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Dr. E.P.C. Ejikeugwu (Ph.D.) Department of Applied Microbiology

Faculty of Science Ebonyi State University, Abakaliki, Nigeria

Website: www.microdok.com Facebook: MicroDok & MicroDok Research Group

AMB 421 (Pharmaceutical Microbiology) NOTE Topics:

Introduction to pharmaceutical microbiology Spectrum of activity and characteristics of antimicrobial agents Sources, clinical application, spectrum of activity, mode of action, resistance, side effects/toxicity,

of chemotherapeutic agents – sulphonamides, penicillins, cephalosporins, chloramphenicol, erythromycin, tetracycline, tuberculosis, therapy, antifungal and anti-protozoal drugs.

Production and synthesis of antibiotics and antimicrobial agents. Quality control of pharmaceutical products. Concept of growth and death of microorganisms. Medical and non-medical uses of antibiotics.

INTRODUCTION TO PHARMACEUTICAL MICROBIOLOGY Pharmaceutical microbiology is the branch of microbiology that focuses on all aspects of pharmacy or pharmaceutical sciences especially as it relates to the discovery, manufacture and quality control of pharmaceuticals and other biological agents such as antimicrobial agent, water for injection and vaccines. It is an applied branch of microbiology that focuses on the study of microorganisms that are directly or indirectly involved in the manufacture of pharmaceutical products. Pharmaceutical microbiologists ensure that starting raw materials for the manufacture of pharmaceuticals including water are sterile enough and free from any form of contaminating microorganisms. They carry out series of tests on starting materials for the manufacture of pharmaceuticals as well as test the finished products to ensure their safety and efficacy. Pharmaceutical microbiologists has contributed significantly in quality healthcare delivery across the world especially in the area of producing, testing, and delivering novel biologicals and medications for the effective management and treatment of infectious and non-infectious diseases globally. Pharmaceutical companies around the world are investing heavily in research and development (R&D); and they are also in high demand for pharmaceutical microbiologists due to the relevance of this branch of microbiology in the manufacture of safe, effective and good-quality pharmaceuticals. Pharmaceutical microbiology also deals with the controlling of microorganisms that cause spoilage of pharmaceutical products, and this area of microbiology is also keenly interested in harnessing the metabolic activities of microorganisms to develop novel and potent medicines and other pharmaceuticals for the healthcare and other related sector. The production of novel drugs from herbal plants and other natural products are also the subject of pharmaceutical microbiology.

SPECTRUM OF ACTIVITY AND CHARACTERISTICS OF ANTIMICROBIAL AGENTS The growth of pathogenic microorganisms is usually accompanied by the synthesis of new molecules including DNA, RNA and proteins – which are critical for the development of the organism. Microorganisms also acquire nutrient molecules from their surrounding environment or growth medium as their cells multiply and divide either in vivo or in vitro. Every drug has particular target site on pathogenic microorganisms when administered. The antimicrobial agent is mainly programmed or designed to disrupt and destabilize these specific target sites especially those that has to do with the growth of the invading pathogen (inclusive of bacteria, viruses, fungi and protozoa). To be more effective, antimicrobial agents also target different metabolic processes in the invading organism such as the inhibition of cell wall development and the blockage of the synthesis of important macromolecules such as DNA, RNA and protein molecules. Antimicrobial agents have different spectrum of activity or action; and this is usually taken into consideration when selecting drugs for a particular infection or disease.

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The spectrum of activity of an antimicrobial agent refers to the range of pathogenic microorganisms to which a particular drug is active against. It is a description of the general activity of an antimicrobial agent against particular microorganisms. Antimicrobial agents are usually divided into two groups based on their spectrum of activity, either as narrow spectrum drugs and broad spectrum drugs. Narrow spectrum agents are antimicrobial agents that have activity against a few groups of pathogenic microorganisms. Such agents can target either Gram-positive bacteria or only Gram-negative bacteria. Narrow spectrum drugs only have antimicrobial activity against a limited number of microorganisms. Broad spectrum agents are antimicrobial agents that have activity against a wide variety of pathogenic microorganisms. They are active against both Gram-positive and Gram-negative bacteria. Antimicrobial agents can also be categorized as “cidal agents” or “static agents” in terms of whether they kill or inhibit the growth of target microorganisms respectively. And the cidal- or static- nature of antimicrobial agents inclusive of antibiotics is usually summarized as the spectrum of activity of the drug. While “cidal agents” kill the target microorganisms, “static agents” only inhibit the growth of the target organisms. The activity of an antimicrobial agent thus describes the nature of the effect of the antimicrobial agent against particular microorganisms. For example, bactericidal agents are antibiotics that can kill bacteria while bacteriostatic agents are antibiotics that only inhibit the growth of bacteria. The term “cidal” and “static” is also applicable to antifungal agents, antiviral agents and antiprotozoal agents.

CHARACTERISTICS OR FEATURES OF ANTIMICROBIAL AGENTS

Antimicrobial agents must possess some features in order to qualify to be used for therapeutic purposes either in vivo or in vitro. Antimicrobial agents must generally be able to reach their target site on the invading pathogen. They must remain stable when administered. They must resist all forms of modification by the host cells or target organisms until their

antimicrobial activity must have been released. They must also have little or no untoward effect (side effects) when used for therapeutic purposes. They must have higher therapeutic index to be clinically effective for treating infectious diseases. They must be selectively toxic in their action. All drugs used for systemic or topical usage including

those that target bacteria, viruses, fungi and protozoa must be designed in such a way that they leave no adverse effect on the recipient host cells.

It should not be easily excreted from the body especially before it has performed its function. It should be chemically-stable and it should have a long shelf-life. It should not eliminate the normal flora of the host. It should have a wide spectrum of activity with the ability to destroy or inhibit many different species

of pathogenic organisms. It should be nontoxic to the host and without undesirable side effects. It should complement the activities of the host’s immune system. It should remain active in tissues and body fluids. Antibiotics should not be too costly so that their prescription should not be biased or based on their

price or cost.

SELECTIVE TOXICITY Selective toxicity is the ability of an antimicrobial agent to be injurious to a target pathogenic microorganism (i.e., kill or inhibit the growth of the microbe) without being detrimental to the recipient host. Normally, the selective toxicity of an antimicrobial agent could be determined by looking for specific targets on the pathogenic microorganism(s) which are actually lacking in the recipient host cell. For example, some drugs (e.g., penicillins) only target bacterial cell walls, and such antibiotics are selectively toxic in action because human cells (eukaryotic cells) do not have cell walls like bacteria (prokaryotic cells). Most antimicrobial agents (particularly drugs) target specific metabolic processes of microbial cells (inclusive of fungi, bacteria, viruses and protozoa) which are not available or obtainable in the normal metabolic activities of the recipient host cells. Such phenomenon makes the agent to be selectively toxic and thus useful for clinical and other therapeutic purposes in humans and/or animals. They must be able to disrupt a microbial function that is lacking in the recipient host taking the drug; and this makes the agent to have a greater selective toxicity than the drug that simultaneously targets a microbial function

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as well as a host cell’s function. Selective toxicity differentiates antibacterial drugs, antifungal agents, antiprotozoal and antiviral drugs from disinfectants which is also an example of antimicrobial agents because disinfectants (which are used mainly on inanimate objects to control microbial growth) are not selectively toxic in action, and may be harmful when used on the human body.

