Anti-biotics and anti-fungals Flashcards
What are the two key targets of antibiotics
Bacterial Protein Synthesis
Bacterial Cell wall synthesis.
Describe the key features of bacteria
Bacteria are single cell micro-organisms surrounded by a lipid membrane, which in turn is surrounded by a cell wall. They have no true nucleus for the storage of genetic information and do not
contain any other membrane-bound organelles. For these reason they are known as ‘prokaryotes’.
1/3 of all bacteria are pathogenic
Which phylogenetic domain do bacteria belong to
They constitute an entire phylogenetic domain along with archaea (e.g. Halobacteria, methanogens) and eukaryotes (e.g. protists, fungi, plants & animals) according to the now generally accepted
three domain system originally proposed by Woese, Kandler & Wheelis (1990).
Summarise the different ways of classifying bacteria
There are various systems used to classify bacteria (e.g. DNA sequence, shape, fastidiousness) but for the purpose of this lecture we will focus on their reaction to Gram staining.
Describe the membrane properties of gram positive bacteria
These bacteria have a prominent peptidoglycan cell wall, which means that they take up the Gram stain in a simple biological test. A common Gram positive bacterial species is Staphylococcus Aureus
Cell membrane is surrounded by peptidoglycan cell wall.
Describe the membrane properties of gram negative bacteria
Outer membrane with lipopolysaccharide
E.g. Escherichia Coli
Far less prominent peptidoglycan- sandwiched between LPS and inner membrane
Describe the membrane properties of mycolic bacteria
This genus of bacteria does not fit into the classical Gram negative and positive classification; although technical they are considered to be Gram positive, since they do take up the stain but are acid-fast. These bacteria have an outer layer of mycolic acid and a common mycobacterial species is Mycobacterium tuberculosis.
Peptidoglycan (may be prominent in some mycolic bacteria)- sandwiched between cell membrane and outer mycolic acid layer
Describe the importance of this classification of the membrane properties of antibiotics for pharmacology
This classification is particularly useful for pharmacological purposes since a number of drugs are only effective against one or the other type of bacteria. Drugs that are effective against both Gram positive and Gram negative bacteria are referred to as broad-spectrum antibiotics.
What is meant by acid-fast bacteria
Once stained, acid-fast organisms can resist the acid or ethanol based decolorization common in many staining protocols.
How do E.coli and Mycobacterium Tuberculosis become resistant
By ‘escape’ mechanisms
State the steps involved in the production of THF from PABA
PABA à DHOp (enzyme = dihydropterase synthase)
DHOp à DHF
DHF à THF (enzyme = DHF reductase
Summarise prokaryotic nucleic acid synthesis
Dihydropteroate (DHOp) Produced from paraaminobenzoate (PABA) Converted into dihydrofolate (DHF) Tetrahydrofolate (THF) Produced from DHF by DHF reductase THF Important in DNA synthesis
Describe the importance of THF in prokaryotic nucleic acid synthesis
Nucleic acids are the building blocks of prokaryotic (and eukaryotic) DNA; therefore their synthesis is essential for cell survival and cell division. Tetrahydrofolate (THF) is a critical co-factor involved in the production of numerous amino acids and thymidylate (which is required for uracil synthesis). And there are subtle differences between THF production in prokaryotes that can be exploited by pharmacological reagents.
Describe the enzyme important for prokaryotic DNA replication
DNA replication is the process by which the nucleotide sequence of DNA is copied into an exact complementary sequence, which is required for cell division. The process requires an elaborate interplay between numerous different proteins but for the purpose of this lecture we will focus on the DNA gyrase enzyme. DNA gyrase is a type II topoisomerase, which cuts DNA strands thus releasing the tension from supercoiling and therefore allowing access to all the proteins involved in transcription.
Describe prokaryotic DNA transcription/RNA synthesis
DNA transcription is the ‘process whereby one strand of a DNA molecule is used as a template for synthesis of a complementary RNA by RNA polymerase’. The complementary RNA (known as messenger (m)RNA) is subsequently utilised for the production of proteins.
In bacteria there is only one RNA polymerase responsible for the entire process (as opposed to eukaryotes, who have multiple types of RNA polymerase)
Define protein synthesis
The process of protein synthesis, or translation, can be defined as ‘the ribosome-mediated production of a polypeptide whose amino acid sequence is specified by the nucleotide sequence in an mRNA.’
Describe prokaryotic protein synthesis
Ribosomes Produce protein from RNA templates Differ from eukaryotic ribosomes Eukaryote = 40S + 60S Prokaryote = 30S + 50S
What are the four key steps in prokaryotic protein synthesis that can be exploited pharmacologically
Nucleic Acid Synthesis
DNA replication
RNA synthesis
Protein synthesis
Describe the antibiotics that target prokaryotic nucleic acid synthesis
The enzyme DHOp synthase is not functional in humans and is targeted by the sulphonamide (e.g. sulfadiazine) group of antibiotics. However, these antibiotics are no longer used readily mainly due to the development of bacterial resistance.
