Microbiology 3 Flashcards
Describe the viral replication cycle
- Virus binds to cell via receptor, enters cell (e.g. via endosome)
- Initiates reverse transcription
- cDNA produced, inserted into chromosome
- cDNA produces RNA strands via transcription of host chromosome
- RNA exits nucleus to produce viral proteins
- Assembled and released as new virus
What stages of the viral replication cycle can be targeted by drugs?
- Every stage
- Entry
- Uncoating
- Replication
- Release
- Immune Response
Give the 3 classes of antiviral therapy
- Drugs which target viral proteins (safe) e.g. inhibit viral replicative enzyme
- Drugs which target host proteins e.g. block receptors
- Drugs which enhance host defences e.g. interferons
What is meant by antiviral selectivity?
The difference between the dose of the drug which inhibits viral protein over any similar cellular protein
Which class of antiviral has the highest specificity?
Drugs targeting viral proteins that have no host analogues, may have absolute specificity. very safe
What are the targets to inhibit attachment and entry be inhibited?
- Viral proteins
- Host cell receptors
Give an example of a antiviral that inhibits viral uncoating
- Influenza A: hydrogen ions cause HA to fuse with membrane, and ions enter virion via M2 channels stimulating release of the nucleoprotein from the virus core
- Amantidine blocks M2, preventing influx of H+ and thus inhibiting release of nucleoproteins
How may viral genome replication be inhibited by antiviral drugs?
- Inhibition of viral polymerase (e.g. herpesvirus DNA polymerase, retrovirus reverse transcriptase)
- Nucleotide analogues
- Antiretroviral drugs
- Integrase inhibitors (HIV)
Explain how nucleotide analogies can inhibit genome replication
- Typically need to be activated by cellular or viral enzymes
- Nucleotide analogue incorporation prevents elongation of DNA and no functioning DNA produced
- e.g. acyclovir (herpesviridae), zidovudine (HIV)
Explain how antiretroviral drugs inhibit genome replication
- Non-nucleotide reverse transcriptase (RT) inhibitors e.g. nevirapine
- Bind near catalytic site of RT
- highly specific
Explain the mechanism of action of integrase inhibitors for HIV
- Bind to viral integrase to prevent viral cDNA from being inserted into the host genome
Describe the mechanism of action of protease inhibitors
- Inhibit virus assembly
- If viral proteins are produced as polyproteins and require cleavage this is a good drug target
- Immature (uncleaved) viruses are inactive
- e.g. HIV, hepatitis C in man
Describe the mechanism of action of antivirals that inhibit viral release
- e.g. Neuraminidase inhibitors
- Prevent release of influenza virus from cells
- Haemaglutinin (binds to sialic acids) important for getting virus into the cell
- Neuraminidase destroys sialic acids on cell allow release of virus
- Neuraminidase inhibitors prevent this destruction and so the virus cannot escape
Outline the use of antivirals in veterinary medicine
- Generally not done in farm/food production animals, often have secondary bacterial infections, expensive, MRLs a concern
- Few antivirals licensed in veterinary species, vaccines are main method
- Biosecurity important for production animals
- Treatment typically requires supportive therapy
Outline antiviral therapy in cats
- Feline interferon omega, nucleotide analogues, human interferon alpha used to treat FeLV and FIV
- Lymphocyte T cell immunomodulator for FeLV (USDA approved but not licensed in UK
- Feline interferon omega for FIP
- trifluorothymidine can be used to treat FIP, also acyclovir ointment, oral acyclovir, oral famicyclovir, L-lysine but these have little proof
List some problems with antiviral therapy
- Drug resistance due to fast development of viral mutants
- More of a worry in RNA viruses due to inherently higher mutation rate
Outline the use of multidrug antiviral therapy
- Combining drugs improves antiviral effect
- But increases complexity and cost, reduces compliance
What are superficial mycoses?
Fungal infections of the skin, hair and nails, mucosal surfaces in upper respiratory tract
What are subcutaneous mycoses?
Fungal infections affecting muscle and connective tissue, below the skin
What are systemic (invasive) mycoses?
Fungal infections of internal organs, may be primary or opportunistic and often relate to immune suppression or other issues
What are some challenges of antifungal therapy?