THERAPEUTIC INDEX Therapeutic index is the ratio of the toxic dosage of an antimicrobial agent compared to its therapeutic dosage. The toxic dosage of a drug is the concentration at which the agent becomes too poisonous to the recipient host, while the therapeutic dosage is the concentration of antimicrobial agent that is clinically relevant for treating a particular microbial disease.

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Both the therapeutic dosage and the toxic dosage of an antimicrobial agent determine the selective toxicity of a drug; and antibiotics with better selective toxicity always have higher therapeutic dosage than toxic dosage.

SOURCES, CLINICAL APPLICATION, SPECTRUM OF ACTIVITY, MODE OF ACTION, RESISTANCE, SIDE EFFECTS/TOXICITY OF CHEMOTHERAPEUTIC AGENTS –

SULPHONAMIDES, PENICILLINS, CEPHALOSPORINS, CHLORAMPHENICOL, ERYTHROMYCIN, TETRACYCLINE, TUBERCULOSIS, THERAPY, ANTIFUNGAL AND ANTI-PROTOZOAL DRUGS.

SULPHONAMIDES

Sulphonamides or sulpha drugs are generally known as folate synthesis inhibitors or antimetabolites because they inhibit the synthesis of folic acid, an important precursor for the synthesis of nucleic acids in pathogenic bacteria. They are antimetabolites; and antibiotics in this category include pyrimethamine, trimethoprim, sulphamethoxazole and sulphamethoxazole-trimethoprim. Antibiotics that are antimetabolites are growth factor analogues because they competitively fight for growth precursors (e.g., folic acid) in the target organism, and thus inhibit or disrupt cell division in bacteria. By inhibiting a key step in the synthesis of folic acid (an important precursor for the biosynthesis of bacterial DNA and RNA), the sulphonamides are generally known as nucleic acid synthesis inhibitors. SOURCES OF SULPHONAMIDES: Sulphonamides are synthetic antibacterial agents. They are generally produced by chemical modifications of sulphanilamide (originally known as prontosil). They are not synthesized by microorganisms as is the case for the other groups of antibacterial agents that are naturally sourced from microorganisms. STRUCTURE OF SULPHONAMIDES: The basic structure of the sulphonamides is sulphanilamide (Figure 1A). Sulphanilamide is a structural analogue of Para-Aminobenzoic Acid (PABA). This implies that sulphanilamide and PABA look much alike structurally. PABA is an important precursor in the synthesis of folic acid (Figure 1B) in bacteria.

Figure 1: General structure of sulphanilamide (A) and PABA (B). CLINICAL APPLICATION OF SULPHONAMIDES: Sulphonamides are clinically used treating urinary tract infections (UTIs), bacterial endocarditis, otitis media, lower respiratory tract infections, rheumatic fever, chlamydial infections and nocardiosis and some infections caused by protozoa. SPECTRUM OF ACTIVITY OF SULPHONAMIDES: Sulphonamides are broad spectrum antibiotics. They are active against pathogenic Gram-positive and some Gram-negative bacteria. Sulphonamides are bacteriostatic in action.

MODE OF ACTION: Sulphonamides are folate inhibitors. They compete for PABA, an important precursor required for the synthesis of folate or folic acid in bacteria. Pathogenic bacteria are inefficient to synthesize folate or folic acid; and thus they derive their folic acid from PABA (a structural analogue of the sulphonamides). Humans derive their folic acid from their daily dietary intake, and this makes the sulphonamides to be selectively toxic in action. PABA is structurally identical to sulphonamides; and thus

B A

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the antibiotic enters into reaction with PABA and competes for the active site of dihydropteroate synthetase. Dihydropteroate synthetase is an important enzyme that catalyzes the combination of PABA with other precursors in the early stages of folic acid synthesis in bacteria. Once the utilization of PABA is competitively inhibited by sulphonamides; the antibiotic becomes incorporated into the metabolic pathway for folate synthesis. This interferes with the biosynthesis of nucleic acids (DNA and RNA) in the bacteria. DNA and RNA direct cell division in bacteria; and when their synthesis is compromised (especially in the presence of antimetabolites such as the sulphonamides), bacterial growth will be inhibited and death of the pathogen may ensue.

BACTERIAL RESISTANCE TO SULPHONAMIDES: The clinical efficacy of the sulphonamides is compromised by the development of resistance in some pathogenic bacteria. Pathogenic bacteria that do not utilize extracellular folic acid but synthesize their own folate are resistant to sulphonamides.

SIDE EFFECT/TOXICITY OF SULPHONAMIDES: The sulphonamides rarely have untoward effects. However, side effects may include mild skin rashes, fever and gastrointestinal disturbances. Some patients develop hypersensitivity or allergic reactions after the oral administration of sulphonamides.

PENICILLINS Penicillins are beta-lactam drugs that inhibit the cross-linking of N-acetyl glucosamine (NAG) and N-acetyl muramic acid (NAM) required for the formation of peptidoglycan in bacterial cells. They are generally bacterial cell wall synthesis inhibitors. They specifically inhibit the activity of transpeptidases, enzymes that catalyzes the final cross-linking step in the synthesis of peptidoglycan or murein in bacteria (especially Gram-positive bacteria). Peptidoglycan or murein, an important component of bacterial cell wall gives bacteria their shape and rigidity; and once their synthesis is interfered with, a bacterium loses its structural integrity and becomes susceptible to harmful substances in its environment including antibiotics. SOURCE OF PENICILLIN: The natural penicillins are derived from moulds such as Penicillium chrysogenum and P. notatum through a fermentation process. Penicillin G (benzyl penicillin) and penicillin V (phenoxymethyl penicillin) are examples of naturally-synthesized penicillins. Ampicillin, oxacillin, methicillin and amoxycillin are examples of semi-synthesized penicillins (or beta-lactam drugs) produced by chemical modification of the 6-aminopenicillanic acid (produced naturally and by the fermentative activity of moulds especially Penicillium). Six (6)- aminopenicillanic acid is the general structure of penicillins. Addition of side chains to the 6-aminopenicillanic acid leads to the formation of semi-synthetic penicillins such as oxacillin.

STRUCTURE OF PENICILLINS: The general structure of penicillins is known as 6-aminopenicillanic acid (Figure 2). The structural integrity of 6-aminopenicillanic acid is important to the biological function of penicillins- whose main antimicrobial activity is to interfere with cell wall synthesis in the target pathogenic bacteria. Once the beta-lactam ring of 6-aminopenicillanic acid is cleaved by beta-lactamase enzymes (or penicillinase), a new compound known as penicilloic acid (devoid of any antibacterial activity) will be formed.

