24. Antibiotics Now most of the antibiotics were discovered from soil microorganisms especially in microorganism Streptomyces spp. Engineered genes into production strains. Modification of existing antibiotics.

Antibiotics are chemicals produced by microorganisms and which in low concentrations are capable of inhibiting the growth of, or killing, other microorganisms. Broadened by some authors to include materials produced by living things – plants, animals or microorganisms – which inhibit any cell activity. Antibiotics may be wholly produced by fermentation. Semi-synthetic processes, in which a product obtained by fermentation is modified by the chemical introduction of side chains.

Some wholly chemically synthesized compounds are also used for the chemotherapy of infectious diseases e.g. sulfonamides and quinolones. Some antibiotics e.g. chloramphenicol were originally produced by fermentation, but are now more cheaply produced by chemical means. Only a small proportion of known antibiotics is used clinically, because the rest are too toxic.

Classification of Antibiotics The classification to be adopted here is based on the chemical structure of the antibiotics and classifies antibiotics into 13 groups. This enables the accommodation of new groups as they are discovered.

Grouping of antibiotics based on their chemical structures

The nomenclature of antibiotics The same antibiotic may have as many as 13 different trade names depending on the manufacturers. Antibiotics are therefore identified by at least three names: The chemical name, which prove long and is rarely used except in scientific or medical literature; The group, generic, or common name, usually a shorter from of the chemical name or the one given by the discoverer; The trade or brand name given by the manufacturer to distinguish it from the product of other companies.

Some Antibiotics Produced By Microorganisms

BETA-LACTAM ANTIBIOTICS The Beta-lactam antibiotics are so-called because they have in their structure the four membered lactam ring. A lactam is a cyclic amide. It is named as such, because the nitrogen atom is attached to the β-carbon relative to the carbonyl. The Beta-lactam antibiotics inhibit the formation of the structure-conferring peptidoglycan of the bacterial cell wall. As this component is absent in mammalian cells, Beta-lactam antibiotics have very low toxicity towards mammal

The Beta-lactam antibiotics include the well-established and clinically important penicillins and cephalosphorins as well as some relatively newer members: cephamycins, nocardicins, thienamycins, and clavulanic acid. Except in the case of nocardicins these antibiotics are derivatives of bicyclic ring systems in which the lactam ring is fused through a nitrogen atom and a carbon atom to ring compound. This ring compound is five-membered in penicillins (thiazolidine), thienamycins (pyrroline) and clavulanic acid (oxazolidine); It is six-membered (dihydrothiazolidine) in cephalosporins and cephamycins.

The commercial production of penicillin

First discovered by Fleming in 1932 19% of worldwide antibiotic market. Superior inhibitory action on bacterial cell wall synthesis Broad spectrum of antibacterial activity Low toxicity Outstanding efficacy against various bacterial strains Excessive use has led to development of resistant pathogens

Characteristics of secondary metabolites: Secondary metabolites (idiolites) are produced from Substrates provided by primary metabolism. Characteristics of secondary metabolites: They are not essential for growth and reproduction. Their formation is extremely dependent on growth conditions. It is possible to get dramatic overproduction of secondary metabolites.

COMMERCIAL PRODUCTION OF PENICILLIN Originally used Penicillium notatum , now use Penicillium chrysogenum. Initially produced via surface mat culture. Problems! Inefficient, slow penicillin synthesis and contamination.

DEEP LIQUID CULTURE   Inoculum prepared until it represents ~ 5-10% of the volume of the fermentor. About 3-5 tonnes of wet mycelial mass will be used to inoculate a 50,000 litre fermentor.  The fermentors vary from 38,000-380,000 litres. Three distinct phases:  1. Trophophase: Rapid mycelial growth (30-40 hrs) Idiophase: Penicillin production via fed batch fermentation (5-7 days).

3. Carbon and nitrogen sources are depleted, antibiotic production ceases, the mycelia lyse releasing ammonia and the pH rises. Fermentor cooled by internal coils or external jackets (25-27oC). The pH is maintained between 6.8-7.4 by the automatic addition of H2SO4 or NaOH as necessary.  Oxygen added and mixed with mycelium.

Composition of early media % corn steep liquor (cotton seeds, peanut, CULTURE MEDIUM   Composition of early media % corn steep liquor (cotton seeds, peanut, Linseed or soybean meals) 2-4 lactose, glucose or beet molasses 2-4 CaCO3 or phosphates (buffer) 0.5-1 precursor 0.1-0.5 Catabolite repression of the enzymes responsible for penicillin biosynthesis occurs in high concentrations of glucose. Use of precursors to increase penicillin yield. 

Precursors of the appropriate side-chain are added to the fermentation. Thus if benzyl penicillin (penicillin G) is desired, phenylacetic acid is added. Phenyl acetic acid is nowadays added continuously as too high an amount inhibits the development of the fungus. High yielding strains of P. chrysogenum resistant to the precursors have therefore been developed.

