Microbiology test 3 - Dr. Collins

Viruses

Major cause of disease

Important members of aquatic world (move organic matter from particulate to dissolved)

Important in genetic variation (transfer genes between bacteria)

What do viruses need to survive?

A living host

What is a virion?

A complete virus particle

Consists of ≥1 molecule of DNA or RNA enclosed in coat of protein

May have additional layers

Cannot reproduce independent of living cells nor carry out cell division

How are virions classified into families?

Based on genome structure, life cycle, morphology, genetic relatedness

What type are most viruses?

Eukaryotic viruses (plants, animals, protists, and fungi), but some are bacterial viruses (“bacteriophages”) and archaeal viruses

What is a nucleocapsid?

Nucleic acid (DNA or RNA) and a protein coat (capsid)

Do all viruses have a nucleocapsid?

Yes

Some are ONLY a nucleocapsid, others have additional components as well

Can you use a normal light microscope to see viruses?

No, you need an electron microscope

What do you call virons with/without envelopes?

Virions having envelopes = enveloped viruses

Virions lacking envelopes = naked viruses

What are capsids?

Large macromolecular structures which serve as protein coat of virus

Made of protein subunits called protomers (capsomers)

Have either a helical, isocahedral, or complex shape

What do capsids do?

Protect viral genetic material and aid in its transfer between host cells

Helical capsids

Shaped like hollow tubes with protein walls

Protomers self assemble

Size of capsid is a based on nucleic acid

What shape is ebola?

It’s like a lil hook lookin thing

Icosahedral capsids

An icosahedron is a regular prolyhedron with 20 equilateral faces and 12 vertices

What are “complex” viruses?

Ones that don’t fit into the category of having helical or icosahedral capsids

Examples:

-poxviruses (largest animal virus)

-large bacteriophages (binal symmetry [head resembles icosahedral, tail is helical])

What is an envelope?

An outer, flexible, membranous layer

Animal virus envelopes (lipids and carbs) usually arise from host cell plasma or nuclear membranes

Viral envelope proteins

May project from the envelope surface as lil spikes

What do viral envelope proteins do?

Involved in viral attachment to host cell (like hemagglutin of influenza virus)

Used for identification of virus

May have enzymatic or other activity (neuraminidase of influenza virus)

May play a roll in nucleic acid replication

Viral genome

A virus may have a single or double stranded DNA or RNA
The size of the nucleic acid also varies from virus to virus

Genomes can be segmented or circular

How does viral multiplication work?

mechanism used depends on viral structure and genome, but the steps are similar:

-Attachment to host cell

-Entry

-Uncoating of genome

-Synthesis

-Assembly

-Release

attachment step of viral multiplication

Attachment is via specific receptors

Receptor determines host preference

-May be specific tissue

-May be more than one host

-May be more than one receptor

-May be in lipid rafts providing entry of virus

Viral entry and uncoating step of viral multiplication

Entire genome or nucleocapsid enters based on naked vs enveloped virus

3 methods used:

-Fusion of the viral envelope with host membrane; nucleocapsid enters

-Endocytosis in vesicle; endosome aids in viral uncoating

-Injection of nucleic acid

Synthesis step of viral multiplication

Genome dictates the events

“ds DNA typical flow”?????

RNA viruses - virus must carry in or synthesis the proteins necessary to complete synthesis

Assembly step of viral multiplication

Assembly is complicated but varies:

-bacteriophages = in stages

-Some are assembled in nucleus

-Some are assembled in cytoplasm

-May be seen as paracrystalline structures in cell

Virion release step of viral multiplication

Nonenveloped viruses lyse (eat) the host cell

-viral proteins may attack peptidoglycan or membrane

Enveloped viruses use budding

-Viral proteins are first incorporated into host membrane

-Nucleocapsid may bind to viral proteins

-Envelop derived from host cell membrane, but may be Golgi, ER, or other

-Virus may use host actin tails to propel through host membrane

Types of viral infections

Infections in bacteria and archaea

Infections in eukaryotic cells

Viruses and cancer

Bacterial and archaeal viral infections

Virulent pahge - one reproductive choice (multiplies immediately upon entry, lyses bacterial host cell)

Temperate phages have two two reproductive options

-Reproduce lytically as virulent phages do

-Remain within host cell without destroying it (many temperate phages integrate their genome into host genome in a relationship called lysogeny)

What is lysogeny

prophage (bacteriophage)

-integrated phage genome

Lysogens (lysogenic bacteria)

-infected bacterial host (appear normal, and can switch from lysogenic to lytic cycle)

Lysogenic vs lytic cycle

Lytic:

-Phage DNA directs the synthesis of many new phages

-Cell lyses and releases the new phages

-New phages can bind to bacterial cells

-Phage injects its dna into cytoplasm

Lysogenic:

-Phage DNA integrates into host chromosome

-Prophage DNA is copied when cell divides

-Exposure to stress such as UV light triggers excision from host chromosome

Lysogenic conversion

-Temperate phage changes phenotype of it’s host (bacteria become immune to superinfection, and phage may express pathogenic toxin or enzyme)

-Advantage to lysogeny for virus (phage remains viable but may not replicate)

-Under appropriate conditions they will lyse and release phage particles (occurs when conditions in the cell cause the prophage to initiate synthesis of new phage particles, a process called “Induction”)

Infection in eukaryotic cells

Cytocidal (WHAT DOES THIS MEAN???) infection results in cell death through lysis

Persistent infections may last years

Cytopahtic effects (CPE’s) - Degenerative changes and abnormalities

Transformation to malignant cell

What is a tumor?

A growth or lump of tissue

Benign tumors remain in place

What is neoplasia?

Abnormal new cell growth and reproduction due to loss of regulation

What is anaplasia?

Reversion to a more primitive or less differentiated state

What is metastasis?

Spread of cancerous cells throughout body

What is carcinogenesis?

A complex, multistep process that often involves oncogenes (cancer causing genes, may come from the virus, may be transformed host proto-oncogenes which are involved in normal regulation of cell growth and differentiation)

Possible mechanisms by which viruses cause cancer

-Altered cell regulation (viral proteins bind host cell tumor supressor proteins)

-Carry oncogene into cell and insert it into host genome

-Insertion of promoter or enhancer next to cellular proto-oncogene

Human papillomavirus (HPV)

Virus belongs to papillomavirus family

Nonevneloped icosehedral capsids

Causes over 120 known viruses (inc. AIDS)

How does HPV infect?

Infects keratinocytes of skin or mucous membrane (most common STD)

Causes warts (HPV type 1, 2, 4, or 63)

Causes cancers (most common is cervical cancer) (caused by types 16, 18, 31, and 45) (it does this by degrading p53, a tumor suppressor protein)

Nonsexual transmission of HPV

Virus attacks through direct contact

•enters through tiny cuts or abrasions (like at the gym! ew.)

Autoinnoculation

•Infecting nearby skin or by infected walked surfaces

Prenatal

•mother to child during birth

•causes warts in the larynx (very bad, child cant breathe)

Sexual transmission of HPV

Genital infections caused by sexual activity

Can be stopped by condoms or like….no sex .-.

Prevention of HPV

Vaccination - gardisil and cervavix (both protect against types 16 and 18, gardisil also protects against types 6 and 11)

Microbicides - topical creams that are applied to skin before sexual contact

HIV

Caused by HPV

Originated in western africa in early twentieth century

First recognized by CDC in 1981

Responsible for 30 million deaths

AIDS

Caused by HIV (RNA virus family retroviridae)

occurs worldwide, causing the great pandemic in second half twentieth century

HIV transmission

CAN get from:

-Infected blood, semen, or vaginal secretions coming into contact w/ person’s broken skin or mucous membranes

-mother to child

-contaminated sharps/needles

CANT get from:

Urine

coughing

sharing utensils

tears, saliva, sweat

casual contact

HIV life cycle

Attachment (attaches to CD4 cells - T helper cells and other cells)

Entry

RNA transformed into DNA via reverse transcriptase

Integration into human genome as provirus

Can remain latent - asymptomatic

Can direct synthesis of viral RNA → synthesis of new viral particles (new virion are assembled and released through budding and eventual lysis)

Course of disease (HIV)

-Some patients rapidly develop clinical AIDS, and die within 2-3 years

-Some patients remain relatively healthy for at least 10 years post infection

-In majority of patients HIV infection progresses to AIDS in 8-10 years

-T helper cell count reduces and opportunistic infections begin

AIDS

Acquired immune deficiency syndrome

When the immune system is no longer able to defend against the HIV virus

You need to have fewer than 200 CD4 T cells/microliter of blood OR a CD4 cell percentage of lymphocytes of less than 14 to be diagnosed with AIDS

Therefore, you can get rid of an AIDS diagnosis

How to diagnose AIDS

Detect reverse transcriptase activity or viral antigens

Detect anti-HIV antibodies in the blood (will only have these if they have HIV)

PCR - genome analysis (amplify a region specific to HIV genome)

Treatment for AIDS

No treatment :)

The only thing you can do is attempt to reduce viral load, disease symptoms, and treating disease and malignancies

Most successful treatment involves a combination of drugs

Prevention of AIDS

Achieved primarily through education

Barrier protection from blood and body fluids

Not sharing intravenous needs or syringes

Continued screening of blood and blood products

Vaccine for AIDS

Non existant yet, but there is ongoing research

Ideal vaccine would:

-Stimulate the production of neutralizing antibodies which would bind to virus preventing it from entering host cells

-Promote formation of cytotoxic T cells capable of destroying cells infected with virus

Problems with coming up with vaccine for AIDS

Envelope proteins of virus continually change their antigenic properties

Sterilization

Destruction or removal of all viable organisms

Disinfection

Killing, inhibition, or removal of disease causing (pathogenic) organisms

Disinfectants

Agents, usually chemical, used for disinfection, usually used on inanimate objects

Sanitization

Reduction of microbial population to levels deemed safe (based on public health standards)

Antisepsis

Prevention of infection of living tissue by microorganisms

Antiseptics

Chemical agents that kill or inhibit growth of microorganisms when applied to tissue

Chemotherapy

Use of chemicals to kill or inhibit growth of microorganisms within host tissue

Cidal vs static agents

Cidal agents kill

Static agents inhibit growth

Cidal agents

-cide (suffix indicating that agent kills)

Germicide - kills pathogens and many nonpathogens but not necessarily endospores

Includes bactericidies, fungicides, algicides, and viricides

Static agents

-static (suffix indicating that agent inhibits growth)

includes bacteriostatic and fungistatic

Conditions influencing the effectiveness of antimicrobial agent activity

Population size (larger populations take longer to kill than smaller populations)

Population composition (microorganisms differ markedly in their sensitivity to antimicrobial agents)

Concentration or intensity of an antimicrobial agent (usually higher concentrations or intensities kill more rapidly. Relationship is not linear)

Duration of exposure (longer exposure = more organisms killed)

Temperature (higher temps usually increase amount of killing)

Local environment (many factors [pH, viscosity, and concentration] can profoundly impact effectiveness. Organisms in biofilms are physiologically altered and less susceptible to many antimicrobial agents

What are the physical control methods?

Heat, Filtration, and radiation

Moist heat

Destroys viruses, fungi, and bacteria

Boiling will not destroy spores and does not sterilize

Degrades nucleic acids, denatures proteins, and disrupts membranes

Steam sterilization

Must be carried out above 100°C which requires saturated steam under pressure

Carried out using an autoclave

Effective against all types of microorganisms including spores

Pasteurization

Controlled heating at temperatures well below boiling

Used for milk, beer, and other beverages

Process does not sterilize but does kill pathogens present and slows spoilage by reducing the total load of organisms present

Dry heat sterilization

Less effective than moist heat sterilization, requiring higher temps and longer exposure times

Items subjected to 160-170°C for 2-3 hours

Oxidizes cell constituents and denatures proteins

Dry heat incineration

Bench top incinerators are used to sterilize inoculating loops used in microbiology laboratories

Filtration

Reduces microbial population or sterilizes solutions of heat-sensitive materials by removing microorganisms

Also used to reduce microbial populations in air

Membrane filters - porous membranes with defined pore sizes that remove microorganisms primarily by physical screning

Filtering air

Surgical masks (not all are equal tho)

Cotton plugs on culture vessels

High-efficiency particulate air (HEPA) filters - used in laminar flow biological safety cabinets

UV radiation

Wavelength of 260 is the most bactericidal (DNA absorbs)

Causes thymine dimers preventing replication and transcription

UV limited to surface sterilization because it does not penetrate glass, dirt, films, water, and other substances

Has been used for water treatment

Ionizing radiation

Gamma radiation penetrates deep into objects

Destroys bacterial endospores

Used for sterilization and pasteurization of antibiotics, hormones, sutures, plastic disposable supplies, and food

Chemical agents

Disinfectant must be effective against wide variety of infectious agents at low concentrations

Must be effective in the presence of organic matter; should be stable in storage

Overuse of antiseptics such as triclosan has been selected for triclosan resistant bacteria and possibly antibiotic resistant (this is bad)

Phenolics

Commonly used as laboratory and hospital disinfectants

Act by denaturing proteins and disrupting cell membranes

Tuberculocidal, effective in presence of organic material, and long lasting

Disagreeable odor and can cause skin irritation

Triclosan is used in hand sanitizers

Alcohols

Among the most widely used disinfectants and antiseptics

Two most common are ethanol and isopropanol

Bactericidal, fungicidal, but not sporicidal

Inactivate some viruses

Denature proteins and possibly dissolve membrane lipids

Halogens

Any of five elements: fluorine, chlorine, bromine, iodine, and astatine

Important antimicrobial agents

Iodine (halogen)

Skin antiseptic

Oxidizes cell constituents and iodinates proteins

At high concentrations may kill spores

Skin damage, staining, and allergies can be a problem

Iodophore - iodine complexed with organic carrier. Released slowly to minimize skin burns

