ANTIBIOTICS Lecture 05

Bio 319: Antibiotics

Lecture Five

Topics: •Mechanisms of Antibiotic Resistance•Production of antibiotics•Commercial production of penicillinsis

Dr. G. Kattam Maiyoh

Tuesday, March 26, 2013 GKM/ANTIBIOTIC/2013 1

• Relative or complete lack of effect of antimicrobial against a previously susceptible microbe

Antibiotic resistance• Relative or complete lack of effect of

antibiotic against a previously susceptible bacreria


Antimicrobial Resistance

Figure 20.20Tuesday, March 26, 2013 GKM/ANTIBIOTIC/2013 3

Antibiotic Resistance

Drugs such as tetracyclines or erythromycins are pumped back out of bacterial cells through efflux pump proteins tokeep intracellular drug concentrations below therapeutic level.

The antibiotic is destroyed by chemical modification by an enzyme that is elaborated by the resistant bacteria. This is exemplified here by the beta-lactamase secreted into the periplasmic space to hydrolyse penicillin molecules before they reach their targets in the cytoplasmic membrane of Gram-negative bacterium.

The aminoglycoside antibiotic kanamycin can be enzymatically modified at three sites by three kinds of enzymatic processing — N-acetylation, O-phosphorylation or O-adenylylation — to block recognition by its target on the ribosome.

The target structure in the bacterium can be reprogrammed to have a low affinity for antibiotic recognition. Here the switch from the amide linkage in the D-Ala-D-Ala peptidoglycan termini to the ester linkage in the D-Ala-D-Lac termini is accompanied by a 1,000-fold drop in drug-binding affinity.


Principal resistance strategies for bacterial survival.


A. Efflux pumps


B. Enzymatic destruction of drug


Enzymatic modification

Aminoglycosides such as Kanamycin


Target modification

target structure in the bacterium can be reprogrammed to have a low affinity for antibiotic recognition


Antibiotic Selection for Resistant Bacteria

• Exposure to sub-optimal levels of antimicrobial – innapropriate antibiotic use (see next page)

• Exposure to microbes carrying resistance genes


What Factors Promote Antimicrobial Resistance?

• Prescribing practices of providers. The use ofantibiotics for viral infections, use of broadspectrum antibiotics and prescribing without a laboratory request or doctor visit.

• Prescription not taken correctly• Antibiotics for viral infections – common cold• Antibiotics sold without medical supervision• Spread of resistant microbes in hospitals due to

lack of hygiene• Concerns of daycare providers (need to restrict

access).


Inappropriate Antimicrobial Use

• Lack of quality control in manufacture or outdated antimicrobial

• Inadequate surveillance or defective susceptibility assays

• Poverty or war

• Use of antibiotics in foods


Inappropriate Antimicrobial Use

– Growth promotion – Disease prevention– Sick animal treatment/plants – very large amounts

– Poultry– Fish farms– Fruit, potatoes, tobacco and others– Ornamental plants


Uses of antibiotics in agriculture?


Should antibiotics for growth promotion and disease prevention be banned?

• Adverse effect on animal industry

• reduced food supply

• increased cost of production

• increased disease incidenceeconomic loss by farmers

• May not be totally necessary

• Might only require ban of specific antimicrobial drugs that could select for resistance to drugs in human medicine.

Consequences of Antimicrobial Resistance

• Infections resistant to available antibiotics

• Increased cost of treatment



Current problems of Resistance/MDR bacteria

Hospital CommunityGram Negative Gram Negative

Acinetobactor sp. E. Coli

Citrobacter sp. Neisseria gonorrhoeae

Enterobacter sp. S. typhi

Klebsiella sp. S. tythimurium

P. aeruginosa

Serratia marcescens

Gram Positive Gram Positive

Enterococcus sp.: vancomycin resistant enterococci (VRE)

Enterococcus sp.: vancomycin resistant enterococci (VRE)