Use of precursors:  

Extraction of penicillin after fermentation The broth is transferred to a settling tank. Penicillin is highly reactive and is easily destroyed by alkali conditions (pH 7.5-8.0) or by enzymes. It is therefore cooled rapidly to 5-10°C. The separation of penicillin is based on the solubility, adsorption and ionic properties of penicillin. Since penicillins are monobasic carboxylic acids they are easily separated by solvent extraction.

The fermentation broth is filtered with a rotary vacuum filter to remove mycelia and other solids and the resulting broth is adjusted to about pH 2 using a mineral acid. It is then extracted with a smaller volume of an organic solvent such as amyl acetate or butyl acetate, keeping it at this very low pH for as short a time as possible. The aqueous phase is separated from the organic solvent usually by centrifugation.

The organic solvent containing the penicillin is then typically passed through charcoal to remove impurities, after which it is back extracted with a 2% phosphate buffer at pH 7.5. The penicillin is then acidified once again with mineral acid (phosphoric acid) and the penicillin is again extracted into an organic solvent (e.g. amyl acetate). The product is transferred into smaller and smaller volumes, the penicillin becomes concentrated several times over, up to 80-100 times.

The penicillin may be converted to a stable salt form in one of several ways which employ the fact that penicillin is an acid: (a) it can be reacted with a calcium carbonate slurry to give the calcium salt which may be filtered, lyophilized or spray dried. (b) it may be reacted with sodium or potassium buffers to give the salts of these metals which can also be freeze or spray dried; (c) it may be precipitated with an organic base such as triethylamine.

NATURAL PENICILLINS They are destroyed by acid in the stomach. Sensitive to the enzyme penicillinase Effective against Gram +ve bacteria only.

SEMISYNTHETIC PENICILLINS

Chemical and Enzymatic Deacylation of Penicillins to 6-APA H R C N S S CH3 CH3 NH2 Penicillin acylase CH3 CH3 O Alkaline N N COOH [Enzymatic] COOH O O Penicillin V or G (6-APA) [R=Ph or PhO] [Chemical] PCl5 ROH H2O Pyridine Me3SiCl H R C N S CH3 CH3 O N COOSiMe3 O

6-Aminopenicillanic Acid (6-APA) Penicillin: 6-APA: Raw material for production of new semisynthetic penicillins (amoxycillin and ampicillin) Fewer side effects Diminished toxicity Greater selectivity against pathogens Broader antimicrobial range including G- -ve Improved pharmacological properties Gastric acid stability & oral absorbability Resistance to beta-lactamases

6-Aminopenicillanic Acid (6-APA) Chemical method: Use of hazardous chemicals - pyridine, phosphorous pentachloride, nitrosyl chloride Enzymatic method: Regio- and stereo-specific Mild reaction conditions (pH 7.5, 37 oC) Enzymatatic process is cheaper by 10% Enzymes: Penicillin G acylase (PGA)- Escherichia coli, Bacillus megaterium, Streptomyces lavendulae Penicillin V acylases (PVA)- Beijerinckia indica var. Penicillium, Fusarium sp., Pseudomonas acidovorans Immobilized Enzyme: Life, 500-2880 hours

Penicillin G Penicillinase (E.coli) 6 - APA Side Chain Modification Amoxycillin AUGMENTIN b-lactamase resistant Clavulanic acid

The Need for New Antibiotics The problem of multiple resistance to existing antibiotics The development of previously non-pathogenic microorganisms into pathogens Need to develop anti-fungal antibiotics Need to develop antibiotics specifically for agricultural purposes Need for anti-tumor and anti-parasitic drugs

Antibiotic Production Development of pilot plant production methods Submission of licence for clinical trials Testing of purified antibiotic Development of plant scale production methods Obtaining a product licence for clinical use Other considerations: Development of methods to control production of antibiotic Development of new applications Development of marketing and distribution system Financing of business Isolation or collection of cultures Screening of cultures to detect those with antimicrobial activity Development of methods for submerged-culture production Development of methods for isolation and purification of antibiotic Determination of antibiotic properties (physical: adsorption and absorption, chemical: reactions, solubility in solvents, stability to acids, alkalis, heat etc.) Evaluation of antibiotic Pharmacological tests Antimicrobial activity Comparison with existing antibiotic

The classical method for searching for antibiotics By random search in the soil. Although the first important commercially produced antibiotic was discovered by chance, most present day antibiotics were discovered by systematic search. The soil is a vast repository of microorganisms and it is to the soil that search is turned when antibiotics are being sought. The stages to be discussed below are not necessarily rigidly followed; they are merely meant to indicate in a general manner some of the activities involved in the development of antibiotics.

(i) The primary screening Several methods have been employed in primary screening. The crowded plate The direct-soil-inoculation method The cross-streak method The agar plug method The replica plating method

(a) The crowded plate A heavy aqueous suspension (1:10; 1:100) of soil is plated on agar. Organisms showing clear zones around themselves are isolated for further study. This method has the disadvantage that slow-growing antibiotic-producing organisms such as actinomycetes are usually over grown and are therefore hardly isolated. The susceptibility of soil organisms to the antibiotics produced in the test, may be unrelated to the susceptibility of clinically important organisms.

