ANTIBIOTICS

HOW DO THEY WORK?

Definitions
Antibiotic- antimicrobial agents produced
by micro-organisms that kill or inhibit other micro-organisms. Chemotherapeutic agentsantimicrobial agents of synthetic origin useful in the treatment of microbial or viral disease. eg. sulfonilamides, isoniazid, ethambutol, AZT, chloramphenicol.

Antibiotics that Inhibit Cell Wall Synthesis

Cell Wall Synthesis

Prokaryotes differ from eukaryotes by having a
cell wall that includes a peptidoglycan layer. Gram ves have a thin layer covered by the lipopolysaccharide (endotoxin). Gram +ves have thick layer with no covering.

Cell Wall Synthesis

Peptidoglycan consists of multiple amino-sugars
that alternate N-acetylglucosamine and Nacetylmuramic acid, which are cross linked to form a lattice. X-linking is essential to risk the high internal osmotic pressures that are generated. Peptidoglycan components manufactured intracellularly and transported across the cell membrane, where they link by transpeptidation.

Cell Wall Synthesis

Beta-Lactam Antibiotics
Penicillins, Cephalosporins, Carbapenems and
monobactams. In 1928, Alexander Fleming observed a fungal contamination of a plate of Staph. Aureus. The fungus was identified as Penicillium notatum, and penicillin was discovered. Florey and Chain were the first to mass produce penicillin.

Penicillins
The beta-lactam ring is derived from the aminoacids valine and cysteine. A secondary amino group (RNH) determines the individual properties of these antibiotics.

Penicillins
They bind to penicillin binding proteins on the
bacterial cell wall and inhibit transpeptidation of the peptidoglycan layer. Active mainly against gram +ves, they are bacteriocidal. Resistance due to -lactamases (Staph.). This can be overcome by Clavulanic acid. Excreted in urine by renal tubular secretion. Hypersensitivity is only real side effect.

Cephalosporins
Cephalosporium fungus from sewer outlet in
Sardinia- shown to inhibit Staph. aureus growth. 3 types of cephalosporins, N,C and P. The type C, is the basis for most of the cephalosporin family by addition of various sidechains to core -lactam nucleus. Action same as penicillins- interferes with peptidoglycan synthesis. Resistant to -lactamases, acid stable.

Cephalosporins
Some are oral, but most are given parentally. Wide distribution after absorption- pleura,
pericardium, joint fluid and across placenta. Cefuroxime, cefotaxime and ceftriaxone also cross the blood brain barrier. Elimination via renal tubular secretion and glomerular filtration, some are eliminated in bile. Side-effects include penicillin type hypersensitivity, 10% cross-reactivity. Nephrotoxicity and alcohol intolerance reported, but uncommon. Diarrhoea with oral route. Broadspectrum, active against most Gram +ves, some Gram ve activity, especially 2nd and 3rd generation.

Other -Lactam Antibiotics

Carbapenems (eg Imipenen)- resistant to most
-lactamases, wide spectrum against gram ve and +ve aerobic and anaerobic organisms.

Monobactams (aztreonam)- derived from the

bacteria Chromobacterium violaceum.

Antimicrobial agents affecting Bacterial Protein Synthesis

Protein synthesis
Ribosomes are the basic units of machinery for protein
synthesis. Bacterial ribosomes have 50S and 30S subunits, mammalian ribosomes have 60S and 40S subunits. mRNA attaches to 30S of ribosome with reads successive mRNA from the right. The next tRNA and attached aa, attach by complementary base-pairing to the mRNA, A site. The P site contains tRNA with its bound peptide chain. The aa on the tRNA in the A site binds to the peptide chain at the P site by transpeptidation, the tRNA at the P site is ejected and the tRNA at the A site translocates to the P site.

Protein synthesis

Tetracyclines
Broad spectrum, original derived from Streptomyces.
More recent compounds are semi-synthetic or synthetic (doxycycline, tetracycline). They block protein synthesis by competing with tRNA for the A site of the ribosome/ mRNA complex.

Tetracyclines
They are bacteriostatic, active against gram +ve/-ve and some protazoa, problems with resitance. Absorbed orally, excreted via bile and glomerular filtration, except doxycycline- GI excretion. Chelate metal ions, bone/teeth/ milk, GI upset.

Chloramphenicol
Originally from Streptomyces, but now
synthetic. Binds to the 50S subunit at same site as erythromycin, and inhibits transpeptidation. Drugs therefore competative.

