ANTIBIOTICS Antibiotic Antimicrobial n Antibiotic Chemical produced by

ANTIBIOTICS

Antibiotic/Antimicrobial n Antibiotic: Chemical produced by a microorganism that kills or inhibits the growth of another microorganism n Antimicrobial agent: Chemical that kills or inhibits the growth of microorganisms

Selective Toxicity n Drugs that specifically target microbial processes, and not the human host cellular processes.

Antibiotics Naturally occurring antimicrobials – Metabolic products of bacteria and fungi – Reduce competition for nutrients and space n Bacteria that produce them: – Streptomyces, Bacillus, n Molds – Penicillium, Cephalosporium n

Fleming and Penicillin

In 1928, British bacteriologist Alexander Fleming (1881– 1955) discovered the bacteriakilling property of penicillin. Fleming noticed that a mold that had accidentally fallen into a bacterial culture in his laboratory had killed the bacteria. Having identified the mold as the fungus Penicillium notatum, Fleming made a juice with it that he named penicillin. After giving it to laboratory mice, he discovered it killed bacteria in the mice without harming healthy body cells. Although Fleming had made an incredible discovery, he was unable to produce penicillin in a form useful to doctors. 7

It was not until 1941 that two English scientists, Howard Florey (1898– 1968) and Ernst Chain (1906– 1979), developed a form of penicillin that could be used to fight bacterial infections in humans. 8

Selman Waksman 9

In 1943, American microbiologist Selman Waksman (1888– 1973) developed the drug streptomycin from soil bacteria. The discovery of streptomycin led to the development of a new class of drugs called aminoglycosides that include neomycin, kanamycin, and gentamicin. These antibiotics work against bacteria that are resistant to penicillin, but they tend to have many of the same side effects as streptomycin and are used only for a short time in cases of serious infection.

ANTIBIOTICS • Selectively toxic for bacteria – bactericidal (killing) – bacteriostatic (growth inhibition) no harm to patient 11

Administration of Antibiotics 12

Microbial Sources of Antibiotics

Spectrum of Activity

Antibacterial Antibiotics Inhibitors of Cell Wall Synthesis n Penicillin (over 50 compounds) – Share 4 -sided ring (b lactam ring) n Natural penicillins • Narrow range of action • Susceptible to penicillinase (b lactamase)

Cell wall synthesis Bactericidal – Penicillin and cephalosporins – binds and blocks peptidases involved in cross-linking the glycan molecules – Vancomycin – hinders peptidoglycan elongation – Cycloserine – inhibits the formation of the basic peptidoglycan subunits

Other Inhibitors of Cell Wall Synthesis n Cephalosporins – 2 nd, 3 rd, and 4 th generations more effective against gram-negatives Figure 20. 9

Cephalosporin n Cephalosporium acremonium (mold) n Widely administered today – Diverse group (natural and semisynthetic) n Structure – similar to penicillin except • Main ring is different • Two sites for R groups

Other Inhibitors of Cell Wall Synthesis n Polypeptide antibiotics – Bacitracin • Topical application • Against gram-positives – Vancomycin • Glycopeptide • Important "last line" against antibiotic resistant S. aureus

Inhibitors of Protein Synthesis Broad spectrum, toxicity problems n Examples n – Aminoglycosides: Streptomycin, neomycin, gentamycin – Tetracyclines – Macrolides: Erythromycin – Chloramphenicol

Tetracycline n Inhibits proteins synthesis n Broad spectrum and low cost n Commonly used to treat sexually transmitted diseases n Minor side effect – gastrointestinal disruption

Erythromycin Inhibits protein synthesis n Broad-spectrum n Commonly used as prophylactic drug prior to surgery n Side effects - low toxicity n

Chloramphenicol Broad-spectrum n Treat typhoid fever, brain abscesses n Rarely used now due to side effects – aplastic anemia n

Injury to the Plasma Membrane n Polymyxin B (Gram negatives)

Inhibitors of Nucleic Acid Synthesis n Rifamycin – Inhibits RNA synthesis – Antituberculosis n Quinolones and fluoroquinolones – Ciprofloxacin – Inhibits DNA gyrase – Urinary tract infections

Inhibition of Nucleic Acid Synthesis n Rifampin binds to DNA-dependent RNA polymerase and inhibits intiation of RNA synthesis

Summary of Targets

Initially penicillin was produced in milk bottles because the technology existed for the handling and filling of these vessels. Over time it became apparent that deep tank fermenters be used as they resulted in a great leapforward in process efficiency and productivity. Almost all antibiotics are produced in mechanically agitated and aerated vessels, the design of which is of crucial importance to the process. 32

Media used for production are extremely varied and most contain both complex nitrogen and carbohydrate sources. EACH PRODUCTION PROCESS IS GENERALLY DIVIDED INTO THE FOLLOWING KEY STAGES. • CULTURE PRESERVATION • INNOCULUM PREPARATION • SEED STAGE • PRODUCTION STAGE • HARVEST, EXTRACTION AND PURIFICATION 33

Culture Preservation Generally this preserved stock is in the form of inert Spores can be stored in dry soil, lyophilised or frozen in a preservation solution. 34

Scale Up It is essential to amplify the starting innoculum through a series of ever increasing volumes until enough material exists to innoculate the production system. A common chain would be 1 ml – 100 ml – 10 L – 1 m 3 - <1000 m 3 Innoculum resuspension – shake flask – pilot plant fermenter – seed fermenter – production fermenter 35

The growth medium is formulated to encourage biomass growth to the detriment of antibiotic production and the production medium simulates the required environmental stress required for the synthesis of antibiotics. Each medium is designed to support a particular stage of the process. Media designed for the early stage of the process will be generally focussed on achieving optimal spore germination and strong vegetative growth. As antibiotics are secondary metabolites, the final production medium will be focussed on the over production of the antibiotic. 36

As with any general growth media it must supply all the basic constituents for growth/product formation. • Carbon source • Nitrogen • Phosphates • Trace Elements

Most production fermenters in antibiotics are of traditional design. • Stainless steel construction • Vessel Height to Tank Diameter 2 -4: 1 • One Rushton Impeller per Tank Diameter in Height. • Air sparged into the vessel below bottom impeller between 0. 3 and 1. 5 VVM. • Power consumption in reactor in the order of 1 -4 W/L 38

Fermentation Fed batch is the most common approach. Lactose used historically as carbon source in batch cultures due to its slow rate of hydrolysis 39

Purification Mycelia are separated from the liquid by filtration occasionally using filter aids or precoats. Penicillin rich filtrate is cooled to 4 C to minimise chemical and enzyme degradation during solvent extraction. In the next stage of the process, penicillin is extracted into amyl acetate or butyl acetate using a continuous countercurrent process. 40

PENICILLIN CRYSTALS ARE WASHED AND PREDIRED WITH ANHYDROUS LPROPANOL, N-BUTANOL OR OTHER VOLATILE SOLVENT. FINAL DRYING IS ACCOMPLISHED USING VACUUM, WARM AIR OR RADIANT HEAT ON LARGE HORIZONTAL BELT FILTERS. 41