(b) The direct-soil-inoculation method This method is used when the aim is to isolate antibiotics against a known organism or organisms. Pour plates containing the test organisms are prepared. Soil crumbs or soil dilutions are then placed on the plates. Antibiotic producing organisms develop which then inhibit the growth of the organisms in the plate. They are recognized by the cleared zone which they produce around themselves and they may then be picked out.

(c) The cross-streak method This method is used for testing individual isolates, especially actinomycetes which may be obtained from soil without any previous knowledge of their antibiotic-producing potential. The organism may come from one of the two methods already indicated above. The purified isolate is streaked across the upper third of plate containing a medium which supports its growth as well as that of the test organisms. A variety of media may be used for streaking the antibiotic producer. It is allowed to grow for up to seven days, in which time any antibiotic produced would have diffused a considerable distance from the streak. Test organisms are streaked at right angles to the original isolates and the extent of the inhibition of the various test organisms observed.

The Cross Streak Method for the Primary Search of Antibiotic Producing Organisms

Testing of Antibiotic Producing Strains

(d) The agar plug method This method is particularly useful when the test organism grows poorly in the medium of the growth of the isolate such as fungi. Plugs about 0.5 cm in diameter are made with a sterile cork borer at progressive distances from the fungus. These plugs are then placed on plates with pure cultures of different organisms. The diameters of zones of clearing are used as a measure of antibiotic production of the isolate. The method may be used with actinomycetes.

(e) The replica plating method If a large number of organisms are to undergo primary screening, one rapid method is the use of replica plating. The method consists of placing a sterile velvet pad on the colonies formed in the crowded plate or soil inoculation plate, or on series of discrete colonies to be tested for antibiotic properties. The pad is thereafter carefully touched on four or five plates seeded with the test organisms. As a landmark is placed on the pad as well as on the plates it is possible to tell which colonies are causing the cleared zones on the tested plates.

Velvet pad

Replica Plating Method of Testing Antibiotic Producing Colonies

Secondary screening Organisms showing suitably wide zones of clearing against selected target organisms are cultivated in broth culture in shake flasks using components of the solid medium in which the isolate grew best. Crude methods of isolating the active antibiotic are developed by extracting the broth using a wide range of extractive methods. With each extraction the resultant material is assessed for activity against the target organisms at various dilutions. The extract is either spotted on filter paper discs placed on agar seeded with the test organism or introduced into wells dug out from the seeded agar with sterile cork borers.

The most efficient extractive methods and the spectrum of activity of the organisms are determined. Secondary screening is aimed at eliminating at an early stage any antibiotic which does not appear promising either by virtue of low activity, other undesirable properties or because it has been discovered previously.

Antibiotic spectrum The minimal inhibitory concentration (MIC) is a means of determining the activity of the isolated antibiotic and comparing this activity with those of existing antibiotics. Tests involving agar diffusion such as filter paper discs or agar wells described above are rapid and very useful for initial screening. Its ability to diffuse through agar. The MIC has the advantage that it is performed in broth thereby eliminating the disadvantage of large-molecule slower-diffusing antibiotics.

Other properties Toxicity to mammals, determined by intra peritoneal injection into animals; Haemolysis is tested by observing the effect on blood agar; Serum binding is tested by adding serum to the broth before testing against susceptible organisms; The inactivation of the antibiotic by several enzymes from various organs is tested by exposing the antibiotic to them; Acid stability is tested if the antibiotic is meant for oral fermentation For plant antibiotics, phytotoxicity as shown by damage to leaves in the laboratory and in the green house, is determined; For feed antibiotics, low absorbability and low toxicity are desirable and are tested.

(iv) Further laboratory evaluation: If and after all the above, the antibiotics is promising, then further experimentation is done in shake flasks as a preparation for pilot production. The optimal conditions of growth are determined; the most suitable medium, optimal pH, temperature, length of fermentation etc. are all determined. (v) Pilot plant production: The results obtained in previous experimentation are fed into the pilot plant. The material produced is subjected to further safety tests and chemical analysis.

(vi) Plant production: The production plant utilizes all the information obtained in the pilot experimentation. (vii) Certification: A government agency must approve the antibiotic before it becomes available for general use. (viii) Marketing and financing

Towards a new definition of ‘antibiotic’ The current definition of the term ‘antibiotic’ which restricts them to chemicals produced by microorganisms However, the higher organisms have been shown to produce anti-microbial substances. Such substances are low molecular weight secondary metabolites in the same way as regular antibiotics are. Due to this, there is now a tendency to extend the term antibiotic to all secondary metabolites, irrespective of their origin, which are able to inhibit various growth processes at low concentration. Even wholly synthetic antimicrobials such as ciprofloxacin are now legitimately termed antibiotics. The word antibiotic derives from two origins, anti (against) and bios (life). Nothing in the word itself restricts antibiotics both in origin or in use to microbial life.