Description
Unit 1 4/18/17 11:50 AM
Microbiology: study of organisms too small to be seen without magnification
Microorganisms include:
• Bacteria
o Prokaryotic
o Cells made of polysaccharide (peptidoglycan)
o Reproduce asexually
o Most much smaller than eukaryotic cells
o Live alone, in pairs, clusters, chains
o Live anywhere w/enough moisture
• Viruses
o Smaller than the smallest prokaryote
o Acellular (not made of cells)
o Has little genetic material
• Fungi
o Eukaryotic
o Get food from other organisms
o Have cell walls
o Microscopic Fungi: Molds & Yeasts
▪ Molds
???? Multicellular
???? Long filaments, intertwine
???? Reproduce w/sexual and asexual spores (make
new individual w/out fusing to another cell)
▪ Yeasts
???? Unicellular
???? Oval – round
???? Reproduce by asexual budding (the daughter cell
grows off the mother), some by sexual spores
• Protozoa
o Single cell eukaryotes
o Similar to animals in nutritional needs & cell structure
o Capable of locomotion
▪ Cilia: Many short protrusions that propel the We also discuss several other topics like What is the definition of macroevolution?
microorganism
▪ Flagella: few extensions, whip-like motion
o Live in water, & inside animal hosts
o Most reproduce asexually, some sexual
• Helminths (worms)
o A parasite that can be seen by the naked eye, studied by microbiologists because eggs are microscopic
• Algae
o Unicellular & Multicellular
o Photosynthetic eukaryotes = make their own food from CO2 + water + energy from sunlight
o Large Algae: kelp and seaweeds, found in oceans, Agar used in lab media comes from these
o Small Algae: found in freshwater and oceans
• Archaea
o Prokaryotic
o Cell walls lack peptidoglycan
o Reproduce asexually
o Live alone/in pairs/chains/clusters
o Live in extreme conditions
▪ Highly saline and arsenic rich Mono Lake CA If you want to learn more check out What causes the trend of electronegativity?
▪ Acidic Hot Springs of Yellowstone National Park
▪ In oxygen depleted mud at bottom of swamps
Microbiology led to:
• Immunology: Study of bodies defense against pathogens • Public Health Microbiology
• Epidemiology: study of the occurrence, distribution, spread of disease in humans
o John Snow studied Cholera spread
• Food, Dairy, Aquatic Microbiology (Food Spoilage)
• Agricultural Microbiology: water pollution caused by microbes • Biotechnology
• Genetic Engineering & Recombinant DNA Technology: Microbes in cheese making, alcohol, microbes interaction with immune system • Environmental Microbiology: Study of Microbes in soil, water, other habitats
• Bioremediation: Use living bacteria, fungi, & algae to detox. Polluted environmentsDon't forget about the age old question of If the government wanted to decrease unemployment, it could be?
• Industrial Microbiology: Microbes used to make products o Pasteurization: Heat something to kill contaminating bacteria Microbes are also involved in flow of Energy and Nutrients • Photosynthesis: convert CO2 to organic material
• Decomposition: Breakdown dead matter & wastes into simple compounds
Microbes involved in infectious diseases
• Pathogens: microorganisms that harm (this is the minority of microorganisms)
o 2,000 dif. Microbes cause harm
o 10 billion new infections worldwide per yr.
o 12 million deaths by infections per yr.
Science according to the organism studied
• Bacteriology: The study of Bacteria
• Phycology: The study of Algae
• Mycology: The study of Fungi
• Protozoology: The study of Protozoa
• Parasitology: The study of Parasites
• Virology: The study of Viruses
History Of Microbiology Don't forget about the age old question of What is the effect of a drug that is an agonist?
• Prominent Discoveries
o Microscopy
o Scientific Method
▪ Make observation
▪ Generate question
▪ Generate hypothesis We also discuss several other topics like What was the cancer rate in 1900?
We also discuss several other topics like What are the two criteria for science?
▪ Experiment (w/control groups)
▪ Accept, reject, or modify the Hypothesis
▪ If hypothesis repeatedly verified = Theory or Law (ex: Theory of Evolution or Selective Pressure)
o Developed Medical Microbiology
o Microbiology Techniques
• Lifestyle of Microorganisms
o Exist freely in soil, water, surface
o Relatively harmless or even beneficial (decomposition)
o If associated with other organisms considered parasites and live in a host
• Early Microscopy
o Robert Hooke - 1660
▪ First compound microscope (microscope w/2 lenses) ▪ Saw cells of cork first and reminded him of a monk’s quarters which were called a “cell” – how we got the
name “cell”
▪ He examined living and non-living things
o Anton van Leeuwenhoek – 1632-1723
▪ Traded in linen & wanted to see the quality so made his own magnifying glasses and achieved the level of
microscopes
▪ Saw things that moved called them Animalcules
▪ When he died never passed on the secret to making the lenses and lost ability to see microscopic things
o Edward Jenner – 1796
▪ Developed first vaccine
???? Knew about variolation (when take puss from
someone w/smallpox & give it to someone else
they either die or are protected)
???? Decided when saw milkmaids hands w/something
that looked like smallpox but they never
contracted it, to take puss from their hands and
give it to a boy
???? The boy survived & was given smallpox, he was
protected, this was considered FIRST
VACCINATION (“vaca,” from “cow”)
o Joseph Lister – 1867
▪ Founder of antiseptic surgery
▪ Opened field of research in antisepsis & disinfection o Ignaz Semmelweis
▪ Introduced hand washing
▪ People not ready for it, didn’t take on even when
proved it worked
▪ Took on when Lister introduced it further
o Spontaneous Generation (Abiogenesis): Commonly held theory that things just started growing randomly, until it was disproved by the following people (proved Biogenesis: creation via biology)
o Louis Pasteur – 1822-1895
▪ Created flasks with bent necks that prevented microbes from falling into sterile solution
???? If broke neck microbes got in and grew
▪ Proved that grapes + yeast = alcohol & grapes + bacteria = acid
▪ Proved Yeast can grow with & w/out oxygen & from other cells
o Robert Koch – 1843-1910
▪ Germ Theory: disease is caused by germs
▪ Pioneer of Etiology: How diseases is caused
▪ Worked w/anthrax in animals & discovered endospores (the resting stage of bacteria) – this was the 1st time bacteria proven to cause disease
▪ Man who worked in lab developed Petri Dishes
▪ Walter Hess developed the media from algae – Agar ▪ Created framework to identify Pathogens: Koch’s Postulates
???? 1. Find evidence of microbe in every case of the disease
???? 2. Isolate microbe from infected subject & grow in lab
???? 3. Inoculate healthy subject & cause same
disease
???? 4. Re-isolate agent from subject
o Alexander Flemming – 1881-1955
▪ Discovered Penicillin 1929 (1st modern antibiotic) ???? Saw that fungi penicillian was killing staph he was growing
???? Penicillin enters widespread US – 1941
▪ Discovered Lysozyme from snot dripping & breaking down bacteria
???? In snot, tears, egg whites
o Selman Waksman – 1944
▪ Discovered Streptomycin – first drug for tuberculosis
▪ Discovered first way to isolate soil bacteria & test
antibiotics
▪ Reference to soil bacteria on grave: “The Earth will
open and bring forth salvation.”
5 I’s of Culturing Microbes
• Inoculation: Introduction of sample into container of media to produce culture of observable growth
• Isolation: Separate one species from another
• Incubation: placed in a temperature controlled chamber and microbe multiples for observable growth
• Inspection: Microscopic and Macroscopic Observation, what you can see upon inspection:
o Pure Culture: growth of 1 single known species
o Mixed Culture: growth of 2 or more identified species
o Contaminated Culture: the mixture was once pure & was mixed w/unwanted microbes growing in it.
• Identification:
o Use micro/macroscopic appearance to identify
o Perform biochemical tests to identify
o Use genetic characteristics to identify
o Use immunological tests to identify
Media to Isolate Microorganisms
• Physical Form of the Media
o Nutrient Agar (made from red algae)
▪ Solid Agar: Solid at room temp and body temp
▪ Holds moisture & nutrients (beef extract, peptone,
agar)
▪ Not digestible for most microbes
▪ Semisolid Agars: help determine if the organism is
motile or not, if it is then it will move in this media
???? Used in clinical tests
o Nutrient Broth: Liquid medium w/beef extract & peptone
• Different Chemical Compositions of Media
o Complex or Non-synthetic: Media has nutrients released by partial chemical breakdown by beef, soy, proteins, has at least 1 ingredient that is not exact (chemically defined)
o Defined: Media in which the exact chemical composition is known
o Synthetic: Contains pure organic and inorganic compounds in an exact amount (chemical formula)
o General Purpose Media: Grows many microbes, considered non-synthetic
o Enriched Media: complex organic substances (blood, serum, hemoglobin, and special growth factors that picky microbe require)
▪ Used to grow less abundant microbes
• Different Functions of Media
o Selective Media: media that contains nutrients that promote the growth of one microorganism and inhibit the growth of others
o Differential Media: media made so there is a change in appearance of media or the organisms that helps the
microbiologists tell the difference between different bacteria ▪ Agar has dye that changes color based on pH
o Blood Agar considered a differential and complex medium
Isolation Techniques
• Streak Plate: Serial dilution on a solid surface, use loop to spread on one quadrant then go on to quadrant 2 and go back into quadrant 1 slightly to dilute the bacteria, repeat 2 more times • Pour Plate Technique:
o Serial dilution of liquids
o Allows growth of different types of microbes because of different environment
o Used for fewer colonies
Colonies: Culture visible on the surface
CFU: Colony Forming Unit which pure culture comes from (comes from a single progenitor)
How to Dispose of Cultures
• Steam Sterilization
• Incineration
Key Characteristics of a Microscope
• Magnification: ability to enlarge objects
• Resolving Power: ability to show detail (Resolution)
o If particle size is smaller it has a better resolution
Compound Microscope
• Hooke used
• Binocular – 2 lenses
o Objective Lens: the lens right above the object, can have a series of lenses (4x, 10x, 40x, and 100x)
▪ Low Power: 10x10=100
▪ High Power: 10x40=400
▪ Oil Immersion: 10x100=1000 used to reduce light
refraction
o Ocular Lens: lens closest to the eye (can be monocular or binocular) – is 10 or 12.5 power (this is the 10 being
multiplied by the objective lens powers above)
• Light needs to be focused on specimen for best resolution Types of Microscopy
• Bright Field Microscopy
o Used in medical labs
o Image made w/light through the object
o Image absorbs light, darker than the background
o Used for live & fixed material
▪ Stain to improve contrast but kills microorganisms
• Dark Field Microscopy
o Illuminates objects from side
o Object white against dark background
o No stain used
o Living objects can be seen
• Fluorescence Microscopy
o UV light used w/filters to protect eyes
o Use dyes that fluoresce when exposed to UV light
▪ Have specificity to certain cell structures
▪ Can be coupled to antibodies to specifically target an
object
▪ Used to target specific bacteria or look for specific viral antigens
• Transmission Electron Microscopy (TEM)
o Uses a wavelength of an electron beam of ~0.5nm and creates a resolution of ~0.3nm = greatest magnifying
resolution
o Use very thin sections (20-100nm) for the beam to go
through specimen
o Use heavy metal salts to stain image & make contrast
o Nuclei diffract electrons
o Max magnification is 120,000x = can see what is inside things • Scanning Electron Microscope (SEM)
o Makes 3D image
o Sample is plated w/thin layer of a metal w/large nuclei (ex. gold)
o Electrons scanned across surface of sample & detector detects reflected electrons
o Image shown on TV screen
o Magnification not as good as TEM, ~10nm
Contrast: difference in intensity between two objects or between the object & the background
• Enhance Contrast by Staining
o Thick cell walls retain blue/purple stain (The Principle stain) o Thin cell walls lose principle/1st stain and allow to see the Secondary Stain = cells are pink
o Staining also deals with charges
▪ Positive Staining: Stains the organism
▪ Negative Staining: Stains the background, acidic dyes
used are repulsed by the negative charges on the cells
and don’t stain them (the capsules of bacteria are
negatively charged) –AKA Capsule Stain
o Simple Staining: made of single basic dye, soak the smear in dye for 30-60 seconds and rinse
o Differential Staining: Use more than one dye to see different cells & structures
Taxonomy
Taxonomy: organizing, classifying, and naming living things, was created by Carl Von Linne (Linneaus)
Classification: The orderly arrangement of organisms into Groups Nomenclature: Assigning names
Identification: determining and recording traits of organisms for placement into taxonomic schemes
Old Whittaker System
• Set up with Binomial (Scientific) Nomenclature, everything had two names – 1st name = Genus & 2nd name = Species
o Ex: Homo Sapiens
• Thought originally only 2 Kingdoms: Plantae and Animalia o Noticed certain organisms didn’t fit and created kingdoms Protista, Bacteria (Monera), and Myceteae for fungi
