Final Exam (all material) - Microbio

what does CRISPR stand for?

clustered regularly interspaced short palindromic repeats

why can’t we use restriction endonucleases to edit DNA in cells?

their recognition sequences occur too frequently in the genome

what is CRISPR?

a form of bacterial adaptive immunity

gives bacteria a way to remember prior, unsuccessful phage infections and defend against them

how did researchers find out about CRISPR loci?

found that bacterial genomes often had arrays of short repeat sequences separated by random-looking spacer sequences—> spacer sequences very important

what are spacer sequences in the CRISPR locus?

sequences acquired from phage DNA after unsuccessful infections

essentially a memory of prior unsuccessful phage infections

how are new spacers formed from the CRISPR system?

CRISPR systems have dedicated enzymes (Cas1, Cas2) that cut out phage DNA and insert them into the bacterial genome

what happens to spacer arrays in the genome in CRISPR

transcribed into a long RNA

RNA is processed into short guide RNAs (gRNAs)—> the other side of gRNA is constant, which helps load it into the main CRISPR enzyme

each gRNA has a segment that is complementary to phage DNA

how does the CRISPR system (Type II), using gRNAs, prevent phage infection

guide RNAs dock onto enzyme Cas9 (DNA nuclease)

gRNA guides Cas9 to complementary sequences of DNA

in subsequent phage infection, the gRNA derived from the prior phage encounter can guide CAs9 to cleave phage DNA

what is Cas9?

a DNA nuclease enzyme

unlike restriction endonucleases, it can’t recognize any DNA sequences on its own—> requires the gRNA as a guide

how can researchers use CRISPR as a super-specific nuclease?

design custom gRNA sequences that target the CRISPR machinery to specific sequences of DNA→ cleave it

cell will try to repair the break using similar sequences as a guide

specialized repair templates cam also be introduced that encourage the cell to make specific edits (can make mutations, deletions, insertions)

what is the purpose of engineering enzymatically defective Cas9 that tightly binds to DNA, but doesn’t cleave?

can be used to block transcription of specific genes

what are some proteins that may be bound to Cas9?

fluorophores, transcriptional activators

chimeric embryo

only certain lineages of cells carry a mutation

can CRISPR/Cas9 enzymes be injected into individual cells?

yes, and it will successfully edit DNA

can be used to create a chimeric embryo, where only certain lineages of cells carry a mutation

what is an example of the first ex vivo CRISPR therapy?

approved in US in 2023 to treat sickle cell disease

hematopoietic stem cells are modified outside the body to correct the mutation, then are reintroduced

how can ex vivo CRISPR be used to treat cancer?

can be used to modify T-cells from a cancer patient

immune cells are modified to target a protein enriched on those specific tumor cells

modified T-cells reintroduced into the patient, directing the immune system to attack the tumor

CAR T therapy

what are the various applications of CRISPR?

-make targeted deletions/mutations/insertions in bacterial and eukaryotic cells for research

-use dCas9 to inhibit expression of essential genes

-construct high-throughput cell-based screens incorporating individual mutations or gene deletions

-rapidly engineer recombinant organisms, eg for agriculture or biofuels

-use in conjunction with gene therapy to edit patient DNA to treat genetic disorders

what are the limitations to CRISPR/Cas9?

there is some rate of off-target Cas9 mutations, so mutations can be made elsewhere in the genome. This is hard to detect, and raises concerns of cancer in therapeutic contexts

what is phenylketonuria?

inherited metabolic disease

the metabolic enzyme in phenylalanine hydroxylase converts the amino acid phenylalanine into tyrosine

if the enzyme is inactive due to a mutation, phenylalanine (and derivatives) accumulate in tissues, especially in the brain, causing developmental and intellectual disorders

treatment usually requires following a modified diet; intervention at birth can prevent symptom onset (injectable enzyme therapy was recently developed)

how might you introduce a functional copy of the gene encoding phenylalanine hydroxylase in a patient with phenylketonuria?

introduce an extra copy of the functional gene to cells in the patient

directly edit the genome of some cells in patient to correct the defect

isolate cells, modify them in vitro, then re-introduce them to a patient

what is gene therapy by a viral vector?

we can engineer viral particles that carry custom genetic information, for example, a functional copy of a non-functional gene

these viral particles don’t carry their normal viral genome and cannot replicate

introduced DNA can persist in treated cells for years, so one dose can have a long effect

what are the concerns of gene therapy by viral vector?

safety→ patient can have a massive immune response to the virus used to transport the gene

ex. Jesse Gelsinger died from a massive immune response to the adenovirus vector → took 15 years for the field to work past these issues

how can we prevent creating a viral infection when using viral vectors in gene therapy?

by using adeno-associated virus (AAV)

AAV is a tiny ssDNA virus that doesn’t encode enough genes to replicate independently

it can only replicate when the cell is co-infected with adenovirus (or some other helper virus)

AAV uses the adeno-virus encoded proteins to replicate

what virus does gene therapy use for viral vectors to prevent infection?

recombinant AAV vectors, since they can’t cause sustained viral infections, even if wild-type AAV were accidentally introduced

what is the ideal disease to treat by gene therapy?

disease caused by a defective copy of a single gene

many metabolic disorders meet this criteria

—many disorders that can be treated are quite rare

other targets: spinal muscular atrophy, T-cell modification to target cancer (CAR-T), mitochondrial disorders, arthritis, angiogenesis to treat circulatory disorders, etc.