- Low susceptibility/high resistance
- Time take for drugs to show effect
- Environmental fomites leading to reinfection or cross infection
- Specificity issues/side effects
- Lack of vaccine
List the main groups of antifungal agents
- Polyenes
- Allylamines
- Azoles
- Echinocandins
- Pyrimidine analogues
- Antimitotics
What is the mechanism of action of polyenes?
- Bind to and disrupt fungal membranes
- Preferentially bind to ergosterol
- Binding alters membrane osmotic regulation, leads to leakage of intracellular potassium and other small molecules
Describe the structure of polyenes
- Composed of hydrophilic polyhydroxyl chain on one side
- Lipophilic polyene hydrocarbon chain on the other
Name some examples of polyenes and their use
- Amphotericin B: systemic infections
- Nystatin: topical and oral (mucosa and GI superficial infections)
What is the main limitation of polyenes?
Toxicity
Describe the formulation of most polyenes
- Poorly soluble in water
- Most formulations are lipid based, reduces intrinsic toxicity of compound
What is the mechanism of action of azoles?
- Interferes with ergosterol synthesis (membrane sterols)
- Fungistatic
- Inhibit 14-alpha-demethylase which depletes ergosterol over time, causes accumulation of methyl-ergostadiene-diol
- Methyl-ergostadiene-diol is toxic
What are the 2 groups of azoles?
- Imidazoles (ketoconazole, clotrimazole)
- Triazoles (Fluconazole, intraconazole)
Describe the rate of action of azoles
Slow, require several generations to act
What fungi are affected by azoles?
- Dimorphic
- dermatophytes
- Aspergillus only susceptible to triazoles, not other azoles
Describe the mechanism of action of allylamines
- Inhibit squalene epoxidase to reduce ergosterol production
- Interfere with ergosterol synthesis (membrane sterols)
- Accumulation of toxis squalene
Describe the spectrum of activity of allylamines
- Broad
- Especially active against dermatophytes
Name some allylamines
Terbinafine, butenafine
Describe the mechanism of action of echinocandins
- Inhibit glycan synthesis
- Affect hyphae growing tips and branching points
- Affect yeasts so that buds fail to separate from mother cell
- Also lead to osmotically sensitive fungal cells
Name examples of echinocandins
- Capsofungin
- Micafungin
- Anidulafungin
Outline resistance to echinocandins
- Intrinsic resistance in some fungal genera/species where other glycans predominate
Describe the mechanism of action of antimitotic antifungals
- Binds to tubulin and interferes with microtubule function
- Thus inhibits mitosis
Name an antimitotic antifungal
Griseofulvin
Describe the administration and distribution of griseofulvin
- Oral dose only, topically inactive
- Drug reaches site of action only when hair or skin replaced by keratin-griseofulvin complex
What is a contraindication for the use of griseofulvin?
Teratogenic, so contraindicated in pregnant animals
Describe the mechanism of action of pyrimidine analogues
- Deaminated in fungal cells by cytochrome deaminase
- Produces fluoorodeoxyuridine monophosphate (FdUMP) and 5-fluorouridine tri-phosphate (FURTP) produced
- FURTP incorporates into fungal RNA in place of uridylic acid, leading to inhibition of protein synthesis
- FdUMP inhibits thymidylate synthetase leading to inhibition of DNA synthesis
Outline the administration of pyrimidine analogues
Parenteral administration, oral rare and only under specific ordering
Give an example of a pyrimidine analogue antifungal agent
Flucytosine
Describe the spectrum of activity of topically administrated azoles
Broad (dermatophytosis, candidiasis, Malassezia and aspergillosis)
Describe the spectrum of activity of orally or IV administrated azoles?
Broad
Which azoles are administered topically?
Clotrimazole (imidazole) and enilconazole
Which azoles are mainly administered orally or IV?
- (Can also be administered topically)
- Ketoconazole (imidazole), fluconazole (triazole), intraconazole (triazole)
Describe the spectrum of activity of flucytosine
- Narrow
- Yeasts, cryptococcossis, candidiasis
Give an example of synergistic antigfungals
Flucytosine and amphotericin B
Describe the spectrum of activity of allylamines
- Narrow
- Dermatophytosis, particularly for oncychomycosis in humans
Outline the use of antifungals in horses
- Dermatophytosis usually self-limiting
- Treatment rare due to cost (may be used to reduce spread, shorten infection time, where animals move a lot, in case of non-self limiting disease)
- Azole wash e.g. imaverol
- Griseofulvin oral treatment
What are the most important control methods for fungal infections of horses?
