Antibiotics PDF

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Dr Loughlin, summary of antibiotics lectures ...

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Dr. Loughlin

Antibiotics 

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One example of industrial production is of penicillin (medicine and fermentation technology) o 50,000 times increase in production from Fleming Stain Improvements: o Largely from soil organisms such as bacteria and fungi o Traditional random mutagenesis and screening for high producers by growing each clone and assaying the culture filtrate o Then if high production was achieved, but with numerous deleterious mutations a backcross parent strain o Works with fungi o Look for hardy fast growing + overproducing antibiotics Mutations o Spontaneous o X-ray o UV o Nitrogen Mustard  Once sequenced much later, found to have 50 copies of the gene required for synthesis, compared to 1 of each in the original  When examined it was found to produce 30% more isopenicillin N than cephalosporin Modern Day production fungal o 40-200k I fermenters stirred for aeration o initial growth, then antibiotic production phase o Oxygen level maintained throughout o Tricky as transfer of material through viscous fluid, increase viscosity as fermentation continues o 25-27˚C o pH 6.65-7-7 maintained by calcium carbonate o Carbon and nitrogen sources can repress production o Precursors for side chains added (often toxic so sub inhibitory) o 5000 spores/ml added to small fermenter until numbers high enough for the larger (varies according to volume) o larger biomass doubles every 6 hours typically and high growth rate maintained for 2 days o Then 6-8 days of low ? feed – (induce penicillin production and release) o Remove myecelium by filters and wash to remove any further penicillin and pulped to use as animals feed or fertilizers o Penicillin extracted by solvent extraction Modern production final part semisynthetic o Many penicillins have activity but some require tweaking o Semi synthetic antibiotics

Dr. Loughlin

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o Penicillin G remove side chain to make 6-APA o The base unit for adding side chains to o Achieved by passing penicillin through a column of immobilised penicillin acylase o In this case leaving 6APA and phenyl aceitic acid o Here you can add side chains to alter activity Case Study: The history of cephalosporins o Beta lactam antibiotic o Attacks cell wall synthesis o Targets penicillin binding proteins that catalyse cross-linking of 2 aminoacids producing the bridge between adjacent NAG-NAM-NAG-NAM chains o Beta lactam ring opens and binds to penicillin binding protein permentantly taking it out of commission

Basis of modifications o Here we see conversion of Ceph. C into the basis for semi synthetic cephalosporins o Straight forward oxidation, decarboxylation and deacylation leads to 7-ACA the basis of all modifications o Here we see the addition of R at the 7 position o But the OCOCH3 is equally able to be altered

Dr. Loughlin

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First generations 1962-65 o Active against gram –ve rods and penicilliase producing staphylococci o More hydrophobic than penicillins so can diffuse through outer membrane quickly o Lower affinity for chromosomal B lactamases o No first generations were stable o Looked for alternatives 2nd generation cephalosporins o Thinking fungi would have similar cephalosporins o Scientists looked to Streptomyces and found cephamycins o Methoxy group at 7-position o Resistant to TEM 3rd Generation o Shortly after introducing cephamycins, P.aeruginosa, E cloacae and s marcesens infections began to be noticed in hospitals and all of them posed a problem as intrinsically resistant to cephmycins o Now synthetic chemistry o Now effective against the above organisms, and with those with inducible Blactamases (organisms resistant to second generation cefoxitin entirely) th 4 Generation o Looked like everything was fine with generation 3 o But then resistance in P.aeruginosa, E cloacae and s marcesens appeared o Mutants producing the inducible beta lactamases at a constitutive high levels Another modification to 4th generation o Cefepime has low affinity for chromosomal B-lactamases so stable in their presence o Good activity against mutants o And fair G+ve activity o Cefepime o Threat to 3rd Generation o CTX cefotaxime hydrolysing B-lactamase o Most B- lactamases have serine at active site o Some have zinc while sensitive to EDTA, they do break most B lactam rings whether cephalosporin, penicillin or carbapenems th 5 Generation o MRSA has an altered PBP so that the beta lactam ring does not bind o MecA o So generate B lactams that target this new PBP o Ceftaroline is a cephalosporin with in vitro activity against Gram positive and Gram negative o Ceftaroline is bactericidal against S. aureus due to its affinity for PBP2a and against Streptococcus pneumonia due to its affinity for PBP2x. o Ceftaroline is not active against Gram-negative bacteria producing extended spectrum beta-lactamases (ESBLs) from the TEM, SHV or CTX-M families,

Dr. Loughlin serine carbapenemases (such as KPC), class B metallo-beta-lactamases, or class C (AmpC) cephalosporinases. o Ceftobiprole similar to above...