7 Main Taxa
• Species: the most specific, organisms that can successfully interbreed
• Genera: Similar species
• Families: Similar genera
• Orders: Similar families
• Classes: Similar orders
• Phyla: Similar classes
• Kingdoms: similar phylas
• The classification is determined by genetic/cellular level, use rRNA to compare things, Woes and Fox were the first to do this
There are 3 Domains which cover all organisms and then they are categorized by the 7 main taxa starting with kingdoms and getting more specific from there
• Archaea: Odd bacteria that live in extreme conditions
• Bacteria: True bacteria (*Prokaryotes fall under Bacteria and Archaea)
• Eukarya: have nucleus and organelles
How to Assign Names
• Binomial (Specific) Nomenclature
o 2 Names
▪ Genus – capitalized
▪ species – lowercase
o Both italicized or underlined
o Sometimes shorten first name with first letter and period. o Inspiration for names varied & imaginative
o Names usually mean something
▪ Ex: Staphylococcus aureus (S. aureus)
???? Staphyl = bunch of grapes (tells plane of division)
???? Coccus = “berry,” spherical shaped cell
???? Aureus = “golden,” yellow color
Evolution of Microorganisms
• Phylogeny: the natural relatedness between groups
• Evolution
o All new species come from an existing
o Close relatives have similar features, evolved from a common ancestor
o Usually progress towards a greater complexity
Characteristics of Cells
• Eukaryotic Cells: (Animals, Plants, Fungi, & Protists)
o Membrane-bound organelles (perform specific functions and compartmentalize cytoplasm)
o Has double-membrane bound nucleus with DNA in
chromosomes
• Prokaryotic Cells: (Bacteria & Archaea)
o No nucleus or other membrane bound organelles
o How to classify Prokaryotes:
▪ Microscopic Morphology: individual cocci or bacilli
appearance
▪ Macroscopic Morphology: Colony appearance
▪ Bacterial Physiology
▪ Serological Analysis: use liquid portion of blood after
clotting factors removed to determine the
microorganisms
▪ Genetic and Molecular Analysis
▪ If something causes a similar disease they are
often named something similar
Bacterial Taxonomy based on Bergey’s Manual of Systematic Bacteriology • Classification based on genetic information = phylogenic • Not all organisms have the same amount of G+C content (chemical content), affects DNA H-bonding
• (Remember that bacteria are Prokaryotic) There are 2 main Domains
o Archaea
o Bacteria
• 5 Major Subgroups w/25 different phyla in each one
*Within the 3 Domains (Eukarya, Archaea, and Bacteria) there are Organelles in the Bacteria Category because of Endosymbiosis (The idea that eukaryotic life evolved from prokaryotic life, will have some of the same characteristics)
Species and Subspecies
• Species: bacterial cells w/overall similar pattern of traits • Strain/Variety: A culture from one parent that differs from the others of that species (different structure or metabolism)
o Ex: Biovars and morphovars
• Type: subspecies w/differences in antigenic
makeup/immunoresponse (serotype or serovar), susceptibility to viruses (phage type) & pathogenicity (pathotype)
Diagnostic Scheme for Medical Use
• Phenotypic qualities to identify
• Limited to bacterial disease agents
• Categorizes bacteria based on shape, arrangement, physiological traits, cell wall structure
Dimensions of Bacteria (Prokaryotic)
Morphology: shape of organism
• Cocci: roughly spherical
o Staphylo: clusters of cocci, form when planes of cell divide randomly
o Strepto: Long chains of cocci, form when cocci cell divides down the center plane & the resulting cells stay connected ▪ Diplococcus: cocci that divide down the center plane &
stay connected in pairs
o Tetrad: the cocci divide in two planes and the four resulting cells stay connected
o Sarcina: Cocci that divide in 3 planes, stay connected to make a 3D tetrad
• Bacillus: non-spore forming rods
o Gram Positive (Thick peptidoglycan = retains purple dye) o Obligate Intracellular Parasite are Bacilli
▪ Can’t grow outside the host
▪ Very small
▪ Pleomorphic (shape varies) or Coccobacilli
▪ Ex: Rickettsia and Chlamydia
• Endospore Producing Bacilli
o Soil organisms
o Gram Positive (Thick peptidoglycan = retains purple dye) o Ex: Bacillus & Clostridium
• Helical Bacteria
o Spirochetes: Flexible twisting bacteria, rod-shaped, have flagella at both ends that tightly spiral around cell
▪ Move by creeping motion
▪ Has 70 turns (wound less tight)
o Spirilla: rigid twisting rod shaped structure
▪ Flagella helps move with swimming motion (like
corkscrew)
▪ Have 20 turns (wound tightly)
Size of microorganism: measured in micrometers
• Largest bacteria 0.75mm, could see w/naked eye
• Smallest bacteria 140 nm
Prokaryotic Structures: cell membrane, cytoplasm, ribosomes, one or a few chromosomes
• External Structures: Appendages and Glycocalyx
o Appendages (2 groups)
▪ Motility
???? Flagella: long structures, extend beyond the surface of the cell and glycocalyx, used to propel through the environment,
• 3 parts
o Filament: long, thin, hollow, helical
structure made of protein called
Flagellin
o Hook: curved structure that connects
the filament to the basal body, made
of different proteins
o Basal Body: Stack of 2 and 4 rings of
protein firmly anchored into cell wall,
varies between gram (-) and gram
(+) cells, it doesn’t extend into the
cytoplasm
• Rotates 360 degrees to move prokaryotic cells like a boat propeller
• Number and arrangement of flagella varies o Monotrichous: one flagella at one end
of the cell
o Lophotrichous: group of flagella at
one end of the cell
o Amphitrichous: one flagella at both
ends of the cell
o Peritrichous: Flagella all around the
perimeter of the cell
• Flagellar Responses: Guide bacteria in
certain direction in response to outside
stimulus
o Chemotaxis: guided by chemical
stimuli (either positive or negative)
o Phototaxis: guided by light stimuli
o Signal sets flagella in rotary motion in
either clockwise or counterclockwise
direction, moving towards nutrients
▪ Counterclockwise: smooth,
linear direction = Run, if
bacteria in high concentration
will run more
▪ Clockwise: Tumbles, if bacteria
in low concentration will tumble
more
▪ Attachments or Channels
???? Fimbriae: fine proteinaceous, hair-like bristles on
cell surface
• Function: Adhesion to other cells and
surfaces
???? Pili: Rigid tubular structure
• Function: join bacterial cell for partial DNA
transfer = Conjugation (an exchange of
genetic material in mating types)
• 1-3 present per cell
• Found only in Gram Negative (-) cells (thin
peptidoglycan layer and outer membrane,
doesn’t hold purple stain = pink cell)
o Glycocalyx: surface coating of cell wall
▪ Capsule: tight grouping
???? Chemicals in these are similar to the host so the
white blood cells don’t recognize them and cant
perform phagocytosis on them
▪ Slime Layer: loose grouping
▪ Functions of both are similar (both made of glycoprotein and polysaccharide)
???? Keep cell from drying out (desiccation)
???? Help bacteria stick together to form Biofilms
(aggregates of cells stuck together in a film)
???? Prevent WBC from phagocytosis
• The Cell Envelope: External covering outside of the cytoplasm, 2 layers, helps maintain cell integrity
o Cell Wall: provides structure, protects from osmotic forces, helps attach to other cells, resists antimicrobial drugs
o Cell Membrane: Selectively permeable, nutrients flow in and wastes flow out
o 2 Groups of Cell Envelopes
▪ Gram Positive (+) Bacteria: Thick cell wall made of peptidoglycan around the cell membrane.
???? Contains Teichoic Acid: in part of peptidoglycan layer, does not extend all the way through
???? Contains Lipoteichoic Acid: extend through the whole peptidoglycan layer & attached to cell
membrane
???? 1 Periplasmic Space
???? Peptidoglycan layer 20-80nm thick
???? Ex: Staphylococcus aureus
▪ Gram Negative (-) Bacteria: Outer cell membrane, thin peptidoglycan layer and then the cell membrane inside ???? Has Porins on the outside
???? 2 Periplasmic spaces
???? Peptidoglycan layer 8-10nm thick
???? Ex: Escherichia coli (E. coli)
o Peptidoglycan Structure:
▪ Polymer of NAM/NAG chains
???? NAM: N-acetylmuramic acid
???? NAG: N-acetylglucosamine
▪ Cross links between adjacent NAMs give strength, these differ between Gram(+) and Gram(-)
???? Gram(+) Cross Linkage
• There is an Interbridge of repeating Glycine
units between the Lysine of one NAM & the
Alanine of the NAM next to it
???? Gram(-) Cross Linkage
• There is a direct bond between the DAP of
one NAM & the Alanine of the NAM next to it
▪ Penicillin affects the direct bonds between adjacent chains, but the outer membrane of Gram(-) make it hard for penicillin to penetrate
▪ Lysozyme also breaks down NAM-NAG chains
o Non-typical Cell Walls
▪ Bacteria that don’t have typical cell wall structure
(Mycobacterium & Nocardia)
???? Gram(+) cell wall with Mycolic Acid give cell wall
waxy nature because it’s a lipid = this wall called
an Acid-Fast Cell Wall
• Gives it pathogenicity & high resistance to
chemicals & dyes
• Must use Acid-Fast Stain to diagnose
infections caused by these microorg.
▪ Bacteria w/No Cell Wall (Mycoplasma)
???? Cell membrane stabilized w/sterols (ex:
cholesterol)
???? Pleomorphic (less structured, more flexible)
• Internal Structures of Bacteria (Reminder: Prokaryotic) o Cytoplasm: gelatinous solution of sugars, amino acids & salts, 70-80% water (solvent used in all cell functions)
o Chromosomes: Single, circular, double-stranded DNA, has all the genetic material, in prokaryotes. not packaged around histones but packaged into Nucleoid
o Plasmids: small circular, double-stranded DNA, not essential for bacteria to growth or metabolism
▪ There are variations in Segregation of these, some linear some circle
▪ Sometimes harbor in antimicrobial resistance genes o Ribosomes: Made of 2 subunits of protein & rRNA
▪ 60% made of rRNA (the biggest portion and most
important component, makes peptide bonds, can be
enzymatic making enzymes included the one that
translates)
▪ Function: synthesis of proteins
▪ Different than Eukaryotic Ribosomes because smaller & sediment faster when centrifuged
▪ Two Types:
???? 30S + 50S = 70S Bacterial Ribosomes, found
inside organelles
???? 40S + 60S = 80S Eukaryotic Ribosomes, found outside organelles
• Prokaryotes have BOTH types of
ribosomes
o Inclusions & Granules
▪ Intracellular storage bodies
▪ Size, number & their content varies
▪ When environmental sources are depleted the bacterial cell can use these
▪ Ex: glycogen, poly-β-hydroxybutyrate, gas vesicles for floating, sulfur & phosphate granules (metachromatic granules), particles of iron oxide
o Cytoskeleton: Internal network of proteins associated w/cell wall
o Endospores: formed by microbes, toughest & longest living form of life
▪ High resistance due to high levels of Calcium & Dipicolinic Acid
▪ Longevity = ~250 million yrs.
▪ Resistant to ordinary cleaning & boiling
▪ Destroyed by pressurized steam @ 120°C for 20-30 min.
▪ When dehydrated = metabolically inactive – is how they live for so long
▪ Sporulation: Formation of an Endospore, depends on the species vegetative cell can form endospore
centrally, subterminally, or terminally
???? Step 1) In a Vegetative Cell (single cell develops into an endospore when one or more nutrients are limited, this endospore reactivates to transform
into a vegetative cell) DNA is replicated
???? Step 2) DNA aligns along cell’s long axis
???? Step 3) The cytoplasmic membrane invaginates to form the Forespore
???? Step 4) the cytoplasmic membrane grows and
engulfs the forespore in a 2nd membrane,
vegetative cell’s DNA disintegrates
???? Step 5) A Cortex of Calcium and dipicolinic acid is
deposited between the two membranes
• the cell holding the Endospore at this point
is called the Sporangium
???? Step 6) Spore coat forms around the endospore
???? Step 7) Endospore matures, spore coat
completed, resistance increases
???? Step 8) Endospore released, divides again under
right conditions
Eukaryotic Structures (Major difference from Prokaryotic is contains a membrane-bound nucleus & has membrane-bound organelles, can also be unicellular or multicellular)
• Organisms Studied
o Protozoa: Always Unicellular
o Fungi & Algae: Unicellular or Multicellular
o Helminths: animals w/unicellular egg or larval forms, animals themselves are multicellular
• External Structures
o Glycocalyx: outermost layer of cell, comes in direct contact w/environment
▪ Made of polysaccharides, is a network of fibers
▪ Can be Slime Layer or Capsule
▪ Eukaryotes not as structurally organized as prokaryotes ▪ Function: adhere cells to one another, strengthen cell
surface, protect against dehydration, cell to cell
recognition & communication
o Under Glycocalyx, some Eukaryotes have Cell Wall some don’t ▪ Fungi & Algae – thick rigid cell wall
???? Cell Wall: Provide support & shape
???? Fungi: thick layer of polysaccharide fibers made of
chitin or cellulose + thin layer of mixed glycans
???? Algae: Variation of chemical composition, some
common ones: cellulose, pectin, mannans, silicon
dioxide, calcium carbonate
???? Some cells that have cell walls can also have
things like Sterols in the cell membrane
▪ Protozoa, some algae, all animal cells – no cell wall, only Cell Membrane
o Cytoplasmic/Cell Membrane:
▪ Bilayer of phospholipids & proteins
▪ Function: selectively permeable barrier for transporting material into & out of the cell, proteins are like
recognition molecules, receptors, carriers, enzymes, or channels.