Cleaning of fomites (tack and self)
What are the key side effects of griseofulvin?
Toxicity - liver damage and carcinogenicity
Outline the use of antifungals in cows
- Rare
- Ringworm common and usually self limiting
- Treat if: serious condition or show animal
- Treatment usually restricted to washes e.g. iodine washes
Outline the use of antifungals in pigs
Very rare
Ringworm very self-limiting, usually no treatment used
What factors may lead to antifungal therapeutic failure?
- Wrong diagnosis and use of wrong antifungal
- Net state of immunosuppression
- High burden of fungus at initiation of treatment
- Strain acquisition of increased virulence
- Pharmacokinetics and/or pharmacodynamics
- Site of infection
- Length of treatment and/or compliance
- Underlying disease
Give potential mechanisms for azole resistance
- Increased efflux
- Altered target
- Upregulation of target
- Development of bypass pathways
Explain why the upregulation of a target would lead to antifungal resistance
More drug required to inhibit the target
Compare the acquisition of resistance in fungi and bacteria
Slower in fungi due to slower growth rate and growth dynamics
What is a potential mechanism for polyene resistance?
Alteration in balance of sterol types so the impact of the drug is less (acts on ergosterol)
What are potential side effects of membrane disrupting antifungals?
- Neuropathy
- GI upset
- Renal damage
- Liver failure
- Rash
Why are fungal membranes good antifungal targets?
Sterols different in fungi and animals
- In fungi: ergosterol
- In animals: cholesterol
Describe the mechanism of action of glucan/chitin synthesis inhibitors
- Lipo-peptide based drug
- Inhibits 1,3-beta-glucan synthase by non-competitive inhibition
- Fungicidal and non-toxic
- Glucan depletion weakens the cell wall
Describe nikkomycins-
- New, chitin targeting
- Nucleoside-peptide antibiotics produced by Streptomyces spp, with antifungal activities
- Structural similarities to natural chitin synthase substrate, uridine diphosphate-N-acetylglucosamine
- Acts as competitive inhibitor
What factors may lead to non-compliance to treatment regimens?
- Regimen complexity
- Side effects
- Method/frequency of administration
- Other lifestyle factors
- Length of time regimen is used
- Cost
- Practical barriers
What is meant by viral fitness?
The ability of a virus to replicate in a given environment
What are the Protect guidelines??
Guidelines for the responsible use of antimicrobials
According to the Protect guidelines, which antibiotics should not be used in veterinary practice?
- Imipenem
- Linezolid
- Teicoplanin
- Vancomycin
Describe the primary antibiotics group under the Protect guidelines?
- Narrow spectrum
- Older
- No less efficacious than newer antibiotics
Describe the secondary antibiotics group under the Protect guidelines
- Broad spectrum
- Newer
- More important in treatment of serious or often resistant infections in humans
- Only to be used where indicated by culture and sensitivity testing
Describe the tertiary antibiotic group under the Protect guidelines
- Broad spectrum
- Very important for human and animal health care
- Efficient against more resistant bacteria
- Only to be used in clinically important infections, shown to be resistant to all primary and secondary drugs
Describe the prohibited antibiotic group under the protect guidelines
- Significant clinical value to human medicine
- Essential for treating resistant infections in humans
Give examples of primary/first line antibiotics
- Penicillin
- 1st gen cephalosporins
- Amoxicillin and clavulanate
- Trimethoprim + sulphonamides
- Tetracyclines
- Lincosamides
Give examples of secondary/second line antibiotics
- Aminoglycosides
- Medtronidazole
- Macrolides
- Chloramphenicol
- Fluoroquinolones
- Cefeovecin
Give examples of tertiary/third line antibiotics
- 3rd and 4th gen cephalosporins
- Rifampicin
- Fosfomycin
Define minimum selective concentration
- The lowest concentration of an antibiotic that still selects for a given resistance mutation
- The lowest concentration of an antimicrobial that gives the resistant strains a competitive advantage based on growth inhibition of non-resistant strains
Give general methods used in disease control
- Accurate identification of infected animals
- Restriction on movement
- Lab testing for diagnosis
- Vaccination, chemophylaxis where appropriate
- Cleaning of environment
What factors affect the survival of a pathogen in the environment?
