Description
Unit 3 2/19/17 7:00 PM
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 If you want to learn more check out What are 3 primary ethical guidelines involved in social research?
tryptophan charged tRNA that the ribosome gets
through the Trp part of the leader region fine and
We also discuss several other topics like What is an example of inoculation?
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 Don't forget about the age old question of What are the stages of general adaptation syndrom (gas)?
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 If you want to learn more check out What is the scope of sexual offense policies in universities?
translation If you want to learn more check out Who won the sino japanese war?
▪ 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 If you want to learn more check out Who is logan and molotch?
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
o Overall condition of the patient – will drugs have a side effect • MIC and Therapeutic Index
o MIC: Minimum Inhibitory Concentration = the smallest concentration of a drug that will visibly inhibit the microbe’s growth
o The in vitro activity of a drug is not always correlated to in vitro effect
▪ If therapy fails consider a different drug, combination of drugs or different administration of drugs
o It is best to choose a drug with highest level of selectivity and lowest level of toxicity (determined by Therapeutic Index)
▪ 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
• Final Points on Antibiotic Resistance
o Antibiotics still valid therapy
o Education is vital – patients can’t demand drugs, need to take their full prescription
o Combinations of drugs & developing new vaccines = slows spread of drug resistant pathogens
Relationships between Microbes and their Hosts
*Human body always in state of dynamic equilibrium, many interactions between human and microbe involve biofilms (on teeth, tongue, periodontal pockets), colonization of the body by microbes is a constant give and take. • Contact, Colonization, Infection, Disease
o Normal (resident) Flora/Microbiota: the normal colonizing bacteria or microbes
▪ Areas in contact w/outside environment typically harbor resident microbes
▪ Internal organs, tissues, & fluids microbe free
▪ Transients: Microbes that occupy body for short periods of time (temporary, a lot of infectious diseases are
transient or temporary)
▪ Residents: microbes that become established
???? Microbial Antagonism: Bacterial Flora benefit the
host by preventing overgrowth of harmful
microbes – simple niche occupancy
???? Endogenous Infections: infections from normal
flora being introduced to a site that was sterile –
there is resistance because you are already
colonized
▪ *Remember how different all the environments of our body are, the norm is that certain microbes live in
specific conditions, it is rare that a microbe lives in
many different conditions in the body.
o Infection: something coming from outside or conditions of being pathogenic
o Pathogen: something that has pathogenic ability
o Infectious: capable of causing infection
• Initial Colonization of the Newborn: before birth you are not colonized, birth is when begin being colonized by mom – being breastfed vs formula leads to biases of different colonies within the child
• Flora of the Human Skin
o Skin is a barrier to infection
o The subcutaneous level is not colonized – this is the boundary to sterile internal organs
• Maintenance of the Normal Resident Flora
o Normal flora essential to health of humans
o Flora creates an environment that prevents infections & enhances host defenses – flora can be altered by antibiotic therapy diet change and disease
o Probiotics: introducing known microbes back into the body • Infection – Fundamental Events in Pathogenesis
o Portal of Entry: entrance site of pathogenic microorganisms (skin, mucous membranes, placenta) – Respiratory tract is the most frequent
o Adherence: process where phagocytes attach to
microorganisms through binding of complementary chemicals on cytoplasmic membrane
o Penetration/Invasion: entrance into the tissue
o Proliferation: rapid increase in numbers/reproduction of a cell, part or organism
o Pathology: infection of target and disease
o Portal of Exit: exit site of pathogenic microorganisms (nose, mouth, urethra)
• Types of Pathogen
o True Pathogen (primary pathogens): these make a healthy individual sick. Ex: influenza virus, plague, bacillus, malarial protozoan
o Opportunistic Pathogens: needs an opportunity to infect, like a cut or weakening of immunity. Ex: Pseudomonas sp & Candida albicans
o The severity of the disease depends on the virulence (ability to cause a disease) of the pathogen – Virulence Factors: part of the microbe that contributes to the ability to cause disease • Factors that Weaken the Host Defenses