Figure 2: General structure of penicillins (6-aminopenicillanic acid).

Beta-lactam ring; the site for β-lactamase

Thiazolidine ring

Side chain

Site of action of amidase

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CLINICAL APPLICATION OF PENICILLINS: Penicillins are general purpose antibacterial drugs. They are used to treat infections caused by Gram positive and Gram negative bacteria including sore throat. SPECTRUM OF ACTIVITY OF PENICILLINS: Generally, the penicillins show both narrow spectrum of activity and broad spectrum of activity since they vary in their antibacterial activity which is dependent on the type of drug used and on the target microbe. For example, ticarcillin, ampicillin and carbenicillin show a broader spectrum of activity and can be used against Gram-negative and Gram-positive bacteria. But penicillin and methicillin show a narrower spectrum of activity and are mainly active against Gram-positive bacteria. Penicillins are bacteriostatic in action.

MODE OF ACTION OF PENICILLINS: Penicillins are cell wall inhibitors. Antibiotics in this category) inhibit the synthesis of cell wall only in actively growing bacteria. They have no activity on wall-less bacteria (e.g., mycoplasmas). The penicillins specifically bind to the penicillin-binding-proteins (PBPs) on the target bacterial cell. PBPs (e.g., transpeptidases and endopeptidases) are series of receptors found on the cell wall and cell membrane of bacteria. The main biological activity of the PBPs is to catalyze the cross-linking of the amino acid side chains with those of the glucan backbone of peptidoglycan. A successful cross-linking reaction catalyzed by the transpeptidases result in the formation of a rigid bacterial cell wall; and this whole process is known as transpeptidation reaction. Penicillins bind to transpeptidases, and this prevents PBPs from carrying out its biological function of cell wall synthesis in bacteria. An incomplete cell wall is synthesized, and this leads to the death of the bacterium due to the influx of water and other harmful substances into the high osmotic pressure of the bacterial cell. BACTERIAL RESISTANCE TO PENICILLINS: Most penicillin-derivatives are inactivated by penicillinases or beta-lactamase enzymes which makes these drugs to be less-effective for clinical applications. Pathogenic bacteria produce antibiotic-degrading enzymes (e.g., beta-lactamase enzymes) which hydrolyze penicillins by binding to the beta-lactam ring of 6-aminopenicillanic acid. Some bacteria mutate and alter their PBPs so that there will be a decreased binding of the antibiotic to the target PBPs. Others use the influx-efflux mechanism to stimulate the exclusion of the drug from their internal environment; and this prevents the intracellular accumulation of the antibiotic necessary to exert their injurious activity inside the bacterial cell.

SIDE EFFECT/TOXICITY OF PENICILLIN: Penicillins are generally nontoxic antibiotics. They are usually the least toxic of all the antibiotics. Untoward effect resulting from the clinical use of penicillins is usually due to hypersensitivity or allergic reaction to the drug. Mild diarrhea can occur in some patients when penicillin is administered orally.

CEPHALOSPORINS Cephalosporins are beta-lactam antibiotics that are penicillinase-resistant, and with related mode of action to the penicillins. Drugs in this category are clinical substitutes for penicillins due to the development of resistance to penicillins by pathogenic bacteria. Cephalosporins are classified into five (5) major generations based on their spectrum of activity, side chain modifications and clinical applications. These five major generations of cephalosporins include first-, second-, third-, fourth-, and fifth- generation cephalosporins.

FIRST GENERATION CEPHALOSPORINS: The 1st-generation cephalosporins are active against Gram-positive bacteria. They include cephalexin, cephalothin, cephapirin, cefazolin, cephadrine and cefadrox. They have a narrow-spectrum of activity against pathogenic bacteria especially the Gram-negative rods (e.g. and Escherichia coli).

SECOND GENERATION CEPHALOSPORINS: The 2nd-generation cephalosporins were designed to counter the resistance of Gram-negative bacteria (mediated by beta-lactamase production) to the 1st-generation cephalosporins. They include cefaclor, cefotetan, cefuroxime, and cefoxitin.

THIRD GENERATION CEPHALOSPORINS: The 3rd-generation cephalosporins include ceftazidime, cefotaxime, and ceftriaxone. Antibiotics in this category are more effective against Gram-negative rods than the 2nd-generation cephalosporins. They are the preferred antibiotic of choice in life-threatening bacterial diseases or infections of yet unknown cause.

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FOURTH GENERATION CEPHALOSPORINS: The 4th-generation cephalosporins have extended antibacterial spectrum. They include cefepime and cefpirome. Fourth (4th)-generation cephalosporins have activity against Enterobacter species, Citrobacter species, Neisseria species, Haemophilus species, Enterobacteriaceae and a wide variety of Gram-negative bacteria.

FIFTH GENERATION CEPHALOSPORINS: The 5th-generation cephalosporins are usually administered intravenously (IV). They are active against a variety of multidrug resistant bacteria pathogens including the dreaded methicillin resistant Staphylococcus aureus (MRSA). The 5th-generation cephalosporins include Ceftaroline Fosamil (IV) and Ceftobiprole (IV).

SOURCES OF CEPHALOSPORINS: Cephalosporins are naturally sourced from the fungus Cephalosporium (e.g. C. acremonium). However, cephalosporins can also be produced semi-synthetically in the laboratory by the addition of substituent groups to the side chains of the 7-aminocephalosporanic acid (7-ACA) structure.

STRUCTURE OF CEPHALOSPORINS: The basic structure of the cephalosporins is known as 7-aminocephalosporanic acid (Figure 3). Addition of substituent groups to this basic structure result in the production of different variants or generations of cephalosporins. Figure 3: Structure of cephalosporins (7-aminocephalosporanic acid) CLINICAL APPLICATION OF CEPHALOSPORINS: Clinically, the cephalosporins are effective against a wide variety of pathogenic bacteria especially the Gram-negative rods to which the penicillins have little or no activity against. 1st-generation cephalosporins are very active against Gram-positive bacteria (e.g. Staphylococcus species). Second (2nd)-generation cephalosporins are effective against Gram-negative rods (e.g. Klebsiella species and Proteus species) but not against Pseudomonas species. Third (3rd)-generation cephalosporins are active against Pseudomonas species and a variety of Enterobacteriaceae. Fourth (4th)-generation cephalosporins are active against a variety of pathogenic bacteria including those that are resistant to 3rd-generation cephalosporins.

SPECTRUM OF ACTIVITY OF CEPHALOSPORINS: Cephalosporins (including the 1st, 2nd, 3rd, 4th, and 5th-generations) have a broader spectrum of activity. They are bacteriocidal in action. Cephalosporins have activity against both Gram-positive and Gram-negative bacteria.

MECHANISM OR MODE OF ACTION OF CEPHALOSPORINS: Cephalosporins like the penicillins are cell wall synthesis inhibitors. They bind to the PBPs on bacterial cell wall. Cephalosporins interfere with the synthesis of peptidoglycan in growing bacteria. They have similar mode of action like the penicillins but cephalosporins have increased stability to beta-lactamases produced by Gram-negative bacteria than the penicillins.

