane contains branched hydrocarbons based on isoprene subunits (not fatty acids)
2. attachment of hydrocarbons to glycerol in the cell membrane via an ether linkage, not an ester linkage as in eubacteria and eukaryotes
3. diglycerol tetraether compounds common in cell membrane
   
4. cell wall functionally similar, chemically different--do not contain peptidoglycan (use pseudopeptidoglycan)
5. ribosomes resistant to chloramphenicol and streptomycin
3. as a side note--many of the known archaeans are from extreme environments (hot springs, acidic waters)
5. structure of eukaryotes
1. size and shape of eukaryotic cells
   
2. subcellular structures
1. cell membrane -- similar to bacterial membranes, but often with cholesterol; inside and outside often contain different types of phospholipids and proteins
2. cell wall -- various or lacking -- chemical composition used to distinguish different groups
3. cytoplasmic structures include
1. membrane-bound nucleus, DNA packaged into linear chromosomes
2. larger ribosomes, not susceptible to streptomycin and chloramphenicol
3. complex system of cytoplasmic membranes (endomembrane system -- endoplasmic reticulum, transport vesicles, Golgi apparatus)
4. complex cytoskeleton responsible for movement of transport vesicles within the cell, endocytosis and exocytosis, mitosis, amoeboid movement, eukaryotic flagella, and, partly, the shape of the cell; prokaryotic cells apparently have a rudimentary cytoskeleton (only recently discovered), not nearly as well-developed or complex at the eukaryotic cytoskeleton
5. mitochondria and chloroplasts
1. involved in ATP formation
2. double membrane
3. 70s ribosomes--sensitive to chloramphenicol
4. circular molecules of DNA
3. groups of eukaryotes important in the study of infectious disease
1. classification systems are a work in progress; used to pay lots of attention to modes of nutrition, movement, now more concerned with cellular structure and relatedness as indicated by ribosomal RNA sequences
   
2. unicellular eukaryotes
   
1. alveolates -- includes dinoflagellates, apicomplexans, ciliates
1. dinoflagellates -- unicellular, photosynthetic, recognized by the arrangement of flagella
1. one of the major groups at the base of aquatic food chains
2. frequently form red tides
3. generally cause harm to humans through intoxicaton -- toxins responsible for symptoms ranging from diarrhea to complete paralysis and death; Pfeisteria may actually infect humans
2. apicomplexans -- intracellular parasites responsible for malaria, toxoplasmosis, cryptosporidiosis; have the remnants of chloroplasts in cell
2. sarcomastigophorans -- old name that includes members of several distinct phyla and possibly kingdoms; generally either flagellate or amoeboid; some do not have mitochondria; diseases include sleeping sickness, leishmaniasis, trichomoniasis, and several intestinal problems
3. multicellular eukaryotes
1. plants -- intoxications, generally no infections
2. fungi -- generally have cell walls with chitin, obtain their nutrients through extracellular digestion then absorption of the small molecules
1. either unicellular (yeasts) or filamentous (molds, mildews, and mushrooms); the filamentous may form complex structures
   
2. produce asexual and sexual spores; classification based on reproduction and the type of sexual spore produced
   
3. cause harm to humans through intoxications or infections (mycoses)
1. toxins cause symptoms ranging from gastroenteritis to neurological impairment; some (ergot-derivatives) used medically
2. infections ranges from ringworm and yeast infections to potenially fatal cases of aspergillosis, cryptococcal meningitis, and histoplasmosis
4. of course many fungi are beneficial -- yeast for beer, bread, and wine, molds for cheese and antibiotics
3. animals -- important as parasites and as vectors for other infectious agents; three phyla generally involved
1. flat worms -- simple body structure without an internal cavity between the gut and the outside
1. trematodes and flukes -- complex life cycles generally involving multiple hosts and/or some sort of movement through the host's body (shistosomiasis)
2. tapeworms -- intestinal parasites with an even simpler, segmented body
2. round worms -- large group with many human parasites; includes pinworms, hookworms, trichella, filaria
3. arthropods -- complex animals with jointed legs and exoskeletons; many are ectoparasites of humans; in this role they may also serve as vectors of disease
1. arachnids (ticks and mites) -- two major body regions, multiple legs
1. ticks transmit the causative agents for spotted fevers, Lyme disease; some cause paralysis while feeding
2. mites are common on humans and other mammals; some cause scabies, others transmit rickettsial diseases
2. insects -- three major body regions and six legs, adults typically, but not always, with wings
1. mosquitoes transmit the agents responsible for malaria, yellow fever, equine encephalitis, etc.
2. fleas can transmit the the agents for black plague and a form of typhus
3. lice can transmit the agents for epidemic typhus and trench fever
   