Chloramphenicol
Broad spectrum and bacteriostatic, crosses
blood-brain barrier. Problems with resistance due to chloramphenicol acetyl-transferase.

Aplastic anaemia (1 in 50,000). Grey-baby

syndrome (40% mortality).

Erythromycin
Inhibits translocation of ribosome along mRNA
chain.

Erythromycin
Spectrum similiar to penicillins, used in
penicillin allergies. Bacteriostatic or bacteriocidal, oral or parenteral, good prostate penetration, poor synovial penetration and does not cross BB barrier. SE rare, can cause hepatitis with prolonged use.

Aminoglycosides
Bind to 30S subunit and prevent complexing of
the 50S subunit. Bacteriocidal and effects enhanced by agents that interfere with cell wall synthesis.

Aminoglycosides
Not absorbed from GI tract. Do not cross BB
barrier. Plasma half-life of 2-3 hours, eliminated entirely by glomerular filtration. Good for gram ve and some gram +ve organisms, no effect on anaerobes. Gentamicin commonest. Some resistance due to microbial inactivating enzymes. Amikacin is resistant to these. Nephrotoxicity and ototoxicity, very rarelyneuromuscular blockade.

Other Antibacterial agents

Fluoro-Quinolones
Synthetic antibiotics which act by inhibition of bacterial
DNA gyrase (topoisomerase II). This is responsible for supercoiling of the DNA helix. Prokaryotic DNA gyrase is structurally different from eukaryotic. Bacteriocidal and early drugs (nalidixic acid) were mainly active against gram ves. Newer forms (ciprofloxacin, ofloxacin and norfloxacin) have wider activity. Absorbed orally with a half-life of 4 hours. GI disturbances, skin rashes, rarely CNS disturbances (confusion, drowsiness, agitation), blood dyscrasias.

Sulphonamides
Discovered in 1930. Prontosil, a dye, could protect
mice from lethal doses of haemolytic streptococci. Found to be pro-drug of sulphanilamide. Landmark discovery as chemical modification of sulphonamide structure has produced thiazides, sulphones and oral hypoglycaemics. Folate synthesised from pABA in bacteria by dihydropteronate synthetase. Bacteriostatic, problems with resistance, liver metabolised. Hypersensitivity, bone marrow depression, crystalluria and methaemaglobinaemia.

Trimethoprim
A folate antagonist. Bacteria must synthesise folate. Folate is converted to
tetrahydrofolate by dihydrofolate reductase. Trimethoprim inhibits this enzyme. There is differential sensitivity to this in humans and bacteria. The IC50(mol/l) is 0.005 and 260, for bacteria and humans. Bacteriostatic, good against gram ves, best given in combination with sulphonamide (sulphamethoxazolecotrimoxazole). However this does not seem to be the case with UTI. Can cause rashes, GI upset and folate deficiency.

Vancomycin
Glycopeptide antibiotic that inhibits cell wall
synthesis, by preventing new disaccharidepentapeptide releasing from carrier lipid, preventing attachment to peptidoglycan layer. Active against gram +ve, including methicillin resistant Staph. aureus. Resistance is rare. Not absorbed from GI tract, excreted by glomerular filtration. Ototoxicity and nephrotoxicity have been reported.

Nitrofurantoin
Synthetic compound with broad spectrum
across gram ve and +ve. Mechanism of action unknown. Rapid excretion in high urine concentrations, so only used for UTI. Works better in acid urine. Side effects rare and mainly GI upset or hypersensitivity skin rash. Bone marrow supression, hepatotoxicity and peripheral neuropathy have been reported.

Metronidazole
Effective against anaerobes and protazoa. Good absorption so oral or IV. Mechanism of action poorly understood, maybe
production of intracellular superoxide and other toxic oxygen free radicals. Does have some nuclease activity, so may cleave bacterial DNA?

Others
Polymixins- Not absorbed from GI tract,
extremely toxic systemically, topical or gut use only. Interrupt bacterial cell wall. Bacitracin- inhibits cell wall synthesis. Not absorbed, systemically toxic, topical use only.

Summary of Antibiotic Actions

Inhibition of cell wall synthesis
Penicillins, Cephalosporins, Carbapenems, Vancomycin

Inhibition of protein synthesis

Aminoglycosides, Tetracycline, Chlorampenicol, Erythromycin

Fluoroquinolones
Prevent coiling of bacterial DNA

Trimethoprim
Inhibits bacterial folate synthesis

Nitrofurantoin
Mechanism of action unknown

Metronidazole
?Cleavage of bacterial DNA

Minimum Inhibitory Concentration

The minimum inhibitory concentration
(MIC) of an antibacterial is defined as the maximum dilution of the product that will still inhibit the growth of a test microorganism.