BACTERIAL RESISTANCE TO CEPHALOSPORINS: Though very effective against a wide variety of pathogenic bacteria, most cephalosporins are fast becoming susceptible to some antibiotic-degrading enzymes such as the ESBLs and other multidrug resistance factors produced by bacteria. SIDE EFFECT OF CEPHALOSPORINS: Untoward effects due to the usage of cephalosporins are attributed to the host’s allergic reactions to the drug as is applicable to the use of penicillins. But hypersensitivity to cephalosporins is fewer compared to that produced by penicillins.

CHLORAMPHENICOL

Chloramphenicol is a protein synthesis inhibitor but the antibiotic unlike other drugs that interfere with bacterial protein biosynthesis (e.g., tetracycline and aminoglycosides) binds to the 50S ribosomal subunit of the target bacterial ribosome the same manner that macrolides (e.g., erythromycin) exhibit their antibacterial activity. SOURCES OF CHLORAMPHENICOL: Chloramphenicol is a naturally synthesized antibiotic. It is naturally biosynthesized by bacteria in the genus Streptomyces (particularly Streptomyces

venezuelae). Chloramphenicol was first isolated in 1947 from a soil sample in Venezuela. It is primarily synthesized by S. venezuelae.

STRUCTURE OF CHLORAMPHENICOL(Figure 4). Chemical modification of the basic structure of chloramphenicol (i.e. the nitrobenzene ring) leads to the production of synthetic forms of the antibiotic. CLINICAL APPLICATION OF CHLORAMPHENICOLchoice for typhoid fever, bacterial meningitis and infectious diseases caused by some intracellular bacterial parasites such as Rickettsial SPECTRUM OF ACTIVITY OF CHLORAMPHENICOLbroad spectrum of activity and it is effective against Gram-negative bacteria. And the antibiotic is mainly bacteriostatic in action.

Figure 4 MECHANISM OR MODE OF ACTION OF CHLORAMPHENICOLtranslation or protein synthesis in pathogenic bacteria by binding to the 50S ribosomal subunit. This binding blocks the activities of peptidyl transferase which is mainly responsible for the elongatpolypeptide bonds during protein biosynthesis in bacteria. The interference of the activities of the enzyme, peptidyl transferase by chloramphenicol generally blocks peptide bond formation, and this also prevents the formation and/or elongation of tbacteria.

BACTERIAL RESISTANCE TO CHLORAMPHENICOLusually mediated by plasmids. Chloramphenicol resistance may be carried on a bacterial plasmid that also codes for resistance to other drug classes such as tetracycline and ampicillin. permeability, mutation of the 50S ribosomal subunit, and the elaboration of chloramphenicol acetyltransferase are other resistance mechanisms SIDE EFFECT/TOXICITY OF CHLORAMPHENICOLuntoward effects which have genermarrow development and aplastic anaemia in patients. Other side effects include respiratory dysfunction and allergic reactions in the recipient host. Chloramphenicol is contraindicated in pregwomen and infants because of its notable toxicity to the unborn

Erythromycin is a protein synthesis inhibitor that binds to the 50S ribosomal subunit of the bacterial ribosome. It is found in the family of antibiotics known as the macrolides. Antibiotics in this group are generally protein synthesis inhibitors. Other macrolides inc SOURCES OF ERYTHROMYCINErythromycin is synthesized naturally from a strain of can also be semi-synthesized by the chemical modification of their general structure

STRUCTURE OF ERYTHROMYCINcontains large lactone rings that are linked through glycoside bonds with amino sugars. They generally possess a macrocyclic (Figure 5). The macrolides structure is usually composed of a largemembered ring that comprises of about 13chemically linked to sugar molecules by glycosidic bonds. The chemical structure of erythromycin is shown in

). Chloramphenicol was first isolated in 1947 from a soil sample in Venezuela. It is primarily

STRUCTURE OF CHLORAMPHENICOL: The structure of chloramphenicol is a nitrobenzene ring ). Chemical modification of the basic structure of chloramphenicol (i.e. the nitrobenzene ring)

leads to the production of synthetic forms of the antibiotic.

CLINICAL APPLICATION OF CHLORAMPHENICOL: Clinically, chloramphenicol is the drug of choice for typhoid fever, bacterial meningitis and infectious diseases caused by some intracellular

Rickettsial species and Chlamydial species.

SPECTRUM OF ACTIVITY OF CHLORAMPHENICOL: Chloramphenicol has a nd it is effective against pathogenic Gram-positive and

negative bacteria. And the antibiotic is mainly bacteriostatic in action.

Figure 4: Chemical structure of chloramphenicol.

E OF ACTION OF CHLORAMPHENICOL: Chloramphenicol inhibits translation or protein synthesis in pathogenic bacteria by binding to the 50S ribosomal subunit. This binding blocks the activities of peptidyl transferase which is mainly responsible for the elongatpolypeptide bonds during protein biosynthesis in bacteria. The interference of the activities of the enzyme, peptidyl transferase by chloramphenicol generally blocks peptide bond formation, and this also prevents the formation and/or elongation of the peptide chain during protein synthesis in the target

BACTERIAL RESISTANCE TO CHLORAMPHENICOL: Bacterial resistance to chloramphenicol is usually mediated by plasmids. Chloramphenicol resistance may be carried on a bacterial plasmid that also codes for resistance to other drug classes such as tetracycline and ampicillin. Reduced membrane

y, mutation of the 50S ribosomal subunit, and the elaboration of chloramphenicol acetylare other resistance mechanisms.

SIDE EFFECT/TOXICITY OF CHLORAMPHENICOL: Chloramphenicol is a toxic antibiotic. Its untoward effects which have generally compromised its clinical usage include suppression of bone marrow development and aplastic anaemia in patients. Other side effects include respiratory dysfunction and allergic reactions in the recipient host. Chloramphenicol is contraindicated in pregwomen and infants because of its notable toxicity to the unborn child in a pregnant woman

ERYTHROMYCIN

is a protein synthesis inhibitor that binds to the 50S ribosomal subunit of the bacterial ribosome. It is found in the family of antibiotics known as the macrolides. Antibiotics in this group are generally protein synthesis inhibitors. Other macrolides include azithromycin and clarithromycin.

SOURCES OF ERYTHROMYCIN: Macrolides are naturally-synthesized by Streptomyces Erythromycin is synthesized naturally from a strain of Streptomyces known as S. erythreus

d by the chemical modification of their general structure

STRUCTURE OF ERYTHROMYCIN: The chemical structure of macrolides contains large lactone rings that are linked through glycoside bonds with amino sugars. They generally possess a macrocyclic lactone structure

). The macrolides structure is usually composed of a large-membered ring that comprises of about 13-15 carbon molecules which are chemically linked to sugar molecules by glycosidic bonds. The chemical

shown in Figure 6. Figure 5: Structure of a macrolide antibiotic.