Trimethoprim targets the bacterial dihydrofolate (DHF) reductase enzyme with a far greater affinity than the human isoform. It is often used in conjunction with the sulphonamide drug (sulphamethoxazole) in the compound preparation co-trimoxazole but is also prescribed alone mainly for the treatment of urinary tract infections
Describe the antibiotics that target prokaryotic DNA replication
DNA gyrase is not present in humans.
Fluoroquinolones (e.g. Ciprofloxacin) inhibit DNA gyrase & topoisomerase IV
Can also use quinolones
They are generally effective against both Gram negative and Gram positive bacteria (broad spectrum) but they are contraindicated in children and individuals with epilepsy.
Describe the antibiotics that target prokaryotic RNA synthesis/DNA transcription
In bacteria there is only one RNA polymerase responsible for the entire process (as opposed to eukaryotes, who have multiple types of RNA polymerase) and this enzyme is targeted by the Rifamycins (e.g. rifampicin), which are primarily used to treat mycobacterial infections (e.g. tuberculosis).
Describe the different antibiotics that can be used to target bacterial protein synthesis
Aminoglycosides
Gentamicin, streptomycin
Broad spectrum. Inactive against anaerobes
Chloramphenicol
Chloramphenicol
Broad spectrum. Eye infections
Macrolides
Erythromycin, azithromycin
Alternatives for penicillin-allergic patients
Tetracyclines
Tetracycline
Broad spectrum. Increased resistance
Target the 70S bacterial ribosome
What can be said about the process of bacterial cell wall synthesis
It is a dynamic process- elements are constantly being removed and added
Synthesis of peptidoglycan begins intracellularly.
Describe the differences in the cell wall between prokaryotes and eukaryotes
Another factor that distinguishes prokaryotes from eukaryotes is the presence of a cell wall situated on the outer surface of the cell membrane. The cell wall is mainly composed of a pentapeptide known as peptidoglycan (aka murein). However, the amount of peptidoglycan in the cell wall varies between Gram positive and Gram negative bacteria.
Gram positive bacteria have a thicker cell wall with more peptidoglycan (PtG) and Gram negative bacteria have a thinner cell wall with less PtG
Summarise the 3 key stages of peptidoglycan synthesis
Assembly in the cytoplasm; transport across the inner membrane & polymerisation in the cell wall
Describe the synthesis of peptidoglycan in the cytoplasm
A pentapeptide is created on N-acetyl muramic acid (NAM)
N-acetyl glucosamine (NAG) associates with NAM forming PtG
Describe the transportation of peptidoglycan from the cytoplasm to the periplasm (between the cell membrane and the cell wall)
PtG is transported across the membrane by bactoprenol
Once the peptidoglycan is incorporated into the cell wall, the bactoprenol can be re-utilised for further PtG transportation.
Describe the incorporation of newly synthesised peptidoglycan into the cell wall
PtG is incorporated into the cell wall when transpeptidase enzyme cross-links PtG pentapeptides
Describe the antibiotics that interfere with peptidoglycan synthesis
Glycopeptides (e.g. vancomycin) – they bind to the pentapeptides and inhibit peptidoglycan synthesis
This is used as a last resort for Gram-positive bacteria that are resistant to other antibiotics
These drugs have poor oral bioavailability and are primarily employed against multidrug resistant infections.
Describe the antibiotics that target peptidoglycan transportation
Bacitracin inhibits bactoprenol regeneration preventing PtG transportation
Targets the recycling step of bactoprenol
However, this drug is only used in topical skin preparations because of its poor bioavailability and toxicity.
Describe the antibiotics that target peptidoglycan incorporation
-lactams bind covalently to transpeptidase inhibiting PtG incorporation into cell wall (also known as penicillin binding proteins).
-lactams include:
Carbapenems
Cephalosporins
Penicillins
Mechanisms vary between classes, propensity for different bacteria varies within classes.
Beta-lactams all contain the beta-lactam ring.
Describe the different classes of beta-lactams
Carbapenems
Imipenem
Broad spectrum.
Cephalosporins
Cefalexin
Broad spectrum. Three ‘generations’
Penicillins
Amoxicillin, flucloxacillin
Broad spectrum. Hypersensitivity
Describe the antibiotics that target cell membrane stability
Lipopeptide - (e.g. daptomycin) disrupt Gram +ve cell membranes
Polymyxins - binds to LPS & disrupts Gram -ve cell membranes
Generally, which antibiotics are poor at treating gram-ve
Those that target cell wall synthesis, opt for antibiotics that target protein synthesis.
Summarise the threat posed by antibiotic resistance
CATASTROPHIC’, ‘APOCALYPTIC’, ‘AS BIG A RISK AS TERRORISM’ UK Chief Medical Officer
~ 70% of bacteria developed resistance
25000 yearly death rate - Europe & US
Outline the causes for antibiotic resistance
Unnecessary prescription
~ 50% of antibiotic prescriptions not required
Livestock farming
~ 30% of UK antibiotic use in livestock farming- this has reduced in recent years- but still very high in the U.S
Lack of regulation
OTC availability in Russia, China, India
Lack of development
Very few antibiotics in recent years - only taken for 3 weeks- not much financial incentive for research.