Luxturna

approved in 2018 to treat a specific form of congenital blindness

AAV vector is delivered by injection to the retina

costs $850,000

Hemgenix

approved in 2022 to treat hemophilia B

AAV vector targets the liver, introducing a functional copy of blood clotting Factor IX

fomites

inanimate objects that may carry microbial contamination

may be treated with more aggressive control methods, or for a longer time, to achieve lower levels of contamination

Disinfection

use on fomites

reduces or destroys microbial load of an inanimate item through application of heat or antimicrobial chemicals

cleaning surfaces like lab benches, clinical surfaces, bathrooms

ex. chlorine bleach, phenols (Lysol) glutaraldehyde

what method for controlling microbial growth should be used for cleaning surfaces like laboratory benches, clinical surfaces, and bathrooms?

disinfection

ex. chlorine bleach, phenols (Lysol) glutaraldehyde

sanitization

use on fomites

reduces microbial load of an inanimate item to safe public health levels through application of heat or antimicrobial chemicals

commercial dishwashing of eating utensils, cleaning public restrooms

ex. detergents containing phosphates, industrial strength cleaners containing quaternary ammonium compounds

what method for controlling microbial growth should be used for commercial dishwashing of eating utensils, cleaning public restrooms?

sanitization

ex. detergents containing phosphates (Finish), industrial strength cleaners containing quaternary ammonium compounds

sterilization

completely eliminates all vegetative cells, endospores, and viruses from an inanimate item

preparation of surgical equipment and of needles used for injection

ex. pressurized steam (autoclave), chemicals, radiation

what method for controlling microbial growth should be used for preparation of surgical equipment and of needles used for injection?

sterilization

ex. pressurized steam (autoclave), chemicals, radiation

what methods for controlling microbial growth should only be used on fomites (as opposed to skin)

disinfection, sanitization, sterilization

what controlling microbial growth techniques can be used on living tissue?

antisepsis, degerming

antisepsis

reduces microbial load on skin or tissue through application of an antimicrobial chemical

cleaning skin broken due to injury; cleaning skin before surgery

ex. boric acid, isopropyl alcohol, hydrogen peroxide, iodine (betadine)

what method for controlling microbial growth should be used for cleaning skin broken due to injury; cleaning skin before surgery

antisepsis

ex. boric acid, isopropyl alcohol, hydrogen peroxide, iodine (betadine)

degerming

reduces microbial load on skin or tissue through gentle to firm scrubbing and the use of mild chemicals

handwashing

ex. soap, alcohol swab

what is an application of degerming?

handwashing using soap or an alcohol swab

aseptic technique

involves a set of protocols that maintain sterility (asepsis), thus preventing contamination of the patient with microbes

what can failure of aseptic technique cause?

can put a clinical patient at risk for sepsis, a systemic inflammatory response to systemic infection

what is sepsis?

a systemic inflammatory response to systemic infection

what are the categories of microbial control methods?

physical→ heat, radiation

chemical→ gas, liquid

mechanical removal→ filtration

biological→ virus, toxin

individual methods are typically tailored to work on either object or living organisms

what are the physical microbial control methods?

heat

refrigeration and freezing

pressure

desiccation

radiation

filtration

what are the chemical microbial control methods?

gas, liquid

what are the mechanical removal microbial control methods?

filtration

what are the biological microbial control methods?

virus, toxin

what represents how treatments don’t kill all microorganisms instantly?

microbial death curve

-killing is a probabilistic process

-microbial populations usually die exponentially (fixed percentage dies per unit time)

-gives a straight line on a semi-log plot

-efficacy of killing agent is measured by D-value→ decimal reduction time; time to kill 90%= 1 Log10 unit

what is the D-value on a microbial death curve?

how the efficacy of a killing agent is determined

stands for decimal reduction time

time to kill 90%= 1 Log10 unit

what is heat killing a function of?