Minimum Inhibitory Concentration

Bactericidal vs bacteriostatic

Bacterial Resistance
Resistance to antibiotics develops by
several different mechanisms. Intrinsic
Natural Resistance

Acquired
Resistance via mutations Transferable resistance

Natural Resistance
Some species of bacteria have inherent
resistance to antibiotics. Increased use of certain antibiotics naturally selects species with inherent resistance.
Proteus is always resistant to Nitrofurantoin. E. Faecalis is resistant to cephalosporins and fluoroquinolones.

Resistance by Mutation
DNA replication 10-9 to 10-10 uncorrected
base substitutions leading to gene alteration.
Drug inactivating enzymes eg penicillase. Alteration in drug target molecule. Decreased uptake eg porins. Increased elimination eg eflux pumps.

Pre-existing mutations, not newly aquired.

Transferable Resistance
Usually via plasmids (R-factor). Commonest form of bacterial resistance. Multiple resistant strains. Very common in Gram +ves, esp. faecal
flora.

Common Resistant Bacteria

MRSA (methicillin resistant Staph. Aureus),
1-3% of Staph. Aureus infections in 19891993, 42% by 2000. VRE (vancomycin resistant enterococcus). ESBLs (Extended spectrum beta-lactamase gram -ve bacteria). Esp. K. pneumonia and E. coli.

Anti-Mycobacterial Agents

Isoniazid

Synthetic compound, very important in TB treatment. It is taken up by mycobacterium, and can penetrate mammalian cells. Mechanism not fully understood, but inhibits mycolic acids, a component of the cell wall specific to mycobacteria. PO or IV, widely distibuted. Inactivated partly by acetylation, and excreted in urine either unchanged or acetylated. Allergic skin eruptions commonest, can cause hepatotoxicity, vasculitis and arthritic symptoms.

Rifampicin
Semi-synthetic that inhibits RNA polymerase
in prokaryotes only. Also active against gram ve/+ve bacteria. Oral, metabolised by liver. Excreted in many body fluids- orange tinge. Very few side effects

Ethambutol
Only effects mycobacteria. Mechanism unknown. Has to be used in combination. Taken orally SE rare but can cause optic neuritis and
disturbances in red-green colour perception.

Pyrazinamide
Only works at acid pH, and is therefore
effective against the mycobacteria in macrophage phagosomes, which are low pH. Oral, excreted by glomerular filtration. Arthalgia, GI upset, decreased uric acid secretion.

Anti-Fungal Drugs

Anti-Fungal Antibiotics
Amphoteracin
Amphoteric polyene Ab. Binds to cell membrane causing pore formation and thus loss of K+. High affinity to membranes of fungi and some protazoa due to ergosterol. Poorly absorbed from GI tract therefore IV.

Nystatin
As above, only for skin and GI tract infections.

Synthetic Anti-Fungals
Azoles
Ketoconazole, miconazole, clotrimazole, fluconazole, etc. Made from azole rings!! Block synthesis of ergosterol.

Anti-Viral Drugs

Viruses
Nucleic acid core enclosed in a
protein coat (capsid) Some have lipoprotein envelope containing viral glycoproteins and also acquired host phospholipids DNA viruses
Pox virus, herpes viruses (chickenpox, shingles, herpes, EBV), adeonviruses and papilloma virus.

RNA viruses
Orthomyxoviruses (influenza), paramyxoviruses (measles, mumps), rhabdoviruses (rabies), retroviruses and hepadnaviruses.

Viruses
Intracellular parasites ie.
no metabolic machinery of their own. Viral DNA is transcribed by host RNA polymerase in mRNA. Viral RNA acts as mRNA in host to synthesise protein. RNA retroviruses contain RT making a DNA copy of viral RNA which inserts into the host cell genome.

Anti-Viral Agents
Inhibition of attachment or penetration of host.
Amantidine - Influenza Gamma globulin hepatitis, polio, rabies, measles.

Inhibition of nucleic acid synthesis

Acyclovir mainly against herpes viruses Zidovudine (AZT) inhibits viral RT. Indinavir proteases inhibitor, prevents HIV forming
protein coat.