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1997 Clinical pharmacology By Duy Thai

ANTIBIOTICS 3 ANTIBIOTIC RESISTANCE 1. In activation of the drug • Production of beta lactamases Which hydrolyses the beta lactam ring of beta lactam antibiotics • Production of specific enzymes which may modify the antibiotic, stuffing it up. • e.g. Modification of aminoglycosides • Covalent modification involves: • Adenylation • Acetylation • Phosphorylation • Any one of these modifications is sufficient to inactivate the drug. This result in the drug no longer being able to pass through the cell membrane. Because aminoglycosides are dependent on their ability to pass through the cell membrane, preventing this step will severely impair their activity. 2. Alteration of the target drug • Bacteria can modify their penicillin binding proteins. e.g. MRSA resistance is mediated by an altered penicillin binding protein so that beta lactams can no longer bind • Vancomycin resistant enterococci mutate the D-ala D-ala terminal region, it becomes D-ala L-lys. The bacterial transpeptidase can still recognise this bond however Vancomycin cannot. • Resistance to erythromycin is via alteration of the 50s subunit • Resistance to streptomycin is via change in the binding site on the 30s subunit • Resistance to trimethoprim is via mutation in DHFR 3. Reduce the importance of the target • If you treat an abscess with sulphonamide , it is not as effective as with a spreading infection. • Sulphonamides inhibit the synthesis of folic acid. Folic acid is required for the synthesis of various nucleotides in DNA synthesis. • In an abscess, there are lots of dead cells, and their contents, including their DNA are spread out everywhere. The bacteria are thus swimming in a sea of nucleotides. They hence do not need to make folic acid because there is an abundance of nucleotides available. 4. Overproduce the target • e.g. To overcome trimethoprim, bacteria can overproduce DHFR to overcome the inhibition of trimethoprim 5. Reduce access of the drug to the target • Bacteria can alter their outer membrane proteins (e.g. porins), which some drugs use to enter the cell. Thus, entry of drugs is restricted. This is not a complete block, since you can still get entry of drug s via other means. This is thus a low level resistance which can be overcome by increasing the dose given. e.g. In gonococcus infection, resistance to penicillin has occurred due to a change in penicillin permeability. That is why, over the years, the dose required to treat gonococcal infection has risen. • Mutation in the OMP’s is not specific, since many drugs can share the same porin to get in. e.g. If the bacteria becomes resistant to streptomycin it will also become resistant to all the other aminoglycosides. • Some drugs are highly lipid soluble and so do not require OMP for access to the cell. e.g. Tetracycline and erythromycin (these drugs are good against intracellular pathogens). In this case. the bacteria have devised specific enzymes to pump the drug outside, before it has had time to act.

6. Failure to activate an inactive prodrug • Loss of a gene which codes for an enzyme which activates a prodrug. eg. These drugs are izoniazid, Metranidazole resistance can occur by the bacteria lacking the enzyme nitro reductase.

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1997 Clinical pharmacology By Duy Thai

Genetic basis of resistance 1. There is intrinsic resistance • The bacteria by their very nature are resistant (no mutations are necessary) • e.g. Mycoplasma do not have a cell wall and hence are resistant to beta lactamases • Enterococci cannot make folic acid and are thus resistant to sulphonamides • Pseudomonas aeuriginosa has a chromosomal beta lactamase (it has always made β lactamase) 2. Acquired resistance • Bacteria can change their genetic makeup via: • Mutation • A certain proportion of bacterial offspring may develop random mutations which may lead to resistance to a certain antibiotic, thus allowing it to survive. • Resistance like this has occurred to nalidixic acid, rifampicin, trimethoprim • This can be prevented by doing combination chemotherapy. The chances of developing dual resistance is very unlikely. • Acquiring resistance genes from other resistant bacteria via: • Transformation • Phage mediated transduction • Plasmid mediated conjugation Transformation 1. A bacteria which is resistant dies. 2. It undergoes lysis, and its DNA is fragmented all over the place 3. The DNA fragments are taken up by another bacterium. 4. There is homologous recombination (the DNA fragment is incorporated into the bacterial chromosome) 5. The particular fragment which is taken up may just happen to code for antibiotic resistance • 2 things need to be fulfilled for this to occur: 1. The donor and recipient bacterium need to be genetically similar (there needs to be homology) 2. The DNA fragment which is taken up needs overcome restriction by endonucleases. Pneumococcal resistance to penicillin by mutation of PBP are acquired via this mechanism

Phage mediated transduction 1. A bacteriophage containing a strand of DNA injects this DNA strand into a bacteria 2. The viral DNA strand is incorporated into the bacterial chromosome by non homologous incorporation. 3. Two things can happen A. Lysogenic infection (temperate phages) • The viral DNA can change the phenotype of a bacteria. The viral DNA codes for a particular protein (e.g. A toxin) but its expression is controlled by the bacteria. B. Lytic infection (virulent phages) • The viral DNA is expressed on its own accord resulting in the production of lots of viral DNA strands which become incorporated into new bacteriophages. These bacteriophages cause lysis of the bacteria from within. • Sometimes, during the packaging of viral DNA into phages, a portion of the bacterial DNA is incorporated instead. This results in a phage which contains bacterial DNA rather than viral DNA • This phage can infect another bacteria and inject this DNA strand into the bacteria, getting homologous recombination. • Bacteriophages tend to infect bacteria which are very related, so that there is no problem of homology • The bacterial DNA strand may just happen to contain a segment coding for antibiotic resistance. • • This is how S. aureus became resistant to many bacteria. S. epidermidus is present on our skins and every time we take an antibiotic, we are selecting for resistant strains of S. epidermidus to continue. S. epidermidus can convey this resistance to S. aureus by phages. 2 things are required: 1. The phage needs to recognise the donor and the recipient 2. Need homology
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1997 Clinical pharmacology By Duy Thai

Plasmid mediated conjugation 1. The donor bacterium contains a plasmid which can encode for many things. One of which is the formation of a conjugation pilus. 2. This bacteria can have sex with another bacteria, resulting in the duplication and transfer of the plasmid into the recipient bacterium. Both bacteria thus have the plasmid 3. This transfer can occur between 2 totally unrelated bacterium, however, it can only occur between Gbacteria 4. E.g. Between E. coli and V. cholerae 5. The plasmid transferred can code for a variety of different things • One plasmid can encode for: • Sulphonamide resistance • Streptomycin resistance • Tetracylin resistance (efflux reaction) • RTF (encodes the conjugation tube - pilus) • Therefore, in one single event, a bacteria can acquire resistance to a whole range of antibiotics The concept of selection • If we have a bacteria which undergoes growth by binary fission, one of its offspring may develop resistance (this bacterium may be one in a million other bacteria). • It would be very easily to find this bacterium. • All you do is add an antibiotic and this will kill all the bacteria, leaving only the resistant bacteria to stay alive. • This bacteria will then start to divide, resulting in a culture which contains 100 % of bacteria, all of which are resistant to the antibiotic.

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