???? Endocytosis: Active Transport, membrane
distends to form pseudopods around a substance
and bring it into the cell
• Phagocytosis: Bringing in a solid substance
• Pinocytosis: Bringing in a liquid substance
• Stuff brought in contained in a Food Vesicle
• Amoeboid Action: some use this for
locomotion by extending pseudopod &
streaming into it
???? Exocytosis: how things exported from the cell,
vesicles w/stuff inside fuse to cytoplasmic
membrane & dump it out
▪ Sterols help with stability
▪ Eukaryotic Cells contain membrane-bound organelles, these are 60-80% of cell volume
• In Cytoplasm/Internal Structures
o In TEM microscopic image, the varying darkness in the cytoplasm is varying density
▪ Euchromatin = light, less dense chromatin fibers
▪ Heterochromatin = dark, more dense chromatin fibers ▪ The more tightly packed DNA (more dense) is being silenced
???? DNA is packaged around Histones ???? beads on a string (Nucleosomes) ???? chromatin fiber ???? form
loops ???? compress to form Chromatid
o Rough Endoplasmic Reticulum:
▪ Ribosomes adhere to it
▪ assemble proteins into their secondary, tertiary, and quaternary structure
▪ transport proteins throughout the cell
▪ modifies protein w/glycoprotein (sugar wrapped around the protein)
o Smooth Endoplasmic Reticulum:
▪ Make Lipids
▪ Release Calcium ions
▪ Detoxify organic chemicals
▪ Produce steroid hormones
o Golgi Complex:
▪ Structure: flattened hollow sacs in a phospholipid bilayer
???? Cisternae: Flattened membrane disk, Golgi
contain 3-20 most contain 6
???? Transitional Vesicles: a sac with molecules that comes from the ER
???? Condensing Vesicles (Secretory Vesicles):
packages secretions into these sacs that fuse to
the cytoplasmic membrane & dump contents
outside cell w/exocytosis
▪ Function: modifies proteins to be sent other places, receives & packages large molecules to export from the cell
o Lysosomes: vesicles with enzymes that come from the Golgi ▪ Involved w/intracellular digestion & protects against microbes
o Vacuoles: membrane bound sacs with particles to be digested, excreted, stored
o Phagosome: A food vesicle (vacuole) that is taken into the cell by endocytosis, when it fuses with a lysosome from the Golgi called a phagolysosome
o Mitochondrion:
▪ Shape: Spherical to elongated, has 2 membranes ▪ Structure:
???? Cristae: Inner bilayer that has many folds to
increase the surface area, where most of ATP
produced in eukaryotic cells produced
???? Matrix: contains (prokaryotic) 70S ribosomes & circular DNA (has genes for some RNA & few
mitochondrial polypeptides made by mitochondrial ribosomes)
▪ Function: Makes ATP, part of the Krebs Cycle (Citric acid cycle), works w/electron transport
o Chloroplast: light harvesting organelle, found in photosynthetic eukaryotes
▪ Structure: two phospholipid bilayer membranes & DNA, make a few polypeptides w/own 70S ribosomes.
???? Thylakoids: membranous sacs that provide a lot of surface area for chloroplasts to
photosynthesize. Chlorophyll found here.
???? Grana: Stacks of Thylakoids
???? Stroma: Space enclosed by the inner membrane, contains mix of metabolic products, enzymes &
ions
o Cytoskeleton
▪ Function: Anchor organelles, help cytoplasmic streaming, move organelles within the cytosol, move cytoplasmic membrane for endocytosis & amoeboid action
▪ Structures:
???? Microtubules made of tubulin, are spindle fibers and are also part of the Eukaryotic Flagella, also
transport things
???? Microfilaments made of actin, used for amoeboid
movement, cytokinesis, cell movement
???? Intermediate filaments made of various proteins
Kingdom Fungi:
• Majority unicellular/colonial, few have cellular specialization • Been around for 650 million yrs.
• Species number difficult because mating forms look different than non-mating forms, could look different & be the same thing • 100,000 species divided into 2 groups
o Macroscopic Fungi (Mushrooms, puffballs, gill fungi)
o Microscopic Fungi (molds, yeast)
▪ Two Morphologies = Dimorphic (Transitions between
two forms to become pathogenic)
???? Yeast: round ovoid shape, reproduce asexually,
the form that is pathogenic
???? Hyphae: long filamentous fungi or molds, the
form that is benign
• When in individuals called “hyphae,” when
make a mat of them called, “Mycelium”
(filamentous fungal growth)
• Fungal Organization
o Grow in loose colonies
o Yeast: soft, uniform in texture & appearance
o Filamentous Fungi: mass of hyphae = Mycelium (cottony, hairy, velvety texture)
▪ Septate: when hyphae is divided with cross walls, each separate part has its own nucleus
???? Aseptate Hyphae: no cross walls dividing
???? Septate Hyphae: cross walls dividing
▪ Vegetative Hyphae: digests & absorbs nutrients
▪ Reproductive Hyphae: makes spores for reproduction
Unit 2 4/18/17 11:50 AM
Microbial Nutrition
Nutrition: the process of acquiring chemical substances or nutrients from the environment and using them in cellular activities
• Essential Nutrients
o Macronutrients: elements that make up the majority of the cell, CHONPS
▪ Carbon
▪ Hydrogen
▪ Oxygen
▪ Phosphorus
▪ Sulfur
o Micronutrients: elements that are required in small amounts and involved in cell function. Ex: Mg used in chlorophyll.
• Nutrients can be:
o Organic: carbon, carbohydrates, proteins
o Inorganic: CO2, water, gases like oxygen
• Microbial cytoplasm made up of
o 70% water
o Proteins
o 96% of cell made up of 6 elements (CHONPS) – this is in a Dry Weight Perspective
• Chemical Composition of E. Coli
o Dry Weight
▪ Proteins: 50%
▪ RNA: 20%
▪ DNA: 3%
▪ Carbohydrates: 10%
▪ Lipids: 10%
o Wet Weight
▪ Proteins: 15%
▪ RNA: 6%
▪ DNA: 1%
▪ Carbohydrates: 3%
▪ Lipids: 3%
• Sources of Essential Nutrients
o Carbon Sources
▪ Heterotroph: must obtain Carbon from organic sources (proteins, carbohydrates, lipids, nucleic acids)
???? Chemoheterotrophs: Microorganisms that use organic compounds for both energy and carbon
(Chemotroph get energy from chemical sources)
▪ Autotroph: organism that gets carbon through inorganic sources, make organic compounds from CO2 –
(photosynthetic organisms = photoautotroph)
???? Chemoautotroph: microorganism that uses CO2 as a source and catabolizes organic molecule for
energy
o Nitrogen Sources
▪ Reservoirs
▪ It is a component of air and chitin
▪ Some organisms can use it as Nitrate and other can make nitrogen through the nitrogen cycle
o Oxygen Sources
▪ It is a component of the air, carbohydrates, lipids, nucleic acids, proteins, salts
▪ It can be directly used
???? Anaerobes don’t get oxygen in the same form (gas) but still use it.
o Hydrogen Sources
▪ Can get it from water, gases and salts
▪ Roles of Hydrogen are to maintain pH and is an acceptor of oxygen during cellular respiration
o Phosphorus/Phosphate Sources
▪ Component of nucleic acids (essential to genetics) ▪ Used in energy transfers (ATP)
o Sulfur Sources
▪ Found in amino acids and vitamins
▪ It is widely distributed in the environment
▪ Used in protein stability
o *Other Nutrients that are Important in Microbial Metabolism ▪ Potassium
▪ Sodium
▪ Calcium
▪ Magnesium
▪ Iron
▪ Zinc, copper, nickel, manganese, etc.
• Classification of Nutritional Types
o Carbon Source
▪ Autotroph: organisms that use inorganic sources of carbon as their only source of carbon and make organic compounds from CO2
▪ Heterotroph: catabolize organic molecules they get from other organisms.
o Energy Source
▪ Chemotroph: organisms that get energy from inorganic and organic chemicals through redox reactions
???? Chemoautotroph: microorganism that use CO2 as
their carbon source but chemical compounds as
their energy source
???? Chemoheterotroph: Microorganisms that use
organic compounds for the carbon source and
chemical compounds as their energy source
▪ Phototroph: organisms that use light as a source of energy
???? Photoautotroph: use CO2 as source of carbon and
light as source of energy
???? Photoheterotroph: use organic compounds as
source of carbon and light as source of energy
• Transport Mechanisms
o Passive Processes – no energy used
▪ Simple Diffusion: net movement of chemical down its concentration gradient, requires no spending of energy ▪ Facilitated Diffusion/Transport: proteins act as channels or carriers to transport down the concentration
gradient, into or out of the cell, no energy spent,
???? Non-specific channel proteins don’t have a
preference for what they transport
???? Permeases: channels that carry only specific
chemicals
▪ Osmosis: diffusion of water across semipermeable
membrane, down concentration gradient
o Active Processes – uses energy, can move against or with the concentration gradient
▪ Active Transport: use transmembrane permease
proteins to move molecules across the membrane, has
to use energy to do so, use gated channels or ports
???? Uniport: one thing moving in one direction
???? Antiport: move two things at the same time but in
opposite directions
???? Symport: move two things at the same time in
the same direction
▪ Group Translocation: The substance transported across the membrane is also chemically changed during the
transport
???? Ex: when take glucose into the cell and it is
phosphorylated at the same time
• Solution Types
o Isotonic: when solutions on both sides of the semipermeable membrane have the same concentration of solutes
o Hypertonic: concentration of solutions unequal, the solution with higher concentration of solutes is called this
o Hypotonic: when concentration of solutions unequal, the solution with the lower concentration of solutes is called this Microbial Growth
*Prokaryotes do not perform phagocytosis, only Eukaryotes do, Prok. send enzymes out to break down things and then import them via active transport and facilitated diffusion
• Population Growth
o Generation Time: the time required for a cell to double
▪ Cells divide by binary fission (form of exponential
growth, to calculate the number of cells that one cell
can produce = 2^n, n being the number of generations,
to calculate the total number of cells = multiply number of original cells by 2^n )
???? Exponential Growth: regular spaced division, very rapid increase in growth
▪ Bacteria Cells synthesize a new cell wall while weakening the outside cell wall to form two new cells from the original.
o Growth Curves: graph that plots # of organisms in a growing population over time
▪ Lag Phase: time period where the cells are adapting to the new environment, do not reproduce right away, synthesize enzymes to use nutrients in the medium
▪ Log Phase: phase when cell population increases logarithmically
▪ Stationary Phase: nutrients become depleted and the number of dying cells equal the number of cells being produced, population remains constant
▪ Death Phase: the number of cell deaths rises about the number of cells produced
o Diauxic Shift: Discovered by Jacques Monod 1941. different sugars produce different growth curves, the growth curves are typically based off of how the bacteria would be grown in a laboratory setting, with a single carbon source. The true growth curves would be different than these standard ones because the organism would have many different carbon sources in real life.
o Methods for Analyzing Growth:
▪ Hemocytometer: sample under a glass slide with an etched grid on the bottom, the grid is 25 squares and designed to suspend the microbes in 0.02mm of liquid, volume of suspension of microbes is 1mm x 1mm x 0.02mm = 0.02mm^3
???? Not good for motile things
▪ Coulter Counter: as cells pass through triggers an electronic sensor that tallies their numbers
???? Useful to count larger cells (yeast, unicellular
algae, protozoa), and for bacterial counts because
of debris in the media.
▪ Flow Cytometer: uses a light sensitive detector, detects changes in light transmission through the tube as cells pass, used to tell between differentially stained cells
that have been stained with fluorescent dyes or tagged w/fluorescent antibodies
???? Detect and sort different populations of cells from
blood
???? Counts bacteria and host cells
???? Used to look at fungal cells to determine if they
are haploid or diploid
▪ Pour Plate Technique: step-wise dilution of a culture, dilution factor the same every time, scientist plates set amount of dilution onto agar plate and counts the
number of colonies resulting on a plate.
▪ Turbidity: cloudiness of the solution, more bacteria the cloudier the solution
???? Spectophotometer measure the amount of light
transmitted through a culture under standard
conditions. Higher concentration of bacteria in a
broth more light will be absorbed and scattered,
less passing through and hitting back sensor.
???? Technique only useful density above 1 million per
mL.
• Environmental Factors Influencing Growth
o Environment – enzymes adapt to whatever condition it grows at
▪ Temperature
???? Psychrophile: microorganism requiring cold
temperatures below 20°C
???? Mesophile: microorganism that requires
temperatures ranging from 20°C to 40°C
• Optimum Growth (can grow at other
temperatures but grows best at optimum
temp) = 37°C
???? Thermophile: grows at temperatures above 45°C • In compost piles and hot springs
▪ Oxygen Requirements
???? Aerobic: organism that uses oxygen as a final electron acceptor in ETC
• Obligate Aerobe: they are obliged to use
oxygen, they can’t survive below depth that oxygen penetrates
???? Microaerophilic: Microorganism that requires low levels of oxygen
???? Facultative Anaerobe: Can live with/without oxygen, prefers oxygen
???? Aerotolerant Anaerobe: microorganisms that prefer anaerobic conditions but can tolerate
exposure to low levels of oxygen.