4. bacterial growth
1. most bacteria grow by binary fission; therefore, when all bacteria reproduce the population doubles
1. counting bacteria
1. direct counts (microscopic or using a cell-counter (such as a Coulter counter))
2. plate counts with serial dilution
3. most probable number (and serial dilution)
4. turbidity
5. total mass
6. DNA, peptidoglycan, etc.
2. in culture, growth follows characteristic growth curve
1. lag phase -- adjust to conditions
2. exponential phase (log phase) -- when doubling with constant doubling time as above; produce normal (primary) metabolites
3. stationary phase -- growth slows, some cells dying, some reproducing
4. death phase -- culture begins to decline
5. prolonged decline phase
3. key number in looking at growth bacteria is the doubling time during exponential growth (time taken for population to double)
1. if you know initial population, the doubling time, and the total time for growth, it is easy to determine the size of the final population:  N_final = N_initial x 2^{number of doublings}  where the number of doublings is found by dividing the total time for growth by the doubling time
2. possible to detemine the doubling time if know the size of the initial and final populations and the time the bacterial were let grow: doubling time =  {time x log(2)}/{log(N_final) - log(N_initial)}
1. simpler if the ratio of the final population to the initial population population is a power of 2; in this case you can compare the exponents directly
1. example:  suppose N_final / N_initial = 1024 = 2¹⁰; then 2¹⁰ = 2^{number of doublings} = 2^{total time/doubling time}; if we compare exponents, 10 = total time/doubling time; solving for the doubling time gives doubling time = total time/10
4. the doubling time depends on the type of bacteria and on the enivornmental conditions
2. key environmental conditions
1. growth range vs optimum conditions
2. temperature conditions
1. temperature affects protein, cell membranes; high temperatures more likely to cause permanent changes in proteins and cell death, low temperatures often just slow processes down; optimum temperatures tend to be near high end of range
2. categories of bacteria
1. psychrophiles -- opt. temp. between -5 and 15
2. psychrotrophs -- opt. temp. between 20 and 30, temperature range goes much lower
3. mesophiles -- opt. temp. between 25 and 45; most common in human infections (although a few disease-causing organisms (syphilis for example) prefer lower temperatures
4. thermophile -- opt. temp. between 45 and 70
5. hyperthermophiles -- opt. temp between 70 and 110
3. oxygen
1. uses of oxygen in cells -- energy metabolism (fermentation vs respiration)
2. harmful effects -- forms hydrogen peroxide, superoxides, which can attack and destroy organic molecules; catalase and superoxide dismutase are regarded as defences against the harmful effects, more common in aerobic organisms than in anaerobic organisms
3. categories of bacteria
1. oligate anaerobes -- cannot grow in the presence of oxygen (Clostridium)
2. aerotolerant anaerobes -- do not use oxygen, but not harmed (Streptococcus)
3. facultative anaerobes -- can grow without oxygen, but does much better with; capable of switching to aerobic respiration if oxygen present, ferments otherwise (Escherichia)
4. microaerophiles -- require small amount of oxygen, but cannot handle high concentrations
5. obligate aerobes -- like us, require oxygen, cannot survive on just fermentation (Pseudomonas)
4. note: oxygen is not the only important atmospheric gas; some bacteria, called capnophiles, require increased carbon dioxide (Neisseria and Haemophilus)
4. pH
1. affects protein structure
2. categories
1. neutrophiles -- optimum between pH 5 and pH 8; most medically important bacteria are neutrophiles, including Helicobacter pylori (survives in stomach by splitting urea into carbon dioxide and ammonium)
2. acidophiles -- optimum pH below 5.5
3. alkaliphiles -- optimum pH above 8.5
5. water availability
1. almost all microbes require liquid water at some concentration; dried foods do not support bacterial growth
2. most bacteria require relatively pure water; adding sugar or salt can control bacterial growth