Coagulase negative staphylococcus Mycobacterium turberculosis

MRSA MRSA

MRSA heterogenously resistant to vancomycin

Streptococcus pneumoniae

Streptococcus pyogenes


Multi-Drug Resistant TB

• Methicillin-Resistant Staphylococcus aureus

• Most frequent nosocomial (hospital-acquired) pathogen

• Usually resistant to several other antibiotics


MRSA “mer-sah”

• Speed development of new antibiotics

• Track resistance data nationwide• Restrict antimicrobial use• Direct observed dosing (TB)


Proposals to Combat Antimicrobial Resistance

• Use more narrow spectrum antibiotics• Use antimicrobial cocktails• Tx only the sick or at risk• Producer education• Further research before imposing bans


Proposals to combat antimicrobial resistance

• Antimicrobial peptides

– Antibiotics from plants and animals• Squalamine (sharks)

• Protegrin (pigs)

• Magainin (frogs)

• DNA technology

• Antisense agents– Complementary DNA or peptide nucleic acids that binds

to a pathogen's virulence gene(s) and prevents transcription

– Phage therapy - use of bacteriophages to treat pathogenic bacterial infections


The Future of Chemotherapeutic Agents

Production of Antibiotics

Tuesday, March 26, 2013 25GKM/ANTIBIOTIC/2013

Production of Antibiotics• The mass production of antibiotics

began during World War II with streptomycin and penicillin.

• Now most antibiotics are produced by staged fermentations in which strains of microorganisms producing high yields are grown under optimum conditions

– nutrient media

– fermentation tanks

– holding several thousand gallons.


Production of Antibiotics• The mold is strained out of the fermentation broth, and

then the antibiotic is removed from the broth by;

– filtration,

– precipitation, and

– other separation methods.

• In some cases new antibiotics are laboratory synthesized, while many antibiotics are produced by chemically modifying natural substances;

• Many such derivative penicillins are effective against bacteria resistant to the parent substance.es are more effective than the natural substances against infecting organisms or are better absorbed by the body.


Raw Materials• The compounds that make the fermentation

broth are the primary raw materials required for antibiotic production.

• This broth is an aqueous solution made up of all of the ingredients necessary for the proliferation of the microorganisms.

• Typically, it contains; – a carbon source like molasses, or soy meal,

both of which are made up of lactose and glucose sugars.

– Other carbon sources include; acetic acid, alcohols, or hydrocarbons

• These materials are needed as a food source for the organisms.

• Nitrogen is another necessary compound in the metabolic cycles of the organisms.

• For this reason, an ammonia salt is typically used.


Other elements


For E.g. Scheme for Penicillin Production


Steps in Production• The production of a new antibiotic - lengthy and costly.

– First, the organism that makes the antibiotic must be identified

– desired microorganism must then be isolated

– Then the organism must be grown on a scale large enough to allow the purification and chemical analysis of the antibiotic

– the antibiotic tested against a wide variety of bacterial species.

– This is a complex procedure because there are several thousand compounds with antibiotic activity that have already been discovered, and these compounds are repeatedly rediscovered.

– It is important that sterile conditions be maintained throughout the manufacturing process, because contamination by foreign microbes will ruin the fermentation.


Commercial Production over view

• After the antibiotic has been shown to be useful in the treatment of infections in animals, larger-scale preparation can be undertaken.

• Commercial development requires a high yield and an economic method of purification.

• Extensive research may be needed to increase the yield by selecting improved strains of the organism or by changing the growth medium.

• The organism is then grown in large steel vats, in submerged cultures with forced aeration.

• The naturally fermented product may be modified chemically to produce a semisynthetic antibiotic.


Large scale antibiotics production

Steel Vats


•The seed tanks are equipped with mixers, which keep the growth medium moving, and a pump to deliver sterilized, filtered air.

•After about 24-28 hours, the material in the seed tanks is transferred to the primary fermentation tanks.

Fermentation• Microorganisms are allowed to grow and multiply. • During this process, they excrete large quantities of the

desired antibiotic. • The tanks are cooled to keep the temperature between

73-81° F (23-27.2 ° C). • It is constantly agitated, and a continuous stream of

sterilized air is pumped into it. For this reason, anti-foaming agents are periodically added.

• Since pH control is vital for optimal growth, acids or bases are added to the tank as necessary.


Isolation and Purification• 3-5days, the maximum amount of antibiotic will

have been produced • The isolation process can begin. • Depending on the specific antibiotic produced,

the fermentation broth is processed by various purification methods.


Water soluble Antibiotic• For example, for antibiotic compounds that

are water soluble, an ion-exchange method may be used for purification.

• In this method, the compound is first separated from the waste organic materials in the broth

• Then sent through equipment, which separates the other water-soluble compounds from the desired one.


Organic Antibiotics• To isolate an oil-soluble antibiotic such

as penicillin, a solvent extraction method is used.

• In this method, the broth is treated with organic solvents such as butyl acetate or methyl isobutyl ketone, which can specifically dissolve the antibiotic.

• The dissolved antibiotic is then recovered using various organic chemical means.

• At the end of this step, the manufacturer is typically left with a purified powdered form of the antibiotic, which can be further refined into different product types.


Refining/Packaging• Antibiotic products can take on many different forms. They can

be sold in solutions for intravenous bags or syringes, in pill or gel capsule form, or they may be sold as powders, which are incorporated into topical ointments.

• Depending on the final form of the antibiotic, various refining steps may be taken after the initial isolation.

• For intravenous bags, the crystalline antibiotic can be dissolved in a solution, put in the bag, which is then hermetically sealed.

• For gel capsules, the powdered antibiotic is physically filled into the bottom half of a capsule then the top half is mechanically put in place.

• When used in topical ointments, the antibiotic is mixed into the ointment.



Antibiotics packaging

Pharmacology and Toxicity

• After purification, the effect of the antibiotic on the normal function of host tissues and organs (its pharmacology), as well as its possible toxic actions (toxicology), must be tested on a large number of animals of several species.

• In addition, the effective forms of administration must be determined..


Production• Once these steps have been completed,

the manufacturer may file an Investigational New Drug Application with the Pharmacy and Poisions Board.

• If approved, the antibiotic can be tested on volunteers for toxicity, tolerance, absorption, and excretion.

• If subsequent tests on small numbers of patients are successful, the drug can be used on a larger group, usually in the hundreds. If all goes well the drug can be used in clinical medicine.

• These procedures, from the time the antibiotic is discovered in the laboratory until it undergoes clinical trial, usually extend over several years.


Quality Control• Quality control is of utmost importance in the production of antibiotics. • Since it involves a fermentation process, steps must be taken to ensure

that absolutely no contamination is introduced at any point during production.

• To this end, the medium and all of the processing equipment are thoroughly steam sterilized.

• During manufacturing, the quality of all the compounds is checked on a regular basis.

• Of particular importance are frequent checks of the condition of the microorganism culture during fermentation.

• These are accomplished using various chromatography techniques. • Also, various physical and chemical properties of the finished product

are checked such as pH, melting point, and moisture content\


The antibiotic substance, named penicillin, was not purified until the 1940s (by Florey and Chain), just in time to be used at the end of the second world war.

Penicillin was the first important commercial product produced by an aerobic, submerged fermentation


Penicillin – Industrial production

When penicillin was first made at the end of the second world war using the fungus Penicilium notatum, the process made 1 mg dm-

3. Today, using a different

species (P. chrysogenum) and a better extraction procedures the yield is 50 g dm-3.

There is a constant search to improve the yield.


Penicilium notatumPenicilium notatum

P. chrysogenumP. chrysogenum

Penicillin is produced by the fungus Penicillium chrysogenum which requires lactose, other sugars, and a source of nitrogen (in this case a yeast extract) in the medium to grow well.

Like all antibiotics, penicillin is a secondary metabolite, so is only produced in the stationary phase.

What sort of fermenter does it require? It requires a batch fermenter. A fed batch process is normally used

to prolong the stationary period and so increase production.


Antibiotic Production Methods

Fed-Batch: based on feeding of a growth limiting nutrient substrate to a culture.