• Expect relatively even growth until it
reaches its point of oxygen tolerance.
???? Anaerobe (Strict): An organism that cannot tolerate oxygen.
▪ pH
???? Acidophile: organisms that grow best in acidic conditions
???? Neutrophile: grow best in a narrow range around a neutral pH (between 6.5 and 7.5, which is also the pH range of tissues and organs in the human body)
???? Alkalophile: organisms that live in alkaline soils and water up to 11.5pH
▪ Osmolarity
???? Halophile: need high salt concentration
???? Osmotolerant: organism can tolerate salt, doesn’t need it to survive
• Ex: Staphylococcus epidermidis
• Ecological Associations
o Symbiotic: organism lives in close nutritional relationship, one or both of the organisms involved require the relationship. ▪ Mutualism: both organisms involved require the
relationship and both benefit from it.
???? Ex: bacteria in cow’s rumen & interaction between
pea plants and Rhizobium & E.coli
▪ Commensalism: one organism benefits from the
relationship, the other is not harmed or benefitted.
???? Ex: Satellitism between Staphylococcus aureus
and Hemophilus influenza
▪ Parasitism: the relationship is not required by both
organisms, the one that requires it is doing damage to
the other
???? The host is always harmed – levels of damage
may differ
???? Ex: any pathogen that can get you sick
o Non-Symbiotic: organisms are free-living, the relationships are not required for survival
▪ Synergism: members benefit from each other but are
still able to survive on their own
???? Works in either biosynthesis of a certain end
product or the breakdown of waste
• Provide extra things to get to the end
product
▪ Antagonism: relationships don’t help survival, compete for nutrition and space
???? Some members are inhibited or destroyed by
others
???? Organisms that produce antibiotics, one microbe
makes antibiotic and the other is hurt by it
• Led to penicillin
Metabolism of Microbes
• Metabolism: chemical and physical workings of a cell
o Anabolism: biosynthesis, forms larger molecules, requires energy input
o Catabolism: degradative, breaks bonds to form smaller molecules, releases energy
o *The intermediates can be used for other reactions at multiple stages in the reaction
• Enzymes (biological catalysts, lower energy of activation to increase the rate of chemical reaction) – in biological reactions o Energy of Activation: the resistance to a reaction, amount of energy needed to trigger a chemical rxn
o Enzymes are Not permanently altered in the rxn
o They promotes rxn by having a physical site for substrates to bind to
o Most are made of protein and may have cofactors
▪ Cofactors could be metallic cofactor like iron in
Hemoglobin or non-metallic cofactors
o They are organic catalysts
▪ Catalyst: chemicals that increase the likelihood of a rxn but are not permanently changed in the process.
o Have unique characteristics (shape, function, specificity) that doesn’t allow them to react with just any type of substrate o Have an active site – where the substrate binds and the reaction happens
o The enzyme itself is not part of the reaction substrate or products
o It is not used up in the reactions or recycled
o The rxns are often controlled mechanisms
o Lock and Key Model: shape of the substrate is complementary to the shape of the active site, describes the enzyme
substrate specificity.
o Induced-fit Model: lock and key analogy not entirely correct because the enzyme’s active site changes shape when the substrate binds, describes enhanced enzyme-substrate specificity
▪ Shape of the substrate is kind of specific to the active site but not exact
o Structure
▪ Apoenzyme: the protein portion of enzyme, enzyme without its cofactor, is inactive unless bound to one or more cofactors.
▪ Holoenzyme (Conjugated): binding of an apoenzyme and its cofactor that makes an active enzyme.
???? Cofactors: inorganic ions or organic molecules that are essential for the enzyme action.
• Coenzymes are an example of organic
cofactors, organic cofactors can also be
vitamin based
o three important ones are the electron
carriers (NAD+, NADP+, FAD)
• Metallic Cofactors
▪ Exoenzymes: enzyme that functions outside the cell that produced it
???? Can secrete things and damage things
???? Eukaryotes and Prokaryotes can secrete things so there are proteins that end up outside the cell for specific reasons
▪ Endoenzymes: enzyme that functions inside the cell that produced it
???? Most enzymes are these, intracellular
▪ Constitutive Enzymes: enzymes that are always present, they are always produced regardless of
physiological demand because their function in cell processes is indispensible.
▪ Induced Enzymes: molecules induce or enhance the production of these enzymes
▪ Synthesis or Condensation Reactions: anabolic rxns, form covalent bonds between smaller substrate
molecules, require ATP, release one molecule of water for each bond formed
▪ Hydrolysis Reactions: Catabolic reactions, breakdown substrates into smaller molecules, need water to break bonds
o Environmental Sensitivities – activity influenced by environment
▪ Labile: describes some enzymes when they are chemically unstable
▪ Increased temperature = Denatured Protein: protein heated up = bonds are broken. If cooled it won’t go back to normal because it didn’t start out complete, they fold as they are made
???? Functional Protein: needs structure to get
catalytic site in place
▪ pH: denatures protein when ions released from acids and bases interfere w/h-bonding and disrupt secondary and tertiary structure
▪ Enzyme/Substrate Concentration: As concentration of substrate increases the enzyme activity increases until all active sites are bound = saturation point, more substrate will not increase rate of activity
o Regulation of Enzymes
▪ Competitive Inhibition: inhibitory molecules, similar in shape to the substrate molecules, compete for & block the active sites. This can be permanent or reversible, if it is reversible it can be overcome by increasing the concentration of substrate = increasing the likelihood that substrate will bind before inhibitor.
▪ Noncompetitive Inhibition: Allosteric, regulatory inhibition
???? Allosteric Inhibition: The regulatory site is
separate from the active site, when the inhibitor binds it distorts the active site and doesn’t allow the substrate to bind
???? Allosteric Activation: the activator reverses this ▪ Feedback Control: when end product of a series of reactions is an allosteric inhibitor of an enzyme in an
earlier part of the pathway, prevents synthesis of more of this end product.
???? There is enough product already made, signals that they don’t need anymore
▪ Control of Gene Synthesis – this is a slower response ???? Enzyme Repression: controls the synthesis of key enzymes and inhibits at the genetic level
???? Enzyme Induction: enzymes are made only when the right substrates are present
o Pursuit and Utilization of Energy
▪ Energy: the capacity of a system to do work
???? Forms of energy: Thermal, radiant, electrical,
mechanical, atomic, chemical
▪ Exergonic: get energy out
???? X + Y ????(Enzyme)???? Z + Energy
▪ Endergonic: not going to happen unless you put energy in
???? Energy + A + B ????(Enzyme)???? C
▪ ATP is the energy currency in the Cell
???? 3 part molecule
• Adenine – nitrogenous base
• Ribose – 5-carbon sugar
• 3 phosphate groups attached
???? using and replenishing of ATP is constantly
happening in the cell
???? Remove terminal phosphate = energy released ???? Formation of ATP through three different
mechanisms:
• Substrate Level Phosphorylation: transfer
phosphate from phosphorylated compound
(substrate) directly to ADP
• Oxidative Phosphorylation: series of redox
reactions occur during respiratory pathway
• Photophosphorylation: ATP is made by using
the energy of sunlight
o Biological Oxidation and Reduction
▪ Redox Reactions: reduction/oxidation reactions, always in pairs (electron donor and acceptor involved), the
process saves electrons and their energy, the energy
that is released can be used to phosphorylate ADP or
another compound
???? Oxidation: rxn where electrons are donated (often in the form of Hydrogen)
???? Reduction: rxn where electrons are gained
???? (OIL RIG = oxidation is loss & reduction is gain)
???? Reduction Potential: measure of tendency of a
chemical species to acquire electrons therefore
being reduced (measured in Volts)
▪ Electron and Proton Carriers: repeatedly accept and release electrons and hydrogen to help transfer redox energy
???? These are Coenzymes (NAD+, NADP, FAD,
coenzyme A, and compounds of respiratory chain)
• Aerobic Respiration: series of enzyme-catalyzed reactions, electrons are passed from fuel molecules to the last electron acceptor, oxygen.
o Glycolysis: glucose is split and produces two molecules of pyruvic acid, while at the same time producing small amounts of ATP, NADH is also generated
▪ Step 1: Glucose is phosphorylated by ATP into glucose 6-phosphate (G6P)
▪ Step 2: G6P is rearranged and another phosphate is added to make Fructose-1,6-bisphosphate (F1,6P)
▪ Step 3: F-1,6-P is divided into DHAP (which is
converted to G3P) and G3P
▪ Step 4: An inorganic phosphate is added to the two G3P molecules to make two molecules of 1,3BPG and a
NAD+ is reduced to NADH (two times)
▪ Step 5: (two molecules so this step happens two times) ADP steals a phosphate from the 1,3BPG and that
becomes 3PG
▪ Step 6: 3PG is rearranged to become 2PG
▪ Step 7: Water molecule released when 2PG is converted to Phospho-enolpyruvic acid (happens 2 times for the two molecules)
▪ Step 8: ADP steal the phosphate off the P-enolpyruvic acid and make pyruvate, and ATP (rxn happens 2x for 2 molecules)
▪ NET TOTAL: 2 ATP and 2 NADH
▪ ALTERNATE PATHWAYS (Instead of Glycolysis)
???? Pentose-Phosphate Pathway
• The enzymatic formation of phosphorylated
pentose sugars from glucose-6-phosphate
• alternate way to breakdown glucose
• yields NADPH instead of NADH
• has different intermediates such as Ribose
5-phosphate which feeds into the synthesis
of nucleotides
• Yields less ATP than glycolysis
???? Entner-Dourdoroff Pathway
• Produces NADPH instead of NADH
• Yields half ATP than glycolysis (1 ATP)
• Pseudomonads use this pathway to
catabolize glucose
o Synthesis of Acetyl-CoA: Decarboxylation, enzymes remove a carbon from the pyruvic acid as CO2. Enzyme then joins the remaining two-carbon molecule (acetate) to coenzyme A to make acetyl-CoA and also produced a molecule of NADH (From NAD+).
▪ Because glycolysis made 2 molecules of pyruvic acid this is done 2 times
o TCA/Citric Acid Cycle/Krebs Cycle: processes pyruvic acid and generates 3 CO2 molecules , NADH and FADH2 are generated ▪ Occurs in the cytosol of prokaryotes and matrix of mitochondria. in eukaryotes. Cells also use for
catabolism of lipids and proteins
▪ Step 1: oxaloacetate combines with acetyl-CoA
▪ Step 2: citrate is converted to isocitrate so the right isomer is in place
▪ Step 3: isocitrate is converted to α-ketoglutarate (NAD+ is converted to NADH and a CO2 molecule is released)
???? *Isocitrate can also take the Glyoxylate
???? Bypass which produces 2 carbon compounds, without this there would not be enough carbon
left to survive at the end of the cycle
▪ Step 4: α-ketoglutarate interacts with CoA to form succinyl-CoA (NAD+ and CoA are used to form Succinyl CoA and NADH and CO2 are released)
▪ Step 5: succinyl-CoA releases CoA to form succinate (two molecules of acetyl-CoA pass through the Krebs cycle from every one glucose molecules creating two pyruvates, from these two molecules two molecules of ATP are produced by substrate level
phosphorylation, GTP is an intermediary at this step) ▪ Step 6: Succinate is converted to fumarate (FAD is reduced to FADH2)
▪ Step 7: fumarate gains water to generate malic acid ▪ Step 8: malic acid is converted to oxaloactetate (NAD +is converted to NADH)
▪ SUMMARY: overall the two molecules of acetyl-CoA that are gained from the original glucose molecule, end up forming: 6 NADH, 2 FADH2, 2 ATP
???? Glycolysis and TCA created ATP, Carbon
intermediates to make other molecules, reduced
electron carriers (NADH, FADH)
o Electron Transport Chain: Final processing of electrons and hydrogen and the major generator of ATP
▪ occurs in the cytoplasmic membranes of prokaryotes and the inner mitochondrial membranes of eukaryotes ▪ Accepts electrons from NADH and FADH2, this generates energy through redox reactions and this
energy is captured by ATP synthase to produce ATP = oxidative phosphorylation
???? Oxidative Phosphorylation: protons flow down their electrochemical gradient through protein channels called ATP synthases and these channels phosphorylate molecules of ADP to ATP.
• Specifically, Hydrogen ions diffuse back
through the ATP synthase complex causing
it to rotate, causing a 3-dimensional change
resulting in the production of ATP
o Form of Chemiosmosis:
▪ as the electron transport
carriers shuttle electrons, they
actively pump hydrogen ions
(protons) across the membrane
setting up a gradient of
hydrogen ions – Proton Motive
Force
▪ Membrane Set Up – membrane includes
???? flavoproteins: integral proteins, FMN is an example of these and is the first carrier molecule of the transport chain that NADH passes electrons to.
???? Ubiquinones are lipid-soluble nonprotein carriers found in the membrane, Coenzyme Q is an
example here.