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). Chloramphenicol was first isolated in 1947 from a soil sample in Venezuela. It is primarily

chloramphenicol is a nitrobenzene ring ). Chemical modification of the basic structure of chloramphenicol (i.e. the nitrobenzene ring)

Clinically, chloramphenicol is the drug of choice for typhoid fever, bacterial meningitis and infectious diseases caused by some intracellular

Chloramphenicol inhibits translation or protein synthesis in pathogenic bacteria by binding to the 50S ribosomal subunit. This binding blocks the activities of peptidyl transferase which is mainly responsible for the elongation of polypeptide bonds during protein biosynthesis in bacteria. The interference of the activities of the enzyme, peptidyl transferase by chloramphenicol generally blocks peptide bond formation, and this also

he peptide chain during protein synthesis in the target

Bacterial resistance to chloramphenicol is usually mediated by plasmids. Chloramphenicol resistance may be carried on a bacterial plasmid that

educed membrane y, mutation of the 50S ribosomal subunit, and the elaboration of chloramphenicol acetyl-

Chloramphenicol is a toxic antibiotic. Its ally compromised its clinical usage include suppression of bone

marrow development and aplastic anaemia in patients. Other side effects include respiratory dysfunction and allergic reactions in the recipient host. Chloramphenicol is contraindicated in pregnant

child in a pregnant woman.

is a protein synthesis inhibitor that binds to the 50S ribosomal subunit of the bacterial ribosome. It is found in the family of antibiotics known as the macrolides. Antibiotics in this group are

lude azithromycin and clarithromycin.

Streptomyces species. S. erythreus. Macrolides

Structure of a macrolide antibiotic.

Figure 6. Chemical structure of erythromycin. CLINICAL APPLICATION OF ERYTHROMYCINof whooping cough (caused by Bordetella pertussisdiphtheria (caused by Corynebacterium diphtheriaediarrhea (caused by Campylobacter infections such as toxoplasmosis (caused by SPECTRUM OF ACTIVITY OF ERYTHROMYCINbacteriostatic in action. They have broad spectrum of activity since they are active against both pathogenic Gram-positive bacteria and Gramaction, macrolides can be bacteriocidal in action to some pathogenic Gram MECHANISM OR MODE OF ACTION OF MACROLIDESgenerally inhibit bacterial protein synthesis by binding to the 50S ribosomal subunit of the bribosome (particularly the 23S rRNA). Binding of the 50S ribosomal subunit by erythromycin inhibits protein elongation (usually mediated by the enzyme, peptidyl transferase). This prevents translocation of the ribosome during protein synthesis. Trthe ribosome (especially the mRNA) to other parts of the ribosome (e.g.synthesis. In particular, genetic information required for the synthesis of a particular protein molecule(as encoded in the gene or DNA) are translocated or moved from one part of the ribosome to another after transcription must have taken place. (mRNA) to the transfer RNA (tRNA), and then to the ribosomacompleted in the ribosome. Erythromycin blockssynthesis when they reversibly bind to the 50S ribosomal subunit.

BACTERIAL RESISTANCE TO ERYTHROMYCINprevents intracellular accumulation of the antibioticalteration of the binding site of the antibiotic on the target organism.

SIDE EFFECT/TOXICITY OF ERYTHROMYCINmacrolides is usually associated with some undesirable untoward effects such as mild stomach or GIT.

Tetracyclines are general purpose antibiotics used for a variety of clinical applications. They include tetracycline, chlortetracycline, oxytetracycline, dimethyl chlortetracycline, minocycline and doxycycline. Antibiotics in this category are protein synthesis inhibitothe 30S ribosomal subunit of bacterial ribosomes during protein synthesis. SOURCES OF TETRACYCLINES: Streptomyces. The species of Streptaureofaciens and S. rimosus which synthesize chlortalso be synthesized by semi-synthetic and other synthetic processes.

STRUCTURE OF TETRACYCLINEStetracyclines is composed of a fourwhich substituent groups are added to form newer tetracyclines such as minocycline and doxycycline.

Chemical structure of erythromycin.

CLINICAL APPLICATION OF ERYTHROMYCIN: Erythromycin is active against the causative agents Bordetella pertussis), pneumonia (caused by Streptococcus pneumoniae

Corynebacterium diphtheriae), syphilis (caused by Treponema pallidumCampylobacter species). Macrolides can also be used to treat some non

infections such as toxoplasmosis (caused by Toxoplasma gondii) and infections caused by mycobacteria.

SPECTRUM OF ACTIVITY OF ERYTHROMYCIN: Erythromycin and the macrolides are generally bacteriostatic in action. They have broad spectrum of activity since they are active against both

positive bacteria and Gram-negative bacteria. Though generally bacteriostatic in des can be bacteriocidal in action to some pathogenic Gram-positive bacteria.

MECHANISM OR MODE OF ACTION OF MACROLIDES: Erythromycin and the other macrolides generally inhibit bacterial protein synthesis by binding to the 50S ribosomal subunit of the bribosome (particularly the 23S rRNA). Binding of the 50S ribosomal subunit by erythromycin inhibits protein elongation (usually mediated by the enzyme, peptidyl transferase). This prevents translocation of the ribosome during protein synthesis. Translocation in this case is the movement of some part of the ribosome (especially the mRNA) to other parts of the ribosome (e.g., tRNA) during protein synthesis. In particular, genetic information required for the synthesis of a particular protein molecule(as encoded in the gene or DNA) are translocated or moved from one part of the ribosome to another after transcription must have taken place. This genetic information flows from the messenger RNA (mRNA) to the transfer RNA (tRNA), and then to the ribosomal RNA (rRNA) where protein synthesis is

. Erythromycin blocks the elongation of the polypeptide chain during synthesis when they reversibly bind to the 50S ribosomal subunit.

BACTERIAL RESISTANCE TO ERYTHROMYCIN: Resistance is by influx-efflux mechanism which ellular accumulation of the antibiotic. Microbial resistance can also occur as a result of

alteration of the binding site of the antibiotic on the target organism.

SIDE EFFECT/TOXICITY OF ERYTHROMYCIN: The clinical usage of erythromycin and other macrolides is usually associated with some undesirable untoward effects such as mild stomach or GIT.

TETRACYCLINES are general purpose antibiotics used for a variety of clinical applications. They include

tetracycline, chlortetracycline, oxytetracycline, dimethyl chlortetracycline, minocycline and doxycycline. Antibiotics in this category are protein synthesis inhibitors like the macrolides, but tetracyclines bind to the 30S ribosomal subunit of bacterial ribosomes during protein synthesis.

: The tetracyclines are naturally synthesized from the bacteria genus Streptomyces that synthesize tetracyclines naturally include

which synthesize chlortetracycline and oxytetracycline. Tetracyclines can synthetic and other synthetic processes.