Which drug class already had resistance against it before it was used clinically
Penicillin- as this is a naturally occurring compound.
Describe the dangers of antibiotic resistance
Since bacteria are able to replicate every few hours genetic changes can occur at an alarming rate and selection pressure means that the ‘antibiotic survivors’ will become the dominant type as they pass on their traits to their progeny.
Bacteria tend to be more pathogenic than viruses
Describe the production of destruction enzymes as a mode of antibiotic resistance
-lactamases hydrolyse C-N bond of the -lactam ring
Compare the mode of action of beta lactamases in gram positive and gram negative bacteria
The mode of action of β-lactamases differs between Gram positive and Gram negative bacteria. The -lactamase is secreted from Gram-positive bacteria, essentially forming a ‘shield’ of protection. In Gram negative bacteria the enzyme has an intracellular location.
Describe the different antibiotics that are both resistant and non-resistant to beta-lactamases
a. b-lactamase resistant – Flucloxacillin & Temocillin.
i. Amoxicillin (broad spectrum AB) combats gram –ve bacteria and is resistant to b-lactamases only when co-administered with Clavulanic acid.
Temocillin- gram -ve
Flucloxacilllin- gram -ve
b. b-lactamase non-resistant – Penicillins G and V (combat gram +ve).
Describe how making additional targets is a resistance mechanism to antibiotics
This type of resistance involves the production of a different enzyme that is able to perform the same function but is no longer a target for the antibiotic. The original drug-susceptible enzyme is still produced to ‘occupy’ the attentions of the antibiotic. A prominent example of this type of resistance is the production of an alternative dihydrofolate reductase enzyme, which is not inhibited by trimethoprim ( E.coli do this).
Describe how alterations in target enzymes is a resistance mechanism to antibiotics
Alteration to the enzyme targeted by the drug. Enzyme still effective but drug now ineffective
Example
S Aureus - Mutations in the ParC region of topoisomerase IV confers resistance to quinolones
bacterial ribosome rendering the macrolides ineffective.
Describe alterations in drug permeation as a method of antibiotic resistance
Reductions in aquaporins & increased efflux systems
Examples
Primarily of importance in gram –ve bacteria
Need aquaporins to get into the bacteria
Increase drug efflux by increasing the production of transporter proteins
Describe hyperproduction as a method of antibiotic resistance
Bacteria significantly increase levels of DHF reductase
Example
E Coli produce additional DHF reductase enzymes making trimethoprim less effective
Demands a lot of energy from the bacteria.
Summarise the key properties of fungi
Fungi are classified as eukaryotes since they contain nuclei and of all the known species of fungi approximately 0.5% are thought to cause human infections. Fungal infections are opportunistic and are predominantly effect immune-compromised individuals or those on antibiotics.
Classify the different fungal infections
These infections have the ability to be lifethreatening and in broad terms can be classified according to the tissue/organs they affect: Superficial – Outermost layers of skin Dermatophyte – Skin, hair or nails Subcutaneous – Innermost skin layers Systemic – Primarily respiratory tract
What are the two main classes of anti-fungals
Azoles (fluconazole and ketoconazole)
Polyenes (amphotericin and nystatin)
15 anti-fungal drugs licensed in the UK
Describe how azoles work
Inhibit cytochrome P450-dependent enzymes involved in membrane sterol synthesis
Fluconazole (oral) candidiasis & systemic infections
Azoles inhibit Cyp51p which converts lanosterol to ergosterol- which a key cell membrane component.
If you lose the cell membrane- you lose cell integrity- cell death.
Describe how polyenes work
Interact with cell membrane sterols forming membrane channels
Amphotericin (I-V) systemic infections - punching holes in the membrane- loses its integrity.
Describe the key properties of the mycobacteria
The outer wall contains a PtG-arabinogalactan polymer that binds mycolic acids, pore forming proteins and a number of extractable lipids giving the bacteria a ‘waxy’ outer layer. The most notable examples of mycobacteria are responsible for tuberculosis (mycobacteria tuberculosis) and leprosy (mycobacteria leprae).
Summarise the treatment for TB
The RNA-polymerase inhibitor rifampicin is one of the first-line drugs recommended for the treatment of tuberculosis but two of the other drugs are specific for mycobacteria and are classified as mycolic acid synthesis inhibitors. Although isoniazid and ethambutol have slightly different modes of action their eventual effect is to prevent the synthesis of mycolic acid. The fourth drug used in the treatment of tuberculosis is pyrazinamide, which is thought to reduce the availability of ribosomes required for protein translation.
What are the two courses of treatment for mycobacterial infections
Isoniazid + Rifampicin (6 months)
Ethambutol + Pyrazinamide (2 months)