???? Metal containing proteins are a mix of integral proteins. Cytochromes are integral proteins
associated with hemes
▪ Bacterial Process – involves 7 enzymes that rapidly undergo redox reactions
???? 1. Complex I – NADH dehydrogenase/FMN ???? 2. Complex II
???? 3. Coenzyme Q
???? 4. Complex III
???? 5. Cytochrome C
???? 6. Complex IV – cytochrome a/a3 and ATP
Synthase
???? The final step: Oxygen accepts 2 electrons from
ETS and forms water with 2 hydrogen ions from
the solution. Oxygen is the final electron acceptor
???? OUTPUT:
• From the 2 NADH from Glycolysis through
the ETS = 6 ATPs
• From the 6 NADH from the TCA through the
ETS = 18 ATPs
• From the 2 FADH2 from the TCA through
the ETS = 4 ATPs
• TOTAL from ETS: 28 ATP
o Complete aerobic oxidation of one molecule of glucose by a prokaryote is 38 molecules of ATP. Eukaryotes have to use 2 ATP to get NADH from the cytoplasm to the
mitochondria so their net ATP is 36.
▪ 10 molecules of NADH from glycolysis, synthesis of acetyl-coA, and TCA = 30 ATP
▪ 2 molecules of FADH2 from TCA = 4 ATP
▪ 2 ATP netted from glycolysis
▪ 2 ATP from TCA
• Anaerobic Respiration: use inorganic chemicals (oxygen containing ions) rather than oxygen as the final electron acceptor in the ETC o Examples of what is used as the final electron acceptor = Nitrate (NO3-), Nitrite (NO2-), Sulfate, Methanogens may use CO2 and end up with methane, some use methyl
o Most obligate anaerobes use H+ made during glycolysis and Krebs cycle to reduce the compound
• Fermentation: partial oxidation of sugars (glucose or other carbohydrates when there is no oxygen present) to release energy o Final electron acceptors in these pathways are organic molecules
o These yield a small amount of ATP
o Examples:
▪ Ethanol Pathway: Decarboxylation of pyruvic acid
produces Acetaldehyde, with oxidation of NADH to
NAD+ ethanol is produced.
???? Yeasts ferment glucose to produce ethanol (then
complete aerobic respiration)
▪ Lactic Acid Pathway: NADH reduces pyruvic acid to create lactic acid
???? Various bacteria acting on pyruvic acid forms acid, gas and other products
▪ Both pathways recycle the electron carrier NAD+ to be used again in Glycolysis to produce more
energy, benefit to fermentation is ATP production without a final electron acceptor or cellular
respiration.
• Amphibolic Pathways: pathways can be catabolic and anabolic o Catabolic pathways can have molecular intermediates that are diverted into anabolic pathways
▪ Pyruvic Acid converted into amino acids through
amination
▪ Amino acids converted into energy sources through deamination
???? Polypeptides can be broken down into their
component amino acids by Proteases, the amino
acids can then be deaminated to create an
intermediate that feeds into the Krebs Cycle
???? Reactions that make and convert amino acids
• Amination: add a free amine to a molecule
to produce an amino acid
o Ex: NH2 + Oxaloacetic Acid ????
Aspartic Acid/Aspartate
• Transamination: switch an amine group
from one molecule to another
o Ex: Glutamic Acid + Oxaloacetic Acid
???? α-ketoglutaric acid + Aspartic
Acid/Aspartate
▪ Glyceraldehyde-3-phosphate converted into precursors for amino acids, carbohydrates, and fats
???? Side note: energy that comes from 6 carbon fatty
acid is about 50 ATP while energy that comes
from 6 carbon sugar is about 38 ATP
Microbial Genetics
• Genome: all of the genetic material in a cell
o Made up of
▪ Chromosomes: A molecule of DNA that is associated
with proteins
???? Prokaryotes: circular and in the nucleoid
???? Eukaryotes: threadlike (chromatin structure,
when packed it is wrapped around histones)
???? Genes: divisions of the chromosome, the unit of
heredity responsible for a given trait, are a
specific sequence of nucleotides that code for RNA
or polypeptide molecules
• Three Categories
o Structural Genes: genes that code for
proteins
o Regulatory Genes: genes that control
gene expression
o RNA Genes: Genes that code for RNA
• Genotype: the actual set of genes in an
organisms genotype
• Phenotype: the physical features &
functional traits of an organism
▪ Plasmids: small circular molecule of DNA, replicates on its own with its own genes, has genes for nonessential
functions like resistance to antibiotics
▪ Mitochondria & Chloroplasts: eukaryotes have
extranuclear DNA in these & plasmids
o Prokaryotic Genome Structure: haploid, chromosomal DNA, circular, all extrachromosomal DNA in the form of plasmids.
o Eukaryotic Genome Structure: nuclear chromosomal DNA, one or more linear chromosomes, plus all extranuclear DNA in mito, chloroplasts, and plasmids
o The genome is a thousand times longer than the cell and must package the DNA efficiently
• DNA: composed of two strands twisted into a double helix, the basic unit is a nucleotide
o Nucleotide has three parts:
▪ 5 carbon sugar - deoxyribose
▪ Phosphate Group
▪ Nitrogenous Base – adenine, guanine, thymine, cytosine o The backbone: the sugar-phosphate linkages
▪ Each sugar attaches to a phosphate at its 5’ carbon and at its 3’ carbon
o The Nitrogenous Bases hydrogen bond with their
complements in the center
▪ Adenine bonds to Thymine with 2 hydrogen bonds, a section of DNA that is AT rich is weaker and easier to
separate
▪ Guanine bonds to Cytosine with 3 hydrogen bonds, a section that is GC rick is stronger and harder to
separate
o As the helix winds it creates a Minor and Major Groove ▪ Minor Groove: information poor
???? Here the proteins can only detect what the base is
▪ Major Groove: information rich
???? Here proteins interact and based on their ability
to form bases can tell the difference between A or
T and G or C, they can also methylate the strand
and use base flips to find a specific sequence
without ever having to separate the strands.
▪ In these grooves there are h-bond donators and h-bond acceptors they are labeled as D and A respectively.
o Watson and Crick – 1953: These are the two scientists given credit for discovering the structure of DNA, however, they
would never have gotten it right if it weren’t for Roslin Franklin’s crystallography data.
o Significance of this structure:
▪ Code must be maintained during reproduction, this constancy of base pairing makes sure the code will be kept (if don’t have constancy then we have mutation) ▪ Order of base pairs provides variety which is
responsible for the unique qualities of organisms
• DNA Replication – takes 30 different enzymes to make exact duplicate
o Begins at Origin of Replication – this is usually an AT rich region because of the weaker hydrogen bonding
▪ Prokaryotes: one origin
▪ Eukaryotes: multiple sites present
o Helicase unwinds & unzips the DNA by breaking hydrogen bonds between complementary bases – creates Replication Forks and Replication Bubble
▪ Theta Structure: when circular DNA is being replicated the fork is opening/replicating in both directions until meeting and forming two new DNA Molecules
o Stabilizing proteins (ssb protein) keep strands from coming back together
o At origin of replication the enzyme RNA Primase makes a short RNA sequence that is complementary to the DNA strand being copied – Primer – provides hydroxyl group for DNA Polymerase III to add nucleotides in the 5’ to 3’ direction (DNA Polymerase III also proofreads – nuclease activity - as it adds bases and can back up to delete incorrect ones and replaces with correct)
▪ Leading Strand: DNA is replicated towards the
replication fork, 5’ to 3’ continuously starting from one primer
▪ Lagging Strand: DNA is replicated away from the
replication fork, synthesized in short 5’ to 3’ segments with many primers (overall the direction is 3’ to 5’)
???? Okazaki Fragments: fragments of replicated DNA,
must be replicated this way on the lagging strand
to maintain 5’ to 3’ direction
o DNA Polymerase I: removes RNA primers and replaces with DNA segments
o DNA Ligase: link the DNA fragments of the lagging strand to complete the synthesis
o The two strands of the original DNA molecule have now separated into two daughter strands each with one original parent strand and one new strand – Semiconservative
• From DNA to Protein – Transcription (DNA into RNA) and Translation (RNA into Polypeptides/Proteins)
o Rules for the Standard Code:
▪ Redundant but not ambiguous: Many codons can code for one amino acid, but each codon only codes for one amino acid.
▪ Code is Universal
o RNA – single stranded molecule made of nucleotides (instead of deoxyribose the 5 carbon sugar is Ribose, and uracil replaces thymine)
▪ mRNA: molecules which carry genetic information from chromosomes to ribosomes as codons (mRNA contains nucleotides and a set of three nucleotides makes up one Codon)
???? In Prokaryotes:
• mRNA carries start codon, codons for other
amino acids in a polypeptide, and one of 3
stop codons
???? In Eukaryotes:
• The mRNA is first called pre-mRNA because
it has to be processed first
• Contains instructions for only one
polypeptide
▪ tRNA: molecules that deliver the right amino acids to the ribosomes based on the sequence of nucleotides in the mRNA.
???? Anticodon: bottom loop of the tRNA that is
complementary to the codon in the mRNA
???? Acceptor Stem: holds the specific amino acid
designated by the anticodon
???? tRNA can recognize more than one codon because of Wobble – a change in the angle of the molecule can cause the third nucleotide to bond with
another besides its normal complement
▪ rRNA: part of ribosomes where protein synthesis occurs o Transcription: Information stored in DNA transcribed into mRNA
▪ Initiation:
???? RNA Polymerase binds to promoter region
???? RNA Polymerase made of 3 subunits: Alpha, Beta, Beta prime
• In Prokaryotes: RNA binds to the promoter
with the help of RNA subunit called Sigma
Factors
o Sigma factors bind to the promoter so
RNA polymerase can recognize it
easier, how tightly the sigma factor
binds varies, this can lead to variation
in amount and kind of polypeptide
formed.
• In Eukaryotes:
o Many Transcription Factors (TFIIH,
TFIID) are used to bind RNA
Polymerase to the promoter
o Have 3 types of RNA Polymerase (kind
to transcribe mRNA, rRNA, tRNA and
other small rRNA)
▪ Elongation:
???? RNA Polymerase adds nucleotides complementary to the template strand of DNA in 5’ to 3’ direction
???? Uracil placed as adenine’s complement
???? Only one DNA strand is transcribed
▪ Termination:
???? RNA polymerase recognizes signals and releases the transcript (100 – 1,200 bases long)
???? In Prokaryotes:
• Self-Termination: RNA polymerase reaches
terminator sequence (rich in GC followed by
rich in AU) , when reaches GC portion
transcription slows and gives time for the
RNA transcript to bind together and form a
hairpin, this causes tension that the AU
portion of the terminator cannot withstand
and the transcript breaks off.
• Rho-dependent Termination: Rho protein
binds to end of RNA transcript and works its
way towards the growing 3’ end, the Rho
protein wedges in between polymerase and
DNA strand and forces them apart,
releasing the transcript, the Rho protein and
the RNA Polymerase.