temperature and time

various microorganisms respond differently to high temps; endospore formers such as Clostridium botulinum are more heat tolerant

boiling does not kill all microbes→ some endospores survive >20 hours of boiling (and it’s less effective at higher altitudes)

methods of heat controlling microbial growth

boiling (100ºC, sea level)→ denature proteins, alters membranes

dry-heat oven (170ºC, 2 hrs)→ denatures proteins, alters membranes, dehydration, dessication

incineration (flame)→ destroy by burning

autoclave (121ºC, 15 min, 15psi)→ denatures proteins, alters membranes

pasteurization (varies, typically 72ºC, 15s)→ denature proteins, alter membranes

autoclaves

rely on moist heat sterilization and raise temps above the boiling pt. of water; considered the most effective method of sterilization

internal indicators used to ensure sterilization—> heat sensitive autoclave tape, biological indicator spore test (determine if endospores are killed)

pasteurization

boiling and autoclaving damage foods—> this method uses heat, but does not render the food sterile (to preserve it)

reduces the number of spoilage-causing microbes while maintaining food quality

Steps:

-LTH (low temp. holding)= 63ºC, 30 min

-HTST (high temp., short time)= 72ºC, 15s → lowers bacterial #s while preserving quality of milk

-UHT (ultra high temp.)= 138ºC >2s → UHT pasteurized milk can be stored for a long time in sealed containers w/o being refrigerated

what are the steps to pasteurization?

-LTH (low temp. holding)= 63ºC, 30 min

-HTST (high temp., short time)= 72ºC, 15s → lowers bacterial #s while preserving quality of milk

-UTH (ultra high temp.)= 138ºC >2s → UHT pasteurized milk can be stored for a long time in sealed containers w/o being refrigerated

refrigeration

0-7ºC

inhibits metabolism (slows/arrests cell division)

preservation of food or laboratory materials (solutions, cultures)

freezing

below -2ºC

stops metabolism, may kill microbes

long-term storage of food, laboratory cultures, or medical specimens

-bacterial cultures and medical specimens requiring long term storage or transport are often frozen at ultra-low temps (dry ice -70ºC or liquid nitrogen tanks -196ºC)

what are methods that use pressure to control microbial growth?

high-pressure processing (100-800 MPa)—> denatures proteins, can cause cell lysis; preservation of food

hyberbaric oxygen therapy (air pressure 3x higher than normal)→ inhibits metabolism and growth of anaerobic microbes; treatment of certain infections (ex. gas gangrene)

how can pressure be used to preserve food?

kills microbes while maintaining food quality and extending shelf life. High pressure between 100 and 800 MPa (sea level is ~0.1MPa) kills vegetative cells by denaturing proteins

how can pressure be used clinically in treatment of infections?

hyperbaric oxygen therapy is used; patient breathes pure oxygen at ~1-3 atmospheres (atm)

inhibits the growth of oxygen-sensitive or anaerobic bacteria

what are forms of desiccation?

simple (drying)→ inhibits metabolism; dried fruits, jerky

reduce water activity (addition of salt or water)→ inhibits metabolism and can cause lysis; salted meats and fish, honey, jams and jellies

lyophilization (rapid freezing under vacuum)→ inhibits metabolism; preservation of food, laboratory cultures, or reagents

what does removing water (simple desiccation) do to bacteria?

slows/halts bacterial growth w/o killing microbes

lyophilization

involves freezing and applying vacuum so that water is lost by sublimation

combines both exposure to cold temperatures and desiccation

how can water activity/water content be lowered?

by adding solutes such as salts or sugars

at very high concentration of salts/sugars, the amount of water in microbial cells is reduced dramatically due to osmosis

ionizing radiation (sterilization)

x-rays, gamma rays, and high E e- beams

penetrates cells, directly damages biological molecules

causes DNA mutations, leading to cell death

x-rays and gamma rays penetrate paper and plastic to sterilize packaged materials

nonionizing radiation

ultraviolet (UV) light

less energetic, less penetrating. Used for surface disinfection

causes thymine dimers to form between adjacent thymines in DNA

disinfection of surfaces in laboratories and rooms in healthcare environment, and disinfection of water and air

filtration

physical separation of microbes from air or liquid

uses filters with pores of specific sizes

ideal when liquids contain heat-sensitive components

ex. High efficiency particulate air (HEPA) filters, membrane filters

High efficiency particulate air (HEPA) filters

pores ~0.3 µm filter out bacteria, endospores, and many viruses

efficiency is 99.97% for particles of 0.3 µm diameter or morem

membrane filters

porous membranes with defined pore sizes

microbes removed by physical screening

-cellular microbes ≥ 0.2 µm

-viruses ≥ 0.1 µm

face masks

surgical and K95 respirators reduce outward particle emission rate by 90% and 74%

for coughing, which produced the highest rates of particle emission for all expiratory activities tested, wearing homemade masks considerably reduced the fraction of large particles (> 0.8 µm)