o Age/Extreme Youth
o Genetic defects & acquired defects - mutations in part of the system makes them more susceptible to the disease
o Surgery
o Disease – cancer, liver malfunction, diabetes
o Chemotherapy/immunosuppressive drugs
o Physical/Mental Stress
o Other infections
• Routes of Entry for Invading Pathogens
o Broken skin or insect bite
o Anus, urethra, vagina or penis
o Placenta
o Mouth, nose, eyes, and ears – mucous membranes
o Some organisms have their own enzymes that facilitate their entry
• Requirements for an Infectious Dose (ID)
o Infectious Dose: minimum number of microbes required for infection to proceed
o Microbes with smaller ID = greater virulence(ability to infect and cause a disease) - can be as small as one cell, others are thousands
o If there is no ID then they will not result in infection ▪ If don’t get proper ID then will not have been exposed and will not get sick
• Surviving Host Defenses
o Once the pathogen is established it is all about avoiding the host cells to get to place of infection
o The initial response of the host defenses come from Phagocytes: cells that take in microbial cells
o Pathogens use Antiphagocytic Factors to avoid phagocytosis o Leukocidins: things that are toxic to white blood cells and are produced by species of Staphylococcus and Streptococcus o Slime layer or Capsule: Makes phagocytosis of pathogen by host cells difficult
o Some pathogens have the ability to survive being
phagocytized
• Exotoxin vs. Endotoxin
o Exotoxin: Secreted by bacteria to breakdown the host cell, it has protein targets and a localized effect to specific tissues ▪ Hemolysins
▪ A-B Toxins (A-active, B-binding)
o Endotoxin: lipopolysaccharide (LPS), part of the outer membrane of gram-negative cell walls. Dead gram-negative bacteria release endotoxin (lipid A) which induces effects such as fever, inflammation, diarrhea, shock and blood
coagulation, not localized, systemic effect
o Toxicity
▪ Exotoxin: high amount
▪ Endotoxin: small amount
o Effects on the Body
▪ Exotoxin: Systemic Effect
▪ Endotoxin: Localized Effect
o Chemical Composition
▪ Exotoxin: protein
▪ Endotoxin: component of LPS
o Heat Denaturation
▪ Exotoxin: protein can be denatured
▪ Endotoxin: heat stable
o Toxoid formation:
▪ Endotoxin: LPS can’t be converted to a toxin
o Immune Response
▪ Exotoxin: can get an immune response
▪ Endotoxin: no immune response from LPS (lipid A
portion)
o *All gram(-) have endotoxin and no gram(+) have it. o *Organisms may or may not have the ability to produce an enzyme (exotoxin)
• Extracellular Enzymes
o Hyaluronidase and Collagenase
▪ Invasive bacteria reach the epithelial surface, the
bacteria produce these enzymes and invade deeper into the tissues.
???? Breaks down hyaluronic acid & collagen, helps get
through the tissue
o Coagulase and Kinase
▪ Bacteria produces coagulase, clot forms around the bacteria so host cells don’t detect it, bacteria later
produce kinase & dissolve the clot, release the bacteria ???? Staphylococcus is a good example of something
that does this
• Stages in the course of infection & disease
o Incubation Period: no signs or symptoms
o Prodromal Period: vague, general symptoms
o Illness: most severe signs & symptoms
o Decline: declining signs and symptoms
o Convalescence: no signs or symptoms
• Signs and Symptoms of Inflammation
o Signs: measurable, objective evidence of disease noted by an observer like a doctor
o Symptoms: subjective evidence of disease sensed by the patient
o Examples of earliest symptoms of disease as a result of activation of the body defenses are fever, pain, soreness, swelling
o Signs of Inflammation:
▪ Edema: accumulation of fluid
▪ Granulomas & abscesses: walled-off collections of
inflammatory cells & microbes
▪ Lymphadenitis: swollen lymph nodes
• Signs of Infection in the Blood
o Change in number of white blood cells
▪ Leukocytosis: increase white blood cells
▪ Leukopenia: decrease in white blood cells
o Septicemia: Microorganisms multiplying in blood, present in large numbers
o Bacteremia: small numbers of bacteria in blood, not necessarily multiplying
o Viremia: small number of viruses, not necessarily multiplying • Infections that go Unnoticed
o Asymptomatic: (subclinical) infections that although the host is infected they don’t show any signs of the disease and don’t know they are infected (inapparent infection) so they don’t seek medical attention
• Portals of Exit – pathogens have a specific exit and this exit influences how the infection spreads
o Normally infectious diseases have a way out but in fungal infections the infected person has to die and decompose for the infection to get into the soil & then spread that way. o Examples of Portals of Exit:
▪ Respiratory: Mucus, sputum, nasal drainage, saliva ▪ Skin scales/flakes
▪ Fecal exit
▪ Urogenital – method for STDs ▪ Removal of blood