???? In Eukaryotes:
• After termination and before Translation the
pre-mRNA must be processed into
mRNA(helps export the RNA from the
nucleus, stabilizes the RNA in cytoplasm,
and aids in translation) – Eukaryotic
transcript more stable than prokaryotic
o Capping: adds Guanine nucleotides to
the 5’ end of mRNA
o Polyadenylation: after termination,
proteins add 100-250 adenine
nucleotides to the 3’ end
o Splicing: spliceosome acts as
ribozyme and removes introns,
splicing together exons
o Translation: mRNA is used by ribosomes to translate the genetic code into polypeptides
▪ Initiation: Initiation complex forms (2 ribosomal subunits, mRNA, several protein factors, the initiator tRNA)
???? In Prokaryotes:
• Might happen while cell is still transcribing RNA from DNA
• Steps:
o Smaller ribosomal subunit attaches to
mRNA at ribosome-binding site near
start codon (AUG)
o Anticodon of initiator tRNA aligns
w/start codon on mRNA, bound in
place w/energy from GTP
o Larger subunit attaches to form
complete initiation complex
???? In Eukaryotes:
• Same process but initiation happens when small ribosomal subunit binds to the 5’ cap
not a specific nucleotide sequence
▪ Elongation:
???? tRNAs deliver amino acids to the A site
(accommodates a tRNA delivering an amino acid) as directed by the codons of the mRNA
???? rRNA (enzymatic RNA molecule – ribozyme) in the large subunit makes a peptide bond between amino acid at A site & growing polypeptide in P site (holds tRNA and growing polypeptide)
???? the ribosome moves ahead one codon & the tRNA that was in the P site exits through the E site, leaving the polypeptide attached to the tRNA that was in the A site but is now in the P site, the A site is now open for the next amino acid to be delivered
???? Elongation Factors escort the tRNA along with a GTP molecule
???? When the empty tRNA is released back into the cytosol an enzyme recharges it with another
molecule
???? As elongation goes on and the ribosome passes over the start codon, it is exposed for other
ribosomes to attach and begin translating
identical polypeptides (a group of these ribosomes = polyribosomal complex)
▪ Termination:
???? Termination factors recognize the stop codon
(UAA, UAG, UGA codons that don’t have a tRNA)
and modify the large subunit to activate another
ribozyme that cuts the polypeptide from the final tRNA – ribosome dissociates
???? The polypeptides released can function alone as proteins or w/others in quaternary proteins
???? *Eukaryotic Note: ribosomes attached to
endoplasmic reticulum can synthesize
polypeptides into the cavity of the RER
▪ Protein Folding
▪ Processing
o Regulation of Protein Synthesis
▪ Rules of Genetic Regulation
???? If the cell doesn’t need it, turn it off
???? If the cell needs it, get an enzyme there as quick as possible
???? The rate of synthesis can be controlled w/DNA sequence
???? Enzymes needed only under certain conditions
???? In prokaryotes regulation pathways can be
controlled as operons
▪ Operons: set of genes that are regulated as a single unit
???? made up of promoter, set of genes that code for enzymes and structures, & an operator
???? Inducible Operons: (Enzyme Induction) operon not normally transcribed must be turned on by inducers
• Ex: Lac Operon – normally off
o Made up of 3 segments
▪ Regulator: gene that codes for
repressor
▪ Control Locus: contains
promoter & operator
▪ Structural Locus: 3 genes that
each code for an enzyme that
catabolizes lactose
???? β-galactosidase:
hydrolyzes lactose
???? Permease: brings lactose
across the cell membrane
???? β-galactosidase
transacetylase: uncertain
fxn
o Activated by:
▪ 1. positive regulation by protein
called CAP
???? When glucose is low
increases [cAMP] which
binds to CAP (catabolite
activator protein), CAP
binds to CAP-binding
sites, RNA polymerase
more attracted to the lac
operon promoter =
increased transcription of
lactose catabolizing genes
▪ 2. deactivation of repressor
molecule
???? When lactose is present
and glucose is absent,
allolactase acts as the
inducer, binds to the
repressor, changes its
shape and causes it to fall
off the operator so RNA
polymerase can bind to
the promoter & lactose
catabolism genes can be
transcribed
???? Absence of lactose =
repressor binds the
operator locus and blocks
RNA Polymerase
transcription of lactose
catabolism genes
o Lac Operon inactivation/activation a
form of Catabolite Repression
(inducible operons involved in
controlling catabolic pathways whose
polypeptides are not needed unless a
particular nutrient is available)
▪ Following is repetitive:
▪ Low lactose & High glucose =
low cAMP = few CAP-cAMP
complexes = inactivation of lac
operon
▪ High lactose & low glucose =
high cAMP = many CAP-cAMP
complexes = activation of lac
operon
???? Repressible Operons: operons that are continually transcribed unless repressor deactivates it
Unit 3 4/18/17 11:50 AM
Repressible Operon: Operon that is always active unless repressed • Tryptophan Operon
o No Tryptophan = inactive repressor and transcription of
operon continues
o Tryptophan present = tryptophan binds to the repressor and activates it, causing it to bind to the operon and block
transcription
o Inside the RNA transcript there is a leader region with two
Tryptophan codons that will need to be translated before
structural genes are translated, keeping this in mind…
▪ If Tryptophan is LOW there isn’t many tRNA charged
with Trp so as transcription moves translation/the
ribosome is close behind but when it gets to the leader
region with 2 Trp codons it stalls because there is not a
lot of charged tRNA. The RNA polymerase/transcription
gets ahead and before the ribosome can catch up to
translate it the mRNA #2 segment and the #3 segment
bind together. This loop is far enough away from where
transcription is happening that it doesn’t cause tension
and transcription can continue.
???? Trp Low = 2-3 bind = Transcription continue
▪ If Tryptophan is HIGH transcription is proceeding and
translation/ribosome is close behind, there is enough
tryptophan charged tRNA that the ribosome gets
through the Trp part of the leader region fine and
instead of 2-3 binding segment #3 and #4 bind
together. This creates a termination loop close to where
transcription is occurring that causes tension on the
transcript and halts transcription.
???? Trp High = 3-4 bind = Transcription stops
Mutations: Change in phenotype due to change in genotype (nucleotide base sequence)
• Wild-type: natural, non-mutated characteristic
• Mutant: organism with a mutation, its morphology, nutritional characteristics, genetic control mechanisms, resistance to chemicals and more vary from the norm.
• Causes of Mutations
o Spontaneous Mutations: Random change in the DNA sequence that happens without a known cause
o Induced Mutations: change in the DNA sequence that is caused by exposure to known mutagens, either physical or chemical agents (Chemicals, nitrogen base analogs, radiation) • Categories of Mutations
o Substitution Mutations
▪ Missense Mutation: change in a base pair that results in a codon that codes for a different amino acid in
translation
▪ Nonsense Mutation: change in a base pair that results in a stop codon and early termination of the polypeptide during translation
▪ Silent Mutation: change in a base pair that changes the codon but still codes for the same amino acid due to the redundancy of the genetic code
o Back Mutation: Reversion, when a mutated gene reverses back to its original base composition
o Frameshift Mutation: insertion or deletion of a single base that causes the nucleotides after the mutation to shift (reading frame of mRNA shifts) resulting in change in codons and different polypeptides
▪ It is a given that stop codons will be produced
downstream in this form of mutation, this happens
whether the bases are added or deleted
• Mutation Repair Mechanisms
o Light Repair: When a thymine dimer (thymines that are right next to each other bind together) is created it causes a mutation in the strand when the strands have to replicate, in light repair the enzyme photolyase gets energy from the sun and breaks the bonds of the dimers to reverse the strand to its normal DNA sequence.
o Base-excision Repair: enzymes (Glycosylases) remove a section of the DNA that has an error in it and DNA polymerase I and ligase fills in the gap
o Nucleotide Excision Repair/Dark Repair: enzymes cut out a short section of the DNA strand (about 12 nucleotides) that contains a dimer and DNA polymerase I and DNA ligase repair the gap with help of the undamaged complementary strand
o Mismatch Repair: enzymes scan newly synthesized DNA strands (they can tell which ones are new and which are old because the new are non-methylated for a short period of time) for mismatched bases and remove and replace them
o SOS Response: Prokaryotic cells with a lot of DNA damage use a variety of processes to induce DNA polymerase to
replicate the damaged strand, replication of this strand may introduce new and possibly fatal mutations but it does allow a few offspring to survive.
*THIS IS THE BEGINNING OF MATERIAL FOR EXAM 3*
• Effects of Mutations
o Cause nonfunctional proteins = could be harmful and even fatal
o Beneficial mutations = organisms can quickly adapt, survive and reproduce (these mutations cause change in populations) ▪ Any change that is advantageous during selection
pressure will be kept by the population
• Genetic Recombination and Transfer
o Genetic Recombination: when an organism gets & expresses genes from another organism, is an exchange of nucleotide sequences between 2 DNA molecules, usually involves
segments that are identical or nearly identical
▪ Three forms of Genetic Recombination in Bacteria:
???? Conjugation: transfer of a plasmid from donor cell
(gram-negative cell donor is a cell with a fertility
plasmid/F+ cell) to recipient cell (cell without a
fertility plasmid/F- cell) with a direct connection
(pilus – tube where DNA passes through). Once the F- cell gets the plasmid from the F+ cell it becomes a F+ cell too.
• In cells where the plasmid transferred doesn’t stay in the cytosol but instead is incorporated into the cellular chromosome = High Frequency Recombination Cells (HFR cells)
o In these cells when they perform
conjugation a portion of the
chromosome and a portion of the
fertility plasmid are transferred to the
recipient.
???? Transformation: organism takes up DNA from its environment, DNA fragments from lysed cells are accepted by the recipient cell and the genetic code is acquired by the recipient
• Donor and recipient cells can be unrelated, is a useful tool in recombinant DNA
technology
???? Transduction: when a bacteriophage injects its DNA into the host cell & degrades the host cells DNA, when creating new phages if one takes up part of the host cells DNA instead of its own it is called a transducing phage and when it injects its DNA into a new cell the old host’s DNA is incorporated into the new cells DNA.
• Two Types:
o Generalized Transduction: random
fragments of host DNA are picked up
during phage assembly, any gene can
be transmitted this way
o Specialized Transduction: A specific
piece of the host genome is
incorporated into the virus
▪ Here the phage goes into
lysogeny & is incorporated into
the bacteria cell’s genome,
something happens to make it
reenter the lytic cycle. The
excised phage DNA contains
some host DNA and when new
phages are produced it has
some bacterial DNA. All phages
can function as phages and
when a new recipient cell is
infected the bacterial DNA is
transferred. The DNA (with
bacterial DNA and phage DNA)
can enter and do lysogeny or
just the bacterial DNA can be
incorporated into the cells
genome.
o Transposition: Mutation in which a genetic segment is
transferred from one location in the genome to another via a Transposon. This is transfer of genetic information within one cell.
▪ Transposons are the segments of DNA that are able to move from one location to another in the genome.
“Jumping Genes”
???? Causes rearrangement of the genetic material
(like a frameshift mutation)
???? Can occur between plasmids and chromosomes
and within and among chromosomes
???? Can be beneficial or harmful, is a way to get
variation in genomes
???? They go to another location but also stay in their
original location
Controlling Microorganisms
• Targets are microorganisms that can infect or cause spoilage. Ex:
o Vegetative bacterial cells & endospores
o Fungal hyphae and spores, yeast
o Protozoan trophozoites & cysts
o Worms
o Viruses
o Prions
• Ranking in terms of Most Resistant to Least Resistant to chemical controls
o Prions – part of microbes, pathogenic proteins that are tolerant to steam
o Bacterial endospores
o Mycobacteria
o Cysts of Protozoa
o Active-stage protozoa (trophozoites)
o Most Gram-negative bacteria – have outer membrane as well as the peptidoglycan layer
o Fungi
o Non-enveloped viruses
o Most gram-positive bacteria
o Enveloped viruses – capsid is not as rigid because of membrane, if it were more tightly packed it would be more resistant
• Terminology and Methods of Control
o Sterilization: process that destroys ALL viable microbes, this includes viruses and endospores
o Disinfection: process that destroys vegetative pathogens but not endospores (this is used on inanimate objects, not the body)
o Antiseptic: disinfectants that are applied directly to the body o Sanitization: cleansing techniques that mechanically remove microbes, this destroys some things
o Degermation: use mechanical means to reduce the number of microbes, example would be hand washing with soap and water
• Microbial Death – how do we tell a living microbe from a dead one?
o Difficult to detect this because microbes don’t reveal vital signs to begin with, can tell w/membrane functions sometimes
o One way to tell a microbe is dead is if it has permanent loss of reproductive capability, even when in optimum growth conditions
o Suffixes:
▪ -cide = the death of something
▪ -static = bacteria are not growing and are also not dying, there is no change.
o Factors that Affect Death Rate (how effective an agent is, is determined by several factors)
▪ Number of Microbes
▪ Nature of Microbes in the population – ex. spores are harder to kill, enveloped viruses are easier to kill than naked
▪ Temperature and pH of the environment - warm disinfectants work better than cool ones (chemicals react faster at higher temperatures), acidic conditions enhance the antimicrobial effect of heat, some
household disinfectant are more effective at a lower pH ???? This affects the denaturing of proteins
▪ Concentration or dosage of the agent
▪ Mode of action of the agent – cellular targets of physical and chemical agents
???? The Cell Wall: wall becomes fragile and cell lyses, done by some antimicrobial drugs, detergents, &
alcohol.
???? The Cell Membrane: membrane loses integrity by influencing the stability of the phospholipid
bilayer, detergent surfactants do this.
???? Protein and Nucleic Acid Synthesis: prevent
replication, transcription, translation, formation of peptide bonds, and protein synthesis. This is done by chloramphenicol, ultraviolet radiation, and
formaldehyde.
???? Disruption or Denaturing of Proteins: Done by alcohols, phenols, acids, and heat.
▪ Presence of solvents, organic matter, or inhibitors o Decimal Reduction: the time it takes to kill 90% of a population, if start with a big number even a 90% reduction would still leave a significant amount.
o Methods of Physical Control
▪ Heat: denatures proteins, interferes with the integrity o of the membrane and walls, disrupts of the function and structure of Nucleic Acids.
???? Moist: Commonly used to disinfect, sanitize,
sterilize, pasteurize, kills cells by denaturing
proteins & destroying cytoplasmic membranes.
More effective than dry heat because water
conducts heat better than air.
• Boiling: kills vegetative cells of bacteria &
fungi, trophozoites of protozoa & most
viruses within 10 min.
o Boil at 100°C for 30 minutes to
destroy non-spore forming pathogens
• Pasteurization: use heat to kill pathogens &
reduce number of spoilage microorganisms
in food & drinks
o A way to control bacterial population
so yeast is the dominating population
without damaging the grapes
o 63°-66°C for 30min – Batch method
that kills a lot of microbes (old
method)
o 71.6°C for 15 seconds – Flash Method
(New method, controls a lot of
microbes)
o Not Sterilization – shelf stable milk –
hit with high temperatures and
changes flavor
• Autoclaving (like a pressure cooker):
sterilize chemicals & things that can tolerate moist heat, prevents the escape of steam increasing the pressure, applying pressure sterilizes because the temperature that
water boils at increases as pressure
increases. Destroys all microorganisms in 15 min.
???? Dry: used for powders and oils, things that can’t be sterilized with steam or boiling, Denatures proteins, fosters oxidation of metabolic & structural chemicals. Requires higher temps for longer times than moist heat because dry heat penetrates slower.