- The environmental conditions i.e. dry, wet, presence of organic material
- The bacterial sensitivity to the environment, stress, spore formation
Compare the environmental sensitivity of Mycoplasmas, some enveloped viruses vegetative bacteria, non-enveloped viruses, endospores of bacteria, prions
- Mycoplasmas (and some enveloped viruses) tend to have poor environmental survival rates
- Average vegetative bacteria and non-enveloped viruses have average survival rates and may persist for months
- Endospores of bacteria and prions survive a long time, years
What factors promote pathogenic persistence in the environment?
- Poor general cleaning routine
- Surface structure i.e. crevices, surface joins
- surface characteristics e.g. absorbent, smooth, rough etc
- Weather
- Local environment i.e. soil pH, faecal contamination
Name the possible methods of sterilisation of media and small equipment
- Filtration
- Heat inactivation
- Irradiation
- Chemical
Describe filtration sterilisation
- Liquids
- Use of filters in the um size can remove pathogens
- Ultrafiltrations using reverse osmotic filters can remove ions, producing ultra-pure water
Describe heat inactivation sterilisation
- Liquids and solids
- Depends on solutions e.g. glucose solutions may caramelise
- Compounds can degrade
- Temperature range of material
- Disinfection (removes residues)
- Some solid substances may clog filters
What is irradiation sterilisation used for?
Medical field plastics and some sensitive material
What is chemical sterilisation used for?
Surface decontamination
What pathogens are killed by a normal autoclave cycle?
- Bacteria
- Viruses
- Parasites
Define disinfection
Destruction of micro-organisms by physical or chemical methods on surfaces or objects
Define antisepsis
Destruction of microorganisms on tissues by chemicals which are non-toxic and non-irritating to the tissue
List the features of the ideal chemical disinfectant
- Broad activity
- Safe
- Stable
- Long shelf life
- Soluble in water
- Compatible with wide range of other chemicals
- Active in organic matter
- Non-corrosive to metal/surfaces
- Active over range of temperatures
- Absence of tainting of food products
- Good price
- Non-polluting/biodegradable
List the groups of chemical disinfectants used in veterinary medicine
- Acids
- Alcohols
- Aldehydes
- Alkalis
- Biguanides
- Halogen compounds
- Chlorine compounds
- Iodine compounds
- Phenolic compounds
- Quarternary ammonium compounds
List the potential modes of action of disinfectants
- Damages nucleic acids
- Direct action on organism surface
- Coagulation of cytoplasmic components
- Affect cell membrane
- Target cell components
- Interaction with essential enzymes
Describe the mode of action of alcohol disinfectants
Denature proteins causing membrane damage
Describe the use of alcohol disinfectants
- Broad action
- Ethyl alcohol and isopropyl based most common
- Rapid activity against vegetative bacteria and some viruses
- Needs to be between 70-90% concentration (water required)
What are some problems with alcohol disinfectants?
- Flammable
- Limited activity in presence of organic material
- Ca damage some plastics and rubber
- Minor irritation to try cracked skin
- Not active against spores or small non-enveloped viruses
What are some practical benefits of alcohol disinfectants?
- Evaporates quickly leaving no residue
- Can be used or combined with other anti-microbial compounds
List the pathogenic groups against which alcohol disinfectants are active
- Mycoplasmas
- Gram +ve and -ve
- Enveloped viruses
- Some fungal spores
- Acid fast bacteria
Describe the mode of action of acid disinfectants
- Function related to pH
- Low pH inhibits growth
- Destroy nucleic acid bonds and precipitate proteins
- Change pH and therefore activity of enzymes etc.
Describe some problems with acid disinfectants
- Concentrated acids can be caustic
- Causes burns
- Caustic fumes with strong acids
- Toxic at high concentrations in air
- Reactive, can damage surfaces
Outline the main uses of acid disinfectants
- Organic acids used in food industry
- Household products
- Cleaning alkaline deposits in machinery such as milking machines
- HCl can destroy endospores
List the microbes against which acid disinfectants are active
- Mycoplasmas
- Gram +ve and -ve bacteria
- Enveloped viruses
- Some fungal spores
- Some bacterial spores