1. osmotolerant microbes can survive concentrations as high at 10% salt (concentrations?)
2. halophiles require 3% or higher salt
6. CHNOPS and energy
1. carbon and energy
1. chemoorganoheterotrophs
2. chemolithoautotrophs
3. photoheterotrophs
4. photoautotrophs
2. major nutrients (NPS)
3. minor nutrients (minerals)
1. growth factors (vitamins, etc.) and fastidious organisms
7. a quick aside: laboratory media
1. complex vs defined
2. selective (only certain types can grow) vs differential (allows you to distinguish between types)
3. controlling bacterial growth
1. general terminology -- review Table 5.1
2. selecting an antimicrobial procedure depends on
1. types of microorganisms expected, especially the degree of resistance
2. numbers of microorganisms expected
3. environmental conditions -- temperature, pH, etc., can influence effectiveness of method
4. potential risk of infection
3. methods of sterilization
1. moist heat
1. pasteurization: depends on the type of material; common protocols include 62C for 30 minutes or 72C for 15 seconds (note the drastic change in the amount of time required with a slightly higher temperature); ultrahigh temperature pasteurization calls for raising the temperature to 140C for only a couple of seconds
2. autoclaves and pressure cookers: standard conditions are 15 PSI, 121C, and 15 minutes or more; flash autoclaving at 135C sterilizes in 3 minutes (but might take hours to destroy prions)
2. dry heat
1. takes longer at higher temperatures--usually 160C for 2 to 3 hours
3. chemicals
1. lots of factors to consider including:
1. mode of action of the chemical (which subcellular structure does it attack)
2. environmental toxicity and risk
3. inactivation by environmental organics
4. residue
5. cost and availability
2. basic categories (review table 5.4)
1. alcohols: 60% - 80% solution of ethanol or isopropanol; coagulates proteins
2. aldehydes: formaldehyde (37% solution, formalin), glutaraldehyde (2% solution); formalin used mostly in vaccine production (and by biologists as a presevative), glutaraldehyde widely used medically
3. chlorhexidine: used in disinfectants and impregnated into catheters
4. ethylene oxide: gas used to sterilize equipment that can't be autoclaved
5. chlorine solutions: sodium hypochlorite--the active ingredient of bleach; a 1/100 to 1/10 dilution of bleach is usually sufficient to sterilize, but lots of organics can be a problem; in addition, reactions with organics may form carcinogens; chlorine oxide gas is used to treat wastewater and swimming pools
6. iodine solutions and iodophore solutions: used frequently as disinfectants; not as effective as chlorine solutions
7. metal compounds: often too toxic to use in medical situations; 1% silver nitrate solution used to be put in neonates eyes; mercury-containing solutions are avialable (mercurochrome, thimerosol, etc.)
8. ozone: gas sometimes used to disinfect drinking water (see Dr. Manning)
9. hydrogen peroxide solutions
10. phenolics: long history (carbolic acid), leave residure on surfaces (good and bad, depending on surface); triclosan and hexachlorophene common ingredients of soaps and lotions, hexachlorophene less so now than in the past (phenolics can have a degree of neurotoxicity)
11. quaternary ammonium compounds (quats): soaps that solubilize phospholipid bilayers
4. filtration
1. filtration works by capturing, not killing, microorganisms
2. two basic approaches:
1. depth filters -- papers, sand, diatomaceous earth
2. membrane filters -- plastic material with defined pore size
5. radiation
1. gamma radiation -- damages DNA and organic molecules
2. ultraviolet radiation -- UVC, with wavelengths between 220nm and 300nm; damages DNA
3. microwaves -- generally do not damage microorganisms directly; instead can raise temperature
4. preservation without sterilization
1. low temperature storage
2. lower water activity drying (and freeze-drying or lyophilization), adding salt, or adding sugar
3. preservatives
1. benzoic, proprionic, and sorbic acids can block membrane-mediated energy transformation
2. nitrates and nitrites block endospore germination; also keep meat red

Continue with Part 2