• Dry Oven: takes longer than an autoclave (15 min. vs. 16 hours), 150°-180°C
coagulates proteins
• Incineration: fosters oxidation, alters protein structure, ultimate means of
sterilization, ex. Heating a loop in a Bunsen burner
???? Thermal Death Measurements
• Thermal Death Time (TDT): shortest length of time to kill all microbes at a certain
temperature
• Thermal Death Point (TDP): lowest
temperature to sterilize something within 10 minutes
• Thermal Death Times of Endospores
o Moist Heat (121°C):
▪ Bacillus subtilis – 1 min
▪ Clostridium botulinum – 10 min
o Dry Heat (121°C and 120°C)
▪ Bacillus subtilis – 120 min
▪ Clostridium botulinum – 120
min
▪ Cold Temperatures: slows microbial growth ???? Refrigeration 0-15°C and freezing below 0°C – preserves microbes, used to preserve food, media and cultures
???? Microbiostatic: slows the growth of microbes ▪ Desiccation: a way to preserve microbes, a gradual removal of water from cells which leads to metabolic inhibition – the cells can’t do anything but they are not dead, preserve them by freeze drying,
???? Can mail them this way & they don’t grow while like this.
???? Not an effective method of microbial control because they can grow again once water is
reintroduced.
???? Lyophilization: freeze drying; preservation ▪ Radiation: release of high-speed subatomic particles or waves of electromagnetic energy from atoms ???? Ionizing: electromagnetic radiation
w/wavelengths shorter than 1nm are ionizing because when they hit molecule have enough energy to eject electrons from atoms create ions • A form of cold sterilization
• This disrupts h-bonding, oxidizes double
covalent bonds, create highly reactive
hydroxyl radicals.
• Ions denature other molecules and cause fatal mutations & cell death.
• Ex: cathode rays, gamma rays, some x-rays • Used to sterilize medical supplies and food products that can’t take heat (like plastics)
???? Non-ionizing: electromagnetic radiation
w/wavelength greater than 1nm, not enough energy to force electrons out of orbit, excites electrons to force creation of new covalent bonds which affects structure of proteins and nucleic acids.
• Ex: UV light, visible light, infrared radiation,
radio waves
o UV light used in microbial control by
making thymine dimers that prevent
replication of genetic material & cause
cells to die. Does not penetrate well,
disinfects air, transparent fluids, &
surfaces
▪ Filtration: passage of fluid through a sieve that traps particles (cells or viruses) and separates them from the fluid.
???? sterilize heat sensitive materials & estimate the number of microbes in a fluid (count how many
on filter after pouring certain amount through)
???? HEPA filters used in labs, filter the air in the hoods so the air in the hood is sterile and the exhaust of the hood is filtered too
• Pressure system is used by these to control
the air flow and control what leaves
o Chemical Microbial Control
▪ Types of Chemicals
???? Disinfectants: do not guarantee all pathogens are eliminated, used on inanimate objects, don’t kill
endospores or some viruses, more concentrated
than antiseptics and can be left on surfaces for
longer
???? Antiseptics: chemical used on skin or other tissue ???? Sterilants: chemicals that sterilize, denature
proteins and DNA by cross-linking organic
functional groups
???? Degermers: remove microbes but don’t kill them ???? Preservatives
▪ Desirable qualities of chemicals:
???? Rapid action at a low concentration
???? Toxic to other things, not you (low toxicity)
???? Doesn’t stain, not corrosive
???? Affordable & readily available
???? Water soluble/Alcohol soluble
???? Stable
▪ Levels of Chemical Decontamination
???? High-level Germicides: kills endospores, use on something that needs to be sterile but can’t be heated
???? Intermediate-level: Doesn’t kill endospores, will kill fungal spores (tuberculosis, virus), disinfects not sterilize, good for things that come into
contact with mucous membrane
???? Low-level: will only kill vegetative cells & normal fungal cells not spores, ok if just touching but not used for things that touch mucous membrane & are invasive
▪ Factors that Affect Germicidal Activity of Chemicals ???? Nature of material being treated – some things can’t handle certain chemicals
???? Degree of contamination – where things are going is important, are they going to be in contact with the mucous membrane etc.
???? Time of Exposure – Ex: Alcohol sanitizer needs to be in contact with your hands for 10 seconds, time microbes are exposed to chemicals affects how effective the chemical is.
???? Strength and Chemical Action of the Germicide ▪ Examples of Chemicals
???? Halogens
• Chlorine: Cl2, hypochlorites (Chlorine
Bleach), chloramines (safer than chlorine),
one of the most highly used
o Method: denatures proteins by
disrupting disulfide bonds
o Intermediate Level
o Unstable in sunlight, inactivated by
organic matter – if something is super
dirty it might not be as effective
against it
o Use: decontaminate water, sewage,
wastewater, inanimate objects
• Iodine: I2, iodophors (betadine)
o Method: interferes with disulfide
bonds of proteins
o Intermediate Level
o Use: milder medical and dental
degerming agents, disinfectants,
ointments
o *Staining is a downside
???? Phenolics
• Method: disrupts cell walls & membranes, precipitates proteins
o At high concentrations – disrupts cell walls and proteins
o At low concentrations – disrupts
critical enzyme systems
• Low to intermediate-level – Bactericidal, fungicidal, virucidal, not sporicidal (Strong antimicrobial but not sporocidal)
• Because of high toxicity don’t use them as antiseptics anymore
• Ex: Lysol, Triclosan – antibacterial additive to soaps
• Use: in lab use phenols to remove proteins from preps
• Lister used carboxylic acid (a types of phenol) to clean skin but was really harsh skin
???? Chlorohexidine – used instead of phenols, low toxicity
• Method: surfactant & protein denaturant w/broad microbicidal properties
• Low to intermediate level – good against bacteria, variable against viruses & fungi, Not good on endospores
• Use: skin degerming agents for
preoperative scrubs, skin cleaning & burns • Ex: Hibiclens & Hibitane (used to clean skin) ???? Alcohols
• Method: acts as surfactants dissolving membrane lipids & coagulating proteins of vegetative bacterial cells & fungi
• Intermediate Level – No impact on
endospores, does impact fungal spores
• Ex: Ethyl, isopropyl in solutions of 50-95% - 70% is more effective than 100% because 100% is dehydrating.
???? Hydrogen Peroxide
• Method: Produces highly reactive hydroxyl free radicals which damage protein & DNA while decomposing to oxygen gas – toxic to anaerobes
• At low concentrations: Antiseptic
• At high concentrations: Sporicidal
???? Aldehydes
• Method: Glutaraldehyde & formaldehyde kill by alkylating protein & DNA
• High level – Glutaraldehyde in 2% solution (Cidex) is used as a sterilant for heat
sensitive instruments
o 2nd chemical to use as a sterilant
• Intermediate level – Formaldehyde is a disinfectant & preservative, limited use due to toxicity, works fast
o Formalin – 37% aqueous solution
???? Gases and Aerosols
• Method: strong alkylating agents
• High Level
• Use: sterilize & disinfect plastics &
prepackaged devices, foods
o Used in a chamber w/temperature,
pressure & oxygen controlled
o 19-3 hours are needed to aerate,
sterile air for hours after
o gas is explosive & can harm our
bodies & cause cancer
• Ex: ethylene oxide and propylene oxide ???? Detergents and Soaps
• Method: quaternary ammonia compounds (quats) work as surfactants (polar
molecules that have hydrophobic and
hydrophilic parts), change some fungi & bacteria membrane permeability
• Very low level
• Use: soaps are a method of degerming, mechanically remove soil & grease that contains microbes
o Good against viruses = Anti-viral
o Most common in Bactene
o Non-germicidal soaps – cause
inhabitant microbes to be pulled out
of skin and after days of scrub water
sitting the number of microbes are
higher with these soaps (which is the
category that antibacterial soaps are
under) than with Germicidal Soap
o Germicidal Soaps – with prolonged
used the number of bacteria
decreases.
???? Heavy Metals
• Method: low concentrations of silver and mercury solutions kill vegetative cells by inactivating proteins
o Oligodynamic action: bind to the final
groups of proteins to inactivate them
o Have antimicrobial properties
• Low level
• Ex: Merthiolate, silver nitrate, silver
???? Dyes as Antimicrobial Agents
• Method: aniline dye very active against
gram-positive species of bacteria and
various fungi
• Low level, narrow spectrum of activity
• Use: antisepsis & treat wounds
???? Acids & Alkalis
• Low level
• Organic Acids: prevent spore germination,
bacterial growth, fungal growth
• Acetic Acid: inhibits bacterial growth
• Propionic Acid: slows molds – this is the
thing that propionic bacteria makes that
gives cheese its flavor
• Lactic Acid: prevents anaerobic bacterial
growth – lactobacillus ferments glycogen
and decreases pH = keeps the vagina acidic
• Benzoic Acid: inhibit yeast
Antibiotics
• Principles of Antimicrobial Therapy
o Give a drug to an infected person, it destroys the infective agent without harming the host’s cells
▪ Therapeutic Index is good when antimicrobial has an
increased toxicity to microbes and decreased toxicity to
us/humans
???? Therapeutic index: the ratio of the dose of the
drug that is toxic to humans as compared to its
minimum effective dose (Ratio= smallest effective
dose/amount that is toxic to humans) ???? want #
on bottom to be smaller
• Higher index is desirable
o Antimicrobial drugs are produced naturally or synthetically ▪ *Side Note: The mortality rate in some countries is the same as before antibiotics because of high rates of
infant mortality due to lack of access to healthcare
• Characteristics of an Ideal Antimicrobial
o Selectively Toxic
o Microbicidal – kills microbes
o Relatively soluble – functions when highly dilute, makes sense because we are made of mostly water
o Remains potent long enough – not broken down or excreted prematurely, with penicillin this was a problem early on because people were peeing it out
o Doesn’t lead to resistance – this is difficult
o Complement or assists the activities of the host defenses o Active in tissues and body fluids
o Can get to site of infection
o Affordable
o Doesn’t negatively affect host’s health…allergies or other infections
• Domagk – showed that red dye called prontosil could be active against bacteria – this was the first sulfur drug
• Paul Erlich – some dyes dye the microbes and not the tissue, he came up with an early chemotherapy for syphilis
• Terminology
o Prophylaxis: Action taken to prevent disease, with a specified means against a specified disease
o Chemotherapeutic drug: drugs that act against diseases o Antimicrobial: any compound used to treat infectious disease, may also function as intermediate-level disinfectant
o Antibiotic: antimicrobial chemicals produced naturally by microorganisms
o Synthetic/Semi-Synthetic: Antimicrobials that are completely synthesized in the lab
o Narrow Spectrum: Drugs that work against only a few kinds of pathogens, target a specific cell component that is only found in certain microbes
o Broad Spectrum: Drugs that work against many different kinds of pathogens, target cell parts that are common to most pathogens (ribosomes)
o Spectrum: range of activity of a drug
• For antibiotics to work must work against the differences in the cells o For example antimicrobial or antibiotics works against the peptidoglycan in bacteria
• Antibiotics come from one genus, are natural in origin – we are not very good at creating drugs on our own
• Sporulation (Life cycle of streptomycin)
o Exospore ???? Spore Germination ???? Vegetative Hyphae Growth (digs into the plate) ???? Aerial Hyphae Growth (microbe grows up and out of the media, produces antibiotic as it grows aerial hyphae, when it does this it breaks down substrate hyphae for nutrients and the nutrients go into the soil) ???? Septation (growth subdivides and twists) ???? Spores maturation
• Theory for why organisms make Antibiotics
o Competitive advantage idea
o Sub-inhibitory levels of antimicrobials cause response to things around it
o Junk Mechanism
o Evolutionary leftover mechanism ???? this is the reason we have resistance ???? prokaryotes are the main producers of
antibiotics, they can kill other things in the same genus, these microbes have a mechanism of resistance ???? can’t get rid of the resistance
• Interactions Between Drug & Microbe
o Antimicrobial drugs should be selectively toxic – drugs should kill or inhibit microbial cells without damaging the host tissue ▪ When the characteristics of the infectious agent become similar to the host cell selective toxicity becomes
difficult to achieve = more side effects
• Mechanisms of Drug Action
o Inhibition of Cell Wall Synthesis
▪ Β-lactams
???? Basic Structure: beta-lactam rings
???? Mode of Action: inhibit peptidoglycan formation by binding to the enzymes that cross-link NAM
subunits (attacks between the NAG/NAM
subunits). This causes the bacterial cells to have
weakened cell walls as they grow and they are
not resistant to osmotic pressure, as water moves into the cell, the membrane bulges through the
weakened part of the cell wall & the cell
eventually lyses.