chemicals that can be used for disinfection

phenolics—> denature proteins, disrupt membranes

metals→ bind to proteins and inhibit enzyme activity

halogens→ oxidation and destabilization of cellular macromolecules

alcohols→ denature proteins, disrupt membranes

surfactants→ lowers surface tension of water to help with washing away of microbes, and disruption of cell membranes

bisbiguanides→ disruption of cell membranes

alkylating agents→ inactivation of enzymes and nucleic acid

peroxygens→ oxidation and destabilization of cellular macromolecules

supercritical gases→ penetrates cells, forms carbonic acid, lowers intracellular pH

chemical food preservatives→ decrease pH, inhibit enzymatic function

natural food preservatives→ inhibition of cell wall synthesis

Chemical control of microbial growth: how do phenolics inhibit microbes?

denature proteins, disrupt membranes

Chemical control of microbial growth: how do metals inhibit microbes?

bind to proteins and inhibit enzyme activity

Chemical control of microbial growth: how do halogens inhibit microbes?

oxidation and destabilization of cellular macromolecules

Chemical control of microbial growth: how do alcohols inhibit microbes?

denature proteins and disrupt membranes

Chemical control of microbial growth: how do surfactants inhibit microbes?

lowers surface tension of water to help with washing away of microbes, and disruption of cell membranes

Chemical control of microbial growth: how do bisbiguanides inhibit microbes?

disruption of cell membranes

Chemical control of microbial growth: how do alkylating agents inhibit microbes?

inactivation of enzymes and nucleic acid

Chemical control of microbial growth: how do peroxygens inhibit microbes?

oxidation and destabilization of cellular macromolecules

Chemical control of microbial growth: how do supercritical gases inhibit microbes?

penetrates cells, forms carbonic acid, lowers intracellular pH

Chemical control of microbial growth: how do chemical food preservatives inhibit microbes?

decrease pH and inhibit enzymatic function

Chemical control of microbial growth: how do natural food preservatives inhibit microbes?

inhibition of cell wall synthesis (Nisin)

disk diffusion method

assesses the effectiveness of chemical microbial agents

filter disks containing chemical placed on an agar plate inoculated w bacterium

compound diffuses and causes zones of inhibition of microbial growth

size of zone correlates w/ potency of compound

in-use test

determines whether an actively used solution of disinfectant in a clinical setting is microbially contaminated

-add an amount of disinfectant to a sterile broth and plate it→ if there are more than 5 colonies growing on either plate that incubate @ 37ºC for 3 days or @ room temp for 7 days, the disinfectant solution is contaminated

Koch’s postulates

link infectious agent with a disease

-causative microbe must be present in diseased organisms, but not healthy organisms

-must be able to isolate the causative organism in pure culture

-must be able to infect healthy organism with the isolated culture

-must be able to re-isolate microbe from experimentally infected organism

do Koch’s postulates hold true for all infectious diseases?

no

-some pathogens are part of the normal microbiome

-some diseases involve multiple agents, or can be caused by different microbes

-some infections have varied symptoms (pleiotropic)

-some infectious agents can’t be cultured

pathogens cause…

infectious disease in host organisms

host is a source of nutrients

primary pathogens

almost always cause disease

ex. some Salmonella enterica strains; enterohemorrhagic E. coli

opportunistic pathogen

common

normally exist outside of the host

cause infection under the right circumstances age, weakened immune system, injury

ex. Staphylococcus epidermis

obligate pathogens

cannot exist outside of host in the natural environment (perhaps can be cultured in lab)

ex. Chlamydia, Rickettsia, Mycobacteria

how do pathogens often exist as?

in a reservoir; natural population outside of the host

-ex. drinking water supply, soil, etc.; animal population—> zoonoses; organisms that spread the infection—> vectors

zoonoses

pathogens existing in an animal population

ex. rabies and influenza

vectors

organisms that spread pathogens

-arthropods like mosquitos carrying malaria

extracellular pathogens

exist on or in host fluids and tissues, but do not enter host cells

may move thru circulatory system or migrate through the matrix between host cells

can directly encounter elements of the immune system

ex. E. coli, Staphylococcus aureus, Helicobacter pylori, Borella burgdorferi

intracellular pathogen

microbes enter and multiply within host cells

allows them to evade many elements of the host immune system

ex. Listeria monocytogenes, Mycobacterium tuberculosis, Salmonella enterica, Legionella pneumophila

what is the characteristic pattern of infection progression?

incubation period, prodromal stage, period of illness, period of decline, convalescence

infection progression: incubation period

pathogen entry, before symptoms

infection progression: prodromal stage

first onset of symptoms

infection progression: period of illness

disease is most severe, symptoms apparent

infection progression; period of decline

body fights off infection