Colony growth and penicillin Production


Downstream processing is relatively easy since penicillin is secreted into the medium

So there is no need to break open the fungal cells.

However, the product needs to be very pure, since it being used as a therapeutic medical drug.

It is dissolved and then precipitated as a potassium salt to separate it from other substances in the medium.

Purification

Purification

Tuesday, March 26, 2013GKM/ANTIBIOTIC/2013

48

Batch-fed fermenter



The resulting penicillin (called penicillin G) can be chemically and enzymatically modified to make a variety of penicillins with slightly different properties.

These semi-synthetic penicillins include penicillin V, penicillin O, ampicillin and amoxycillin.

Products

1.1. What is the Carbon source?What is the Carbon source?2.2. What is the nitrogen source?What is the nitrogen source?3.3. What is the energy source?What is the energy source?4.4. Is the fermentation aerobic or anaerobic?Is the fermentation aerobic or anaerobic?5.5. What is the optimum temperature?What is the optimum temperature?6.6. Is penicillin a primary or secondary metabolite?Is penicillin a primary or secondary metabolite?7.7. What volume fermenter is used?What volume fermenter is used?8.8. Why isn't a larger fermenter used?Why isn't a larger fermenter used?9.9. When is penicillin produced?When is penicillin produced?10.10.How long can it be produced for?How long can it be produced for?11.11.What was the first fungus known to produce penicillin?What was the first fungus known to produce penicillin?12.12.What species produces about 60mg/dm3 of penicillin?What species produces about 60mg/dm3 of penicillin?13.13.How did scientists improve the yield still further?How did scientists improve the yield still further?14.14.What is the substrate?What is the substrate?15.15.Why is batch culture used?Why is batch culture used?16.16.What are the processes involved in down-stream processing?What are the processes involved in down-stream processing?

a)a)b)b)c)c)

17.17.Why can't penicillin be taken orally?Why can't penicillin be taken orally?18.18.Name the form of penicillin which can be taken orally.Name the form of penicillin which can be taken orally.19.19.How does Penicillin kill bacteria?How does Penicillin kill bacteria?20.20.Why are Gram negative bacteria not killed by penicillin?Why are Gram negative bacteria not killed by penicillin?


lactoselactoseyeastyeast

glucoseglucoseaerobicaerobic

25 - 2725 - 27ºCºCsecondarysecondary

40 – 200 dm40 – 200 dm33

Too difficult to aerateToo difficult to aerate40 hours – after main increase in fungal mass40 hours – after main increase in fungal mass

140 hours (180 – 40 hours)140 hours (180 – 40 hours)Penicillin notatumPenicillin notatum

Penicillin chrysogenumPenicillin chrysogenumGenetic modificationGenetic modification

Corn steep liquorCorn steep liquorSecondary metaboliteSecondary metabolite

Filtration of liquidFiltration of liquidExtraction from filtrate by counter current of butylacetateExtraction from filtrate by counter current of butylacetatePrecipitation by potassium saltsPrecipitation by potassium salts

Destroyed by stomach acidDestroyed by stomach acidPenicillin V, ampicillinPenicillin V, ampicillin

Stops production of cell wallStops production of cell wallDifferent cell wallDifferent cell wall


Biosynthesis of Penicillin•Three main and important steps to the biosynthesis of penicillin G (benzylpenicillin)

1.Condensation of three amino acids L-α-aminoadipic acid, L-cysteine, L-valine into a tripeptide

2.ACV will undergoes oxidation which then allows a ring closure so that a bicyclic ring is formed

3.Exchange the side chain group so that isopenicillin N will become penicillin G

•The alpha-aminoadipyl side chain of isopenicillin N is removed and exchanged for a phenylacetyl side chain

δ-(L-α-aminoadipyl)-L-cysteine-D-valine

δ-(L-α-aminoadipyl)-L-cysteine-D-valine synthetase (ACVS)

isopenicillin N acyltransferase (IAT)

isopenicillin N synthase

http://en.wikipedia.org/wiki/Penicillin_G

http://en.wikipedia.org/wiki/Tripeptide

ANTIBIOTICS Lecture 05

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