• For something that attacks to the forming
bonds we need something to be growing
rapidly for this to work (aka cause cells to
lyse)
???? Effectiveness:
• Penicillinase or B-lactamase
???? Drugs within this group:
• Penicillins – Penicillin chrysogenum is a
major source
o Consists of three parts:
▪ Thiazolidine Ring
▪ Beta-lactam ring
▪ Variable side chain dictating
microbial activity – affect the
ability to get across the outer
membrane
o Subgroups and Uses of Penicillins
▪ Penicillin G and V most
important natural forms
▪ Drug of choice for Gram-Positive
cocci (streptococci), some
Gram-Negative bacteria
(meningococci and syphilis
spirochete)
▪ Semisynthetic Penicillins –
ampicillin, carbenicillin,
amoxicillin broader spectra –
Gram-Negative infections
▪ Penicillinase-resistant –
methicillin, nafcillin, cloxacillin
(resistant to enzyme that
breaks down penicillin)
▪ Primary problems: allergies to
penicillin and resistant strains of
bacteria
• Cephalosporins: Targets the building of peptidoglycan, 1/3 of all antibiotics
administered
o Relatively broad spectrum, resistant to most penicillinases, cause fewer
allergic reactions
o Some given orally, many parentally o Generic Names have root – cef, ceph, kef
o 4 Generations Exist (each group more effective against gram-negatives than the previous one, better dosing
schedule and less side effects) –
become more broad range as
progress:
▪ First Generation: cephalothin,
cefazolin – most effective
against gram-positive cocci and
few gram-negative
▪ Second Generation: cefaclor,
cefonacid – more effective
against gram-negative
▪ Third Generation: cephalexin,
ceftriaxone – broad-spectrum
activity against enteric bacteria
w/beta-lactamases
▪ Fourth Generation: cefepime,
widest range, both gram
negative & gram-positive
• Carbapenems: Imipenem – broad spectrum drug, used for infections w/aerobic &
anaerobic pathogens, low dose,
administered orally, few side effects
• Monobactams: Aztreonam – narrow
spectrum drug, used for infections by gram
negative aerobic bacilli, used by people
allergic to penicillin
▪ Non beta-lactam Cell Wall Inhibitors
???? Vancomycin: Narrow-spectrum, used for
staphylococcus infections when there is resistance to penicillin & methicillin or if patient is allergic to penicillin, is toxic and hard to administer, use is restricted
• Disrupts formation of G-positive cell wall by interfering with alanine-alanine crossbridges linking NAG subunits
• Used to treat MRSA
• Now some things are resistant to this
???? Bacitracin: narrow-spectrum, made by strain of Bacillus subtilis, used in topical ointments
• Blocks NAG/NAM secretion from the
cytoplasm
• Main ingredient in neosporin
???? Isoniazid (INH): interferes w/mycolic acid synthesis, treats infections w/Mycobacterium tuberculosis
• Disrupts the formation of arabinogalactan mycolic acid by mycobacteria
• Used in a triple therapy to treat
Tuberculosis
o Breakdown of cell membrane structure or function: cell dies from disruption in metabolism or lysis
▪ Cant carry out chemical rxns without intact membrane ▪ These drugs have Specificity for particular microbial group based on differences in types of lipids in
membranes
▪ Polymyxins: interact w/phospholipids, cause leakage, specifically in gram-negative bacteria
▪ Amphotericin B and Nystatin: make complexes with sterols (ergosterol) on fungal membranes ???? disrupts the membrane and causes lysis = leakages (fungicidal)
???? The difference between fungal cells and your cells are the sterols, membranes without cell walls
have sterols to maintain rigidity.
???? Human cells somewhat susceptible because
cholesterol is similar to ergosterol (but don’t bind as well)
o Inhibition of nucleic acid synthesis, structure or function ▪ Block synthesis of nucleotides, inhibits replication, stops transcription
▪ Chloroquine: binds and cross-links the double helix, inhibit DNA helicases
▪ Antiviral drugs, analogs of purines and pyrimidines insert in viral nucleic acids, prevents replication
▪ Rifampin: bind and inhibit the action of RNA polymerase during synthesis of RNA from DNA, binds more readily to prokaryotic than eukaryotic so more toxic to those. o Drugs that Act on DNA or RNA
▪ DNA Gyrase Inhibitors
???? Fluoroquinolones: synthetic drugs that are active against bacterial DNA, work by binding to DNA
gyrase (enzyme necessary for correct coiling and uncoiling of replicating bacterial DNA) and
topoisomerase IV, may act against replication of
mitochondrial DNA in some Eukaryotes.
• Broad spectrum effectiveness
• Concerns w/overuse of quinolone drugs –
recommend careful monitoring to avoid
ciprofloxacin-resistant bacteria
o Inhibiting Protein Synthesis – Ribosomes of euk. differ from prok. Antimicrobics selective action against prokaryotes but can also damage the euk mitochondria
▪ Aminoglycosides: ex. Streptomycin (treats TB), gentamycin. These insert on sites on the 30S subunit, cause misreading of mRNA
▪ Tetracyclines: 4 ring structure, block attachment of tRNA on A acceptor site & stop further synthesis, used against lime disease, Typhus, some STD’s, has side effects
▪ Chloramphenicol: phenol ring in structure, affects peptide bonding, attaches to 50S subunit and prevents peptide bond formation
???? Broad spectrum
???? Nitrobenzene type structure
???? Harms bone marrow
???? Used for rickettsia and chlamydia
▪ Macrolides – Erythromycin: binds to 50S subunit blocking proper movement of mRNA through ribosome, synthesis stops
???? Used prophylactically – before someone has an infection
???? Used for penicillin resistant organisms
o Blocks key metabolic pathways
▪ Sulfonamides & Trimethoprim block enzymes required for tetrahydrofolate synthesis that is needed for DNA and RNA synthesis
???? Methods
• Competitive inhibition: the drug competes
with normal substrate for the enzyme’s
active site
• Synergistic Effect: drugs work better
together than on their own
▪ *Remember that the problem with broad spectrum antibiotics is that they kill normal microbiota too, which can be good/helpful to us
• Agents to Treat Fungal Infections
o Fungal cells eukaryotic, drug toxic to fungal cells also toxic to human cells = hard to treat, bad side effects, remember antibiotics don’t work on fungal organisms
o Five Antifungal Groups:
▪ Macrolide polyene
???? Amphotericin B: mimic lipids, most versatile &
effective, topical and systemic treatments
???? Nystatin: topical treatment and oral swishes
• Again the function of these make complexes
w/sterols (ergosterol) on fungal membranes
???? disrupts the membrane & causes lysis =
leakages (fungicidal)
▪ Griseofulvin: used for stubborn cases of dermatophyte infections, is nephrotoxic (damaging to the kidneys)
▪ Synthetic azoles: broad spectrum, ketoconazole,
clotrimazole, miconazole
▪ Flucytosine: analog of cytosine, used against cutaneous mycoses (disease of the hair, skin, and nails) or used with Amphotericin B for systemic mycoses
▪ Echinocandins: damages cell walls, used against
capsofungin, these are the reason they stopped
antifungal research because they are very good against dominant fungal infections.
• Antiparasitic Chemotherapy
o Antimalarial drugs: quinine, chloroquinine, primaquine, mefloquine
o Antiprotozoan drugs: metronidazole (Flagyl), quinicrine, sulfonamides, tetracyclines
o Antihelminthic drugs: these immobilize, disintegrate, inhibit metabolism
▪ Mebendazole, thiabendazole –broad spectrum- inhibit function of microtubules, interfere w/the usage of
glucose & disables them
▪ Pyrantel, piperazine – paralyze muscles
▪ Niclosamide – destroys scolex
• Antiviral Chemotherapy
o Selective toxicity hard because of obligate intracellular parasitic nature of viruses
o Methods
▪ Inhibition of virus entry or release (interfere with fusion of virus to the membrane)
???? Fuzeon: blocks HIV infection
???? Amantidine: blocks influenza virus
???? Tamiflu and Relenza: stops actions of influenza
neuramidase required to enter the cell
▪ Block replication, transcription, or translation of viral genetic material – Inhibition of nucleic acid synthesis
???? Nucleotide analogs
• Acyclovir – used against herpes virus
(terminates DNA replication)
• Ribavirin – a guanine analog used against
RSV, hemorrhagic fevers
• AZT – thymine analog used against HIV
(HIV is an RNA based virus, if block reverse
transcriptase, which converts RNA to DNA,
then can’t produce viral DNA to enter the
host DNA)
▪ Prevent maturation of the viral particles – Inhibition of Effective Viral Assembly and Release
???? Protease inhibitors – used against HIV (inserts
into HIV protease an enzyme that clips viral
proteins into functional pieces)
• Interferons (INF) – human-based glycoproteins, made mostly by fibroblasts and leukocytes
o Therapeutic benefits:
▪ Decrease healing time and complications of infections – antiviral & anticancer properties
▪ Prevents/reduces symptoms of cold & papilloma virus
▪ Slows progress of some cancers, leukemia, &
lymphomas
▪ Treats hepatitis C, genital warts, Kaposi’s sarcoma
Drug Resistance – an adaptive response when microbes begin to tolerate an amount of drug that would normally kill them, because of genetic versatility or variation (can be intrinsic or acquired)
• Acquired Resistance
o spontaneous mutations in critical chromosomes
o get new genes/sets of genes from resistance factors (R plasmids) encoded w/drug resistance, transposons
▪ Antibiotic Resistance Transfer – based on R plasmids
(plasmids carrying resistance genes)
• Mechanisms of Drug Resistance
o Mechanism 1: limiting access of the antibiotic due to
decreased permeability to drug/increased elimination of drug from cell – acquired/mutation
▪ Outer membrane porins
▪ Active Efflux: resistant cells can pump the antimicrobial out of the cell before the drug can act. Some cells are
able to pump more than one antimicrobial from the cell.
▪ Reduced uptake across cytoplasmic membrane
o Mechanism 2: Drug Inactivation due to acquired enzymatic activity – penicillinases – acquired mutation
▪ B-lactamases: enzymes break the beta-lactam rings of penicillin & similar molecules making them inactive
???? In Penicillin the efficacy of it as an antibiotic is
dependent on the Lactam Ring and penicillinases
breaks that ring to make it inactive
▪ Modifying Enzymes
o Mechanism 3: Modification or protection of target -
acquired/mutation
▪ Resistant cells may alter the target of the drug so that the drug either cannot bind to it or binds less effectively ???? change in drug receptors
o Mechanism 4: Change in metabolic Patterns – mutation of enzyme
▪ Alter the metabolic chemistry or abandon sensitive metabolic step altogether, cell could become more
resistant to a drug by producing more enzyme
molecules for the metabolic pathway & reducing the
power of the drug
▪ Trymethoprin and Sulfonamides
• Development of Resistance
o Spontaneous Mutation
o Transfer of Resistance – 3 Methods
▪ Transduction: when a bacteriophage injects its DNA into the host cell and degrades the host cells DNA, when
creating new phages if one takes up part of the host
cells DNA instead of its own it is called a transducing
phage and when it injects its DNA into a new cell the
old host’s DNA is incorporated into the new cells DNA.
▪ Transformation: organism takes up DNA from its
environment, DNA fragments from lysed cells are
accepted by the recipient cell and the genetic code is
acquired by the recipient
▪ Conjugation: transfer of a plasmid from donor cell
(gram-negative cell donor is a cell with a fertility
plasmid/F+ cell) to recipient cell (cell without a fertility plasmid/F- cell) with a direct connection (pilus – tube where DNA passes through).
• Factors that Contribute to Resistance
o Natural Selection
▪ Big populations of microbes are likely to have drug resistant cells from prior mutations or transfer of
plasmids, there is no growth advantage to these until they are exposed to a drug.
▪ Once exposed sensitive cells die & resistant cells survive
▪ Population becomes resistant from selective
pressure/natural selection
▪ Indiscriminate use of antimicrobials worldwide has led to many drug resistant microorganisms.
o Health care mentality
o Agriculture
o Worldwide Resistance
• How to Limit Drug Resistance
o Drug Usage
▪ Physicians – accurate diagnosis, give right drug
▪ Patients – comply w/guidelines
▪ Combined Therapy – more than 1 antibiotic works to kill off microbes, remember dead cells can still give other cells nucleic acids
o Drug Research
▪ Develop shorter duration, higher dose antimicrobials ▪ Find drugs whose structures are not inactivated by enzymes & not readily circumvented
o Long-term
▪ Educate Healthcare workers – reduce the abuse of antibiotics
▪ Restrict use of antibiotics
▪ Stop using antibiotics in animal feed
▪ Come up w/programs that have effective therapy
available to low income populations
▪ Vaccine where possible – there is nothing out there supporting a link between autism & vaccines
???? Some components of vaccines work better with
others, they are synergistic so vaccines work
better if taken together
• Interactions Between Drug & Host
o ~5% of everyone taking antimicrobials will experience serious adverse reaction/side effects
o Major Side Effects:
▪ Damage to tissue because of drug toxicity
???? If use Tetracycline when child is developing =
tooth discoloration
???? Flagyl - hairy tongue is a side effect of this
antibiotic because it kills good microbes that were
stopping that before
▪ Allergic Reactions
▪ Disrupts balance of normal flora = superinfections
possible (Secondary infections that are caused by
treatment of antibiotics which are killing microbes and opening niches)
• What to Consider when Selecting a Drug
o Identify microorganism causing infection – restrict the use of anything that might work against an ideal organism
▪ Identify as soon as possible
▪ Specimens should be taken before starting
antimicrobials
o Test microorganism’s susceptibility to drugs in vitro when indicated
▪ Essential for bacteria that are commonly resistant
▪ Kirby-bauer disk test: create lawn of microbe and
inoculate with a disk of antibiotic, read the zone of
inhibition to determine if the microbe is sensitive or
resistant
???? Resistant: If something is resistant then we can’t
get the antibiotic to a high enough concentration
in the patients to safely kill the microbes
???? Intermediate: Can use in patients but will have
side effects
???? Sensitive: a low enough concentration of the
antibiotic kills the microbe that it could be used in
a patient
▪ E-test diffusion test
▪ Dilution Test: conducted to find the MIC (minimum inhibitory concentration) – the smallest concentration of a drug that will visibly inhibit the microbes growth
▪ Provide profile of drug sensitivity