Chlorine (halogen)

Oxidizes cell constituents

Important in disinfection of water supplies and swimming pools, used in dairy and food industries, effective household disinfectant

Destroys vegetative bacteria and fungi

Chlorine gas is sporicidal

Can react with organic matter to form carcinogenic compounds

Heavy metals

Eg. Ions of mercury, silver, arsenic, zinc, and copper

Effective but usually toxic

Combine with and inactivate proteins; may also precipitate proteins

Quarternary ammonium compounds

Detergents that have antimicrobial activity and are effective disinfectants

(amphipathic organic cleansing agents. act as wetting agents and emulsifiers)

Cationic detergents are effective disinfectants (kill most bacteria, but not mycobacterium tuberculosis, or endospores) (safe and easy to use but inactivated by hard water)

Aldehydes

Commonly used agents are formaldehyde and glutaraldehyde

Highly reactive molecules

Sporicidal and can be used as chemical sterilants

Combine with and inactivate nucleic acids and proteins

Sterilizing gases

Used to sterilize heat-sensitive materials

MIcrobicidal and sporicidal

Ethylene oxide sterilization is carried out in equipment resembling an autoclave

Betapropiolactone (BPL) and vaporized hydrogen peroxide - BPL = blood products and vaccines, H2O2 = operating rooms

Combine with and inactivate DNA and proteins

Action of antimicrobial drugs

Inhibiting cell wall synthesis

Inhibiting protein synthesis

Injuring plasma membrane

Inhibiting nucleic acid synthesis

Inhibiting the synthesis of essential metabolites

Penicillins

Inhibit cell wall synthesis by preventing the crosslinking of peptidoglycan during the synthesis (primarily of gram+ bacteria, and in actively growing cells) - it makes them pop like in the videos :)

Include a beta-lactam ring (nucleus) and a side chain

Natural penicillins

Extracted from culture of penicillium

-Penicillin G

•Narrow spectrum of activity

•best drug against staphylococci, streptococci, and several species of spirochetes

•acidity of stomach diminishes its concentration

-Penicillin V

•Stable in stomach

Disadvantages to natural penicillins

Narrow spectrum and susceptibility to penicillinases (beta-lactamases)

Semisynthetic penicillins

Created to overcome disadvantages of natural penicillins

What are the two ways semisynthetic penicillins are created?

-Interrupt synthesis of molecule by Penicillium and only obtain nucleus

-Remove side chain from natural molecule and add new side chain to make it more resistant

Extended-spectrum penicillins

Effective against many gram- and gram+

Ampicillin and amoxicillin

Carbenicillin and ticarcillin (greater activity against g- like P. aeruginosa)

Penicillin resistance

Resistance to Staphylococcal infections

-Plasmid with gene encoding for beta-lactamase

Introduction of methicillin

-MRSA

Combat Penicillinase

Penicillins plus beta-lactamase inhibitors (potassium clavulanate, a product of streptomycete that has no antimicrobial activity of it’s own)

Augmentin

-Amoxicillin and beta-lactamase inhibitor

Inhibitors of cell wall synthesis besides penicillin

Other beta-lactam antibiotics:

-Carbapenems (primaxin, active against 98% of all organisms isolated from hospitals, and CRE [carbapenems-resistant enterobacteriacae])

-Cephalosporins

Polypeptide anitbiotics:

-Bacitracin (effective against mainly g+, and it’s topical)

-Vanomycin (used to address problem with MRSA[which is a big problem tbh, the thing’s resistant to everything lol])

Antibiotics that mess with protein synthesis

Idk bruh, something about 80S and 70S?????

But chloramphenicol, erthromycin, streptomycin, and tetracycline all fit in here or something

How does streptomycin stop protein synthesis? EXTRA CREDIT QUESTION

Changes shape of 30S portion, causing code on mRNA to be read incorrectly

How does chloramphenicol inhibit protein synthesis? EXTRA CREDIT QUESTION

Binds to 50S portion and inhibits formation of peptide bond

How does tetracyclines inhibit protein synthesis? EXTRA CREDIT QUESTION

Interferes with attachment of tRNA to mRNA-ribosome complex

What antibiotics cause injury to plasma membrane?

Fatty acid synthesis attractive target for new antibiotics (biosynthesis distinctly different in bacteria than in humans)

Weakness - some bacterial pathogens can take up fatty acids from serum

Polymyxin B - bactericidal against g- (topical treatment. neosporin = polymyxin B + bacitracin + neomycin)

How did bacitracin get it’s name?

Baci - from “bacilus subtilus”

Tracin - from the patient “tracy”