TETRACYCLINES: The general structure of the tetracyclines is composed of a four-fused benzene ring (Figure 7) to which substituent groups are added to form newer tetracyclines such

Deoxy SugarResidues

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Erythromycin is active against the causative agents Streptococcus pneumoniae), Treponema pallidum) and

species). Macrolides can also be used to treat some non-bacterial infections caused by mycobacteria.

Erythromycin and the macrolides are generally bacteriostatic in action. They have broad spectrum of activity since they are active against both

negative bacteria. Though generally bacteriostatic in positive bacteria.

Erythromycin and the other macrolides generally inhibit bacterial protein synthesis by binding to the 50S ribosomal subunit of the bacterial ribosome (particularly the 23S rRNA). Binding of the 50S ribosomal subunit by erythromycin inhibits protein elongation (usually mediated by the enzyme, peptidyl transferase). This prevents translocation

is the movement of some part of tRNA) during protein

synthesis. In particular, genetic information required for the synthesis of a particular protein molecule (as encoded in the gene or DNA) are translocated or moved from one part of the ribosome to another

genetic information flows from the messenger RNA l RNA (rRNA) where protein synthesis is

the elongation of the polypeptide chain during

efflux mechanism which . Microbial resistance can also occur as a result of

inical usage of erythromycin and other macrolides is usually associated with some undesirable untoward effects such as mild stomach or GIT.

are general purpose antibiotics used for a variety of clinical applications. They include tetracycline, chlortetracycline, oxytetracycline, dimethyl chlortetracycline, minocycline and doxycycline.

rs like the macrolides, but tetracyclines bind to

The tetracyclines are naturally synthesized from the bacteria genus that synthesize tetracyclines naturally include S.

Tetracyclines can

Deoxy Sugar Residues

9

Figure 7: General structure of tetracyclines. CLINICAL APPLICATION OF TETRACYCLINES: Tetracyclines are usually used for blind treatment in clinical medicine. Blind treatment is a type of treatment administered prior to formal laboratory test results. They are often the drug of choice for some bacterial infections such as those caused by Brucella, Chlamydia, Vibrio, Francisella and Yersinia, Chlamydia and Rickettsia and some protozoa.

SPECTRUM OF ACTIVITY OF TETRACYCLINES: The tetracyclines are broad spectrum antibiotics. They have antibacterial activity against pathogenic Gram-positive bacteria and Gram-negative bacteria. They are bacteriostatic in action. MECHANISM OR MODE OF ACTION OF TETRACYCLINES: Tetracyclines are protein synthesis inhibitors. They act by preventing the binding of aminoacyl tRNA to the acceptor (A) site on bacterial ribosome. The binding of tetracyclines to the 30S ribosomal subunit blocks the attachment of aminoacyl-tRNA to the acceptor site on mRNA (i.e., the 30S ribosomal subunit).This interferes with protein synthesis by inhibiting the elongation of the polypeptide chain. BACTERIAL RESISTANCE TO TETRACYCLINES: Pathogenic bacteria that fail to concentrate the antibiotic intracellularly become resistant to the agent. The alteration of the cell membrane of the target bacteria and inactivation of the antibiotic by bacterial enzymes can compromise the clinical efficacy of the tetracyclines. SIDE EFFECT/TOXICITY OF TETRACYCLINES: The untoward effect associated with the clinical usage of tetracyclines is minimal, and the antibiotic has a low toxicity compared to other antibacterial agents. However, the use of tetracyclines in children (between ages 0-8 years old) has been associated with tooth decolouration (seen as yellowing of the tooth) and impairment of bone development due to the high affinity of the antibiotic for calcium, the main component of bones and teeth. Tetracycline is also contra-indicated for pregnant women because the antibiotic reacts with calcium phosphate when administered for a long time; and this generally affects bone development in the foetus. Other side effects include nausea, vomiting, skin rashes, diarrhea, and disruption of the normal bacterial flora in the intestines. Alteration of the normal flora of the intestines by the overuse or prolonged usage of tetracyclines predisposes the recipient human hosts to other bacterial-related infections and opportunistic diseases (e.g., candidiasis).

OVERVIEW OF ANTI-TUBERCULOSIS (TB) DRUGS AND ANTIFUNGAL AGENTS

Anti-tuberculosis drugs are groups of antimicrobial agents that are used for the management and treatment of infections and/or disease caused by the Gram-negative, acid-fast and rod-like bacterium known as Mycobacterium tuberculosis. M. tuberculosis is the main causative agent of tuberculosis (TB). Prevention is usually better than cure when it comes to TB. BCG vaccine which consists of a live attenuated strain derived from Mycobacterium bovis is used in immunization to contain the development and spread of TB disease in the human race. BCG is the acronym for: Bacillus of Calmette and Guerin, which was named after the two Frenchmen that developed the vaccine. Isoniazid (INH), rifampin (RIF), pyrazinamide (PZA) and ethambutol (EMB) and streptomycin are examples of anti-TB drugs used for clinical management and treatment of tuberculosis (TB).

ISONIAZID Isoniazid or isonicotinyl hydrazine (INH) is a first-line antibiotic used for the treatment of tuberculosis (TB), a droplet bacterial infection caused by Mycobacterium tuberculosis. TB is a highly infectious diseases that is mainly spread via the respiratory tract of infected persons to susceptible human hosts through sneezes, cough and saliva or aerosols that contain the infectious particle of the pathogenic organism. Other first-line anti-tuberculosis drugs include ethambutol, streptomycin, pyrazinamide and rifampicin. The second-line drugs used for the treatment of tuberculosis especially in cases of drug resistance or when antimicrobial synergistic effect is warranted include para-aminosalicylic acid (PAS), cycloserine, kanamycin (an aminoglycoside), ethionamide and fluoroquinolones (e.g., ciprofloxacin). Isoniazid is a cell wall synthesis inhibitor, but it is only effective

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on the cell wall of Mycobacterium species (particularly those that cause TB) which contain a different type of element in their cell wall (i.e., mycolic acid). The presence of high content of mycolic acid exterior to the mycobacterial peptidoglycan layer makes the mycobacteria to be resistant to most antibacterial drugs inclusive of some anti-TB agents.

SOURCE AND STRUCTURE OF ISONIAZID: INH and other first-line anti-TB drugs are chemically synthesized antibacterial drugs. Anti-TB drugs are also produced by semi-synthetic processes. But rifampicin is a semi-synthetic derivative of rifamycin, an antibiotic produced by Streptomyces mediterranei. The structure of isoniazid (INH) is a benzene nucleus (Figure 8).

Figure 8: Chemical structure of isoniazid (INH). CLINICAL APPLICATION OF ISONIAZID: Anti-TB drugs are used in combination with each other for the effective treatment of TB, in order to avoid the development of resistance due to the use of a single anti-TB drug. But multidrug resistant tuberculosis (MDR TB) and extensively drug resistant TB (XDR TB) now exist. These pathogens (MDR TB and XDR TB microbes) complicate the effective treatment of TB because they are virtually resistant to some of the known available drugs used for the clinical management of TB infections. The standard TB treatment according to WHO is normally administered in two phases viz: an initial phase which last for about 2-3 months using a combination of 3 anti-TB agents from the first-line TB drugs (inclusive of INH, RIF and EMB) and a continuation phase which spans a period about 8 months, and in which two of any of the first-line drugs are co-administered to the patient. Both the initial stage and continuation stage are administered under a procedure known as directly observed therapy (DOT). DOT is a therapeutic procedure in which TB patients are given drugs based on the supervision of a health personnel or physician in a health center. DOT ensures that the TB patients follow the actual course of their therapy, as the possibility of not adhering strictly to it may arise due to the large number of drugs involved in TB treatment.

SPECTRUM OF ACTIVITY OF ISONIAZID: Isoniazid and other anti-TB drugs are bacteriocidal in action. They are broad spectrum drugs.

MECHANISM OR MODE OF ACTION OF ISONIAZID: INH acts on the cell wall of mycobacteria by inhibiting the synthesis of mycolic acid, a fatty acid found mainly in mycobacteria. PAS is an antimetabolite that inhibits the biosynthesis of folate or folic acid in mycobacteria the same way that the sulphonamides act in other pathogenic bacteria. EMB inhibits the incorporation of mycolic acids into the mycobacterial cell wall. RIF is a protein synthesis inhibitor in mycobacteria; and it is also effective against some pathogenic Gram-positive bacteria and some Gram-negative rods. Streptomycin like rifampin is a protein synthesis inhibitor. They interfere with the protein synthesis machinery of the mycobacterial cell wall and thus stop protein synthesis.

BACTERIAL RESISTANCE TO ISONIAZID: Mycobacteria easily develop resistance to antimicrobial agents and the mycolic acid content of their cell wall is a formidable force that expels most antibiotics. Mutation in the active sites of anti-TB drugs on mycobacteria is one of the factors that mediate resistance in M. tuberculosis.

ANTI-FUNGAL AGENTS Antifungal agents are antimicrobial agents that kill or inhibit the growth of pathogenic fungi. Fungal infection is treated using antifungal drugs. Some of the drugs used for the treatment of mycoses in humans include polyenes (e.g., amphotericin B and nystatin) which are cidal in action and binds to the fungal ergosterol membranes to disrupt the integrity of the fungal cell membrane; azoles (e.g., itraconazole, ketoconazole, voriconazole, fluconazole, miconazole) which are static in action and inhibit the synthesis of ergosterol; griseofulvin which is static in action and inhibits fungal growth by binding to microtubules during mitosis; 5-fluorocytosine or flucytosine which is an antimetabolite and inhibits nucleic acid synthesis and protein synthesis; echinocandins (e.g., caspofungin, micafungin) which are static in action and inhibits the synthesis of chitin and glucan in fungal cell wall and allylamines (e.g., terbinafine) which are static in action and inhibit the synthesis of ergosterol like the azoles. Cycloheximide is an antifungal agent that inhibits the growth of saprophytic fungi (i.e., non-pathogenic

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fungi) found in the environment. They are usually incorporated into fungal culture media (e.g., Sabouraud dextrose agar) to prevent the growth of saprophytic fungi. Antimicrobial agents that inhibit the growth of pathogenic fungi are generally known as fungistatic agents while those that kill fungi are known as fungicidal agents. There are fewer antifungal agents than antibacterial agents because fungi like the mammalian cells are eukaryotic organisms. Drugs used for the treatment of fungal infections or human mycoses have profound untoward effects on their recipient human hosts because both humans and fungi have eukaryotic cells. Since fungi and human cells share similar cellular and metabolic similarities because they are both eukaryotes, it is difficult to find suitable antifungal agents with little or no toxicity. Many antifungal agents are too toxic for the clinical management of human mycoses. Antifungal agents have poor selective toxicity compared to antibacterial drugs which target prokaryotic cells (e.g., bacteria); and a considerable amount of antifungal agents have adverse pharmacological features which compromises their usage in clinical medicine. They also have low therapeutic index compared to antibacterial agents with higher therapeutic index. This is because most antifungal agents disrupts metabolic functions found not only in pathogenic fungi but also in the animal or human host cells taking the antifungal agent; and this is because of similarities in the cellular makeup of fungi and animal or human cells. Therefore, most antifungal agents are produced in topical application forms – in which they are applied on the skin. And such topical applications are used to treat fungal infections caused by dermatophytes. Antifungal agents like antibacterial agents target specific sites of their target pathogenic fungi including the fungal cell wall, nucleic acid and cell membranes, even though some of these agents have low selective toxicity when used for treatment.

NUCLEIC ACID SYNTHESIS INHIBITORS

Some fungal agents are classified as nucleic acid inhibitors because they interfere with the biosynthesis of DNA and RNA which are both critical for the overall development of the target pathogenic fungi. Flucytosine or 5-fluorocytosine is a typical example of antifungal agent that inhibits the synthesis of nucleic acids in pathogenic fungi. Flucytosine or 5-fluorocytosine (5-FC) is a synthetic antifungal agent that is used for the treatment of some fungal infections. Flucytosine (Figure 9) is an oral antifungal agent that acts as an antimetabolite. Figure 9: Chemical structure of 5-FC. The drug is an analogue of cytosine, and 5-FC inhibits the synthesis of nucleic acids (e.g., DNA) and protein synthesis in fungal cells. CLINICAL APPLICATION OF 5-FC: 5-FC has in vitro and in vivo activity against Candida and Cryptococcus. 5-FC is clinically used for the treatment of systemic mycoses, and the agent is active against Candida species including C. albicans and Cryptococcus neoformans that cause candidiasis and cryptococcosis respectively. It is usually used in combination with other antifungal agents (e.g., amphotericin B) for treating deep or systemic mycoses. Flucytosine is mainly used for treating yeast infections, and the drug has little or no activity against dimorphic fungi and moulds. MECHANISM OF ACTION OF 5-FLUOROCYTOSINE: Flucytosine is a nucleoside analogue of cytosine, and 5-FC is deaminated or converted to 5-fluorouracil (a false nucleotide) by cytosine deaminase in the target fungi. The formation of 5-fluorouracil and its incorporation by the pathogenic fungi inhibits the activity of thymidylate synthetase, which is the enzyme that directs DNA synthesis in the organism. Interference of the activities of thymidylate synthetase limits the supply of nucleotides (e.g. thymidine) which is required for the synthesis of DNA in fungi. Flucytosine is a narrow spectrum antifungal agent, and it is only used to treat human mycoses caused by yeasts especially in systemic fungal infections. SIDE EFFECTS AND FUNGAL RESISTANCE TO 5-FC: The clinical usage of flucytosine causes the depression of bone marrow development in recipient hosts. Pathogenic fungi develop resistance to the agent especially in cases where the drug is used alone. Mutation in the target pathogenic fungi could also lead to resistance of the organism to the antifungal agent.

CELL MEMBRANE INHIBITORS The cytoplasmic membrane of fungal cell is vital to the sustenance and development of the fungal organism. This is because this part of the cell helps to maintain a constant internal environment. It also

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helps to regulate the inflow and outflow of materials from the cell. Antifungal agents that interfere with the synthesis of fungal cytoplasmic membrane include amphotericin B and nystatin. Drugs that perform this vital function are generally known as polyenes. Amphotericin B is a widely used polyene that is naturally produced by Streptomyces species. Both nystatin and amphotericin B disrupt the structural integrity of fungal cytoplasmic membrane. Nystatin is also synthesized naturally by Streptomyces species.

INHIBITORS OF ERGOSTEROL BIOSYNTHESIS

Antifungal agents that inhibit the biosynthesis of ergosterol (fungal sterol), a major component of fungal cell wall are generally known as azoles because they contain the imidazole group from which other agents in this category are chemically derived. Typical examples of antifungal agents that are azoles include fluconazole, ketoconazole, itraconazole, miconazole, voriconazole and clotrimazole. A handful of the azoles or imidazoles as they are often called are used topically as antimicrobial creams or solutions to treat superficial mycoses while the others are either used orally (e.g., ketoconazole) or intravenously (e.g., fluconazole) to treat a wide variety of human mycoses inclusive of superficial mycoses, cutaneous mycoses and systemic mycoses. This group of antimicrobial agent known as azoles or imidazoles also contains agents that have activity against non-fungal organisms such as helminthes (e.g., mebendazole), parasites or protozoa (e.g., metronidazole) and pathogenic bacteria (e.g., metronidazole).

OVERVIEW OF ANTI-PROTOZOAL AGENTS Antiprotozoal agents are antimicrobial agents or drugs used to treat infections caused by parasites in humans. Parasites are organisms that live in or on the body of humans and other animals; and they include helminthes and worms. Some notable parasitic or protozoal infections in humans include malaria, leishmaniasis, trichomoniasis, giardiasis, amebiasis, and toxoplasmosis amongst others. A wide variety of protozoal infections are prevalent in subtropical and tropical regions of the world especially in places where public hygiene, water supply and environmental sanitation are still at a pitiable state. Metronidazole (flagyl), pentamidine, Ivermectin, pyrantel, albendazole, primaquine, and artemisinin, quinine and mefloquine amongst others are some antiprotozoal agents used to treat different parasitic infections in humans. Some antibacterial agents such as tetracycline, sulpha drugs and doxycycline are also co-administered with some antiprotozoal agents to treat parasitic diseases in humans. For some parasite diseases such as malaria (caused by Plasmodium species) prophylactic measures and other preventive practices are most appropriate to the containment of the disease rather than treatment. Vector control for human parasitic infections is also critical to the prevention of a wide variety of parasitic diseases since most of their causative agents are transmitted to humans via insects and other animal carriers. Most antiprotozoal drugs act by interrupting key stages in the lifecycle of the parasite in vivo; and the mechanism of action of these drugs varies from each other, and they should always be used with caution. The search for potent antiprotozoal agents with minimal or lesser toxicity on human hosts is still ongoing.

OVERVIEW OF ANTI-VIRAL AGENTS Antiviral agents are antimicrobial agents that specifically inhibit the replication of viruses in living cells (inclusive of human cells affected by viral particles). These antimicrobial agents like antifungal agents have poor or low selective toxicity. This is because viruses only live in living cells i.e. they are obligate intracellular parasites and cannot survive outside a living system. Because of their intracellular nature, antiviral drugs targeted at pathogenic viruses not only inhibit viral replication but they also interfere with the cellular and metabolic activities of the recipient human or animal cells. Viruses live inside their host cells; and they depend mainly on the biosynthetic and enzymatic machinery of their host cells which they overwhelm to direct their own replication processes in vivo. This is why most of the antiviral agents end up harming the recipient host aside their normal antimicrobial onslaught on their target viruses. The number of antiviral agents like antifungal agents is very small, and this is because most drugs used for antiviral therapy also adversely affect the animal or human host taking them since the organism lives inside the cells of the recipient animal or human host. Since viruses are intracellular parasites and lives inside the host cells, most antiviral drugs target specific phases in the life cycle and/or replication processes of the viral particle while others exhibit

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their antimicrobial activity by targeting the nucleic acids of the viruses (either DNA or RNA) or by preventing the adherence of the viral agent to the host cells. Apart from synthetic antiviral agents used for the clinical management of some viral infections, the human body produces chemical substances that possess antiviral activity. These natural substances produced by the body and which inhibit the nefarious activities of viruses in vivo are generally known as immunomodulators. Immunomodulators are chemical substances which boosts the body’s some components of the immune system (particularly the T-cells) in order to direct the host’s immunological response to an invading viral agent. Immunomodulators o immunomodulants can also be produced artificially, and their main function in the body is to interfere with viral replication processes in vivo i.e. inside the infected cells of living organisms. Interferon (IFN) is a typical example of an immunomodulant. IFN is a proteinous substance produced by viral infected cells in response to viral invasion of host cells. Interferons help to reduce the spread of viruses in the body by interfering with their replication, and they also boost the body’s response to antigenic substances (e.g. microorganisms). Unlike other microorganisms (inclusive of bacteria, fungi, algae and protozoa) which contain both DNA and RNA in their genome as nucleic acids, a viral particle (virion) either contains DNA or RNA as its genetic makeup or genome. The development of viral-specific antiviral agent is complicated and difficult because drugs used for the treatment of viral infections (which are small at the moment compared to the many antibacterial agents) apart from targeting the virion also adversely leave untoward effects in the recipient host cells which harbours the viral particle. Most antiviral agents target cellular DNA in their quest for invading viral particles, and this limits the number of effective viral agents with higher selective toxicity used in clinical medicine. Despite their low selective toxicity, antiviral agents target specific sites and functions supposed to be more prevalent in the virion than in the host cell. For example, the rate at which viruses synthesize their nucleic acids (DNA or RNA) is faster compare to human or animal hosts whose cellular DNA synthesis may be slower. Viruses are fast replicating organisms and they also mutate in these processes into various forms that may be missed by antiviral agents. The various target sites and/or mode of action of antiviral agents are briefly highlighted in this section.

Viral attachment inhibition: Viral attachment to host cells is often the first prerequisite to their entry into infected cells. Once they attach to specific receptors on their target host cell, viruses penetrate the outer cell structure and start to uncoat within the host cell. Viral attachment, penetration and uncoating are often the first step in the replication of viruses within living host cells. Amantadines, rimantadine and enfuvirtide are examples of antiviral agents that inhibit or interfere with viral penetration and uncoating.

Viral nucleic acid synthesis: Antiviral agents that interfere with the synthesis of viral nucleic acids (either DNA or RNA) act as nucleoside analogues i.e. they resemble the nucleoside molecules (inclusive of guanosine, adenosine, cytidine and thymidine) required by potential viral particles for the synthesis of their own nucleