MicroBiology Uiowa Exam 2

Exam 2 material

DNA Recombination

A donor bacterial cell transfers its DNA to a recipient cell

Plasmids

Non-essential DNA

Conjugation

Bacteria must have a direct connection (pilus) to transfer genetic material

Resistance (R) Plasmids

Contain genes that can be used to resist antibiotics

Transformation

A cell nonspecifically accepts small fragments of soluble DNA

Transduction

Viruses pick up DNA from donor cells during viral assembly and release it during viral entry of the recipient cell

Transposons

Can help move DNA within an organism

Mutations

Random changes to the nucleotide sequence

Spontaneous Mutation

Change in the DNA arising from errors in replication

Occurs randomly

Induced Mutations

Mutations that result from exposure to known mutagens that disrupt DNA

Point Mutations

Addition, deletion, or substitution of single bases

Missense mutation

Any change that leads to the placement of a different amino acid

Nonsense Mutation

Changes a codon into a stop codon which will stop the production of the protein early

Silent Mutation

Alters a nucleotide but does not change the amino acid formed

Frameshift Mutation

An insertion or deletion of one or more base pairs which shifts the reading frame of the mRNA

Genetic Engineering

Altering genes to introduce new traits

Goal is to alleviate human disease

Restriction Endonucleases

Used to cut DNA at sites with a particular nucleotide sequences

Comes from bacteria that used it as a defense mechanism

Polymerase Chain Reaction (PCR)

Works by making copies of genes present in a sample

So sensitive that you don’t need to culture microbes to detect them

Pulsed Field Gel Electrophoresis

Restriction endonucleases cut the whole genome at certain sites

Each organism will have different lengths of DNA pieces

Organisms with similar banding patterns are from the same source

DNA Sequencing

DNA genome is broken into smaller fragments

Nucleotide sequence is determined

Recombinant DNA Technology

By adding foreign DNA we can make cells do foreign things

Gene Therapy

Goal is to replace defective genes with working copies

Gene Addition

Non-mutated genes are added to the cell with a mutated genome

Gene Editing

The mutated gene is corrected in place

CRISPR can direct a cut site in the DNA

Gene Delivery

Done with viruses, nanoparticles, or membrane bound vesicles

Viruses are used to get to hard-to-reach areas but only for specific cell types

Clinical Microbiology

Used to determine if there is a pathogen present

Correctly identify which pathogen is responsible

Characterize the pathogen

Specimen collection

One of the most important steps towards identification

Phenotypic

Observe cell morphology and bacterial physiology or biochemistry

Genotypic

Identify microbes through genetic techniques

Some microbes cannot be cultured

Immunologic

Serological analysis to identify microbes

Microscopic

Cell shape size and arrangement, gram stain reaction, acid fast reaction

Macroscopic

Colony color, size, texture, and shape

Physiological/biochemical

Enzymes produced, antimicrobial sensitivity, growth in selective or differential media

Catalase Test

This test uses hydrogen peroxide (H2O2) to test for an enzyme that produces Oxygen gas

Hybridization

Genotypic identification

Uses DNA or RNA probes

If probes bind the genome it confirms the identity

FISH

Fluorescent in situ Hybridization

Positive fluorescence indicates hybridization

Serological Testing

Testing using Patient’s antibodies and microbial antigens

Can also identify previous infections

Latex Agglutination

Positive result → Clumping of colonies

Negative result → no clumping typical colony formtion

Immunofluorescence

Tests for antigen

Patient’s sample is mixed with an antibody labeled with a fluorescent tag

Western blots

Identifies proteins using antibodies

ELISA

Enzyme linked immunosorbent assay

Indirect → Tests for antibodies

Direct → Tests for antigen

Microbial Control

The process of killing, removing, or impeding growth of microorganisms

Sterilization

The process by which all living cells, spores, viruses, and prions are destroyed or removed from an object or habitat

Disinfection

The killing, inhibition, or removal of microorganisms that may cause disease

Sanitization

The reduction of microbial population to levels that are considered safe by public health standards

Antisepsis

The destruction or inhibition of microorganisms on living tissue

Prevention of infection or sepsis

-cide

Indicates a method that kills a microorganism

-static

describes a method that inhibits the growth of a microorganism

Mechanical Barries

Does not kill microorganism but instead removes them through filtration

can be used on liquids and gases

HEPA Filters

High efficiency particulate air may remove particles smaller than .1 um

Will sterilize air

N95 masks

Remove 95% of particles larger than .3 um

Heat

Heat can be used to destroy cells and viruses by degrading nucleic acids, denaturing proteins, and disrupting cell membranes

Boiling

10 minutes of water boiling will destroy all vegetative cells and viruses but endospores can live on for hours

Pasteurization

Heat sensitive substances are briefly subjected to temperates below boiling, only reduces the number of microbes that contribute to spoilage

Autoclave

Steam under pressure

Can destroy endospores

Gold Standard of Sterilization

Dry Heat

Less effective than moist heat

Higher temperatures and longer exposures are needed

Dry oven sterilizes 

Incinerator

Commonly used to get ride of biological waste

Reduces microbes to ash

Ionizing Radiation

Dislodges electrons from atoms or molecules, producing chemically reactive free radicals which destroy nearby matter

Uses gamma and beta radiation 

Non-Ionizing Radiation

UV radiation

260 nm waves cause thymine-thymine dimerization of DNA, preventing replication and transcription

Chemical

Can be used for sterilization, disinfection, and antisepsis

Ideal to find a balance to kill a variety of microbes and also be non toxic to people and materials

Phenols

Denature proteins and disrupt cell membranes of microbes

Alcohols

Denature proteins and may dissolve membrane lipids

Widely used for disinfecting, sterilizing, and antisepsis

Iodine

Often used for antiseptics

works by oxidizing cell constituents and iodinating proteins

Chlorine

disinfectant for water and food

Works by oxidizing cellular materials

Heavy Metals

Historically, mercury, silver, arsenic, zinc, and copper were all used as germicides

Very toxic, but modern versions have been developed are less toxic

Detergents

Organic cleansing agents that are amphipathic

They disrupt microbial membranes and denature proteins

often used as disinfectants

Aldehydes

Inactivate nucleic acids and proteins by cross linking molecules

Sterilizing gases

Used to sterilize heat sensitive items such as petri dishes and sutures

Vaporized Hydrogen peroxide

Commonly used to decontaminate facilities

Harmless because it breaks down into water and oxygen

Microbial Death Curves

Microbes are not killed instantly

Some microbes may still remain

to test if an organism is dead it must not grow when inoculated

Population size

More microbes means it will take longer for all microbes to die

Population Composition

Microbes differ in their susceptibility to each method

Concentration or intensity

Can change how quickly microbes are killed

Contact time

Longer exposure means more microbes killed

Temperature

Increases in temperature often increase the activity of an antimicrobial

local environment

Environmental factors surrounding microbes may offer protection or aid in destruction

Phenol Coefficient Test

Test your measure alongside phenol and compare

In Use Tests

Test your measure in the environment it will be sued then determine the presence of viable microbes

Kirby-Bauer

Bacteria of Interest are spread on plate

Discs with antimicrobial drugs are placed on top

Incubate and then measure the zone of inhibition around the discs

E-Tests

Bacteria of Interest are spread on plate

Strip of plastic with a gradient of antimicrobial concentrations is placed on top

After 48 hours of incubation look for susceptibility

Minimum Inhibitory Concentration

(MIC) The smallest concentration of drug that visibly inhibits growth

E-Test

Therapeutic Index

Compares the toxic dose to the minimum effective dose

Drug Failure

Drug did not reach affected area

Resistant microbes were missed during testing

more than one pathogen responsible for the disease

Patient did not take antimicrobials as prescribed

Drug Interaction with Microbes

Attacks specific structures and pathways specific to microbial cells

Goal is to disrupt the structure or function to the point where the microbe can no longer survive

Drug Action Mechanisms

1. Inhibit cell wall synthesis

2. Inhibit nucleic acid structure and function

3. Inhibit protein synthesis

4. Interfere with cytoplasmic membrane

5. Inhibit folic acid synthesis

B-Lactam Antibodies

Block the last stage of cross linking peptides in peptidoglycan synthesis

More effective on Gram Positive Bacteria

Vancomycin

Important for role in treating methicillin-resistant Staphylococcus aureus (MRSA)

Quinolone Antibiotics

Inhibit DNA transcription and replication by inhibiting microbe specific enzymes

Tetracycline Clindamycin

Block successful translation at the ribosome/mRNA level

Polymyxin Daptomycin

Interact with phopholipids causing distortions and leakage

More effective on Gram-Negative Bacteria

Folic Acid

An essential vitamin required for DNA synthesis and cell replications

Bacteria synthesize folic acid

Humans acquire it in diet

Sulfonamides

Inhibit enzymes required for folic acid synthesis

Fungal Treatments

Similarities between fungal and human cells makes treatment toxic to humans

Helminth Infections

Most difficult to treat because they are most similar to humans

Blocking reproduction doesn’t eliminate adult worms

Goal is to destroy all life cycle stages

Viral Infections

Viruses use our cellular pathways to replicate so treatments often include inhibiting host cell pathways

Vaccines are the best defense against viral infections

Prevent Viral entry, production of viral proteins/genomes replication, virion maturation

Biofilm Considerations

Antimicrobials may not be able to penetrate the sticky layers of biofilms

Longer treatment periods, mechanical disruption or removal of biofilm and adding additional antimicrobials may help

Antimicrobial Resistance

1. Synthesis of new enzymes which inactivate the drug

2. Microbial cell des not let drug in

3. Drug is eliminated

4. Binding sites for drug are decreased

5. Microbe uses an alternate pathway

Beta-lactamases

Degrade beta-lactam Antibiotics

MecA

Antibacterial resistance gene used by MRSA

Penicillin attacks peptidoglycan by binding to its target, PBP

MecA allows the bacteria to make another version of the target protein, called PBP2a which pencillian cannot bind to

Toxicity to Organs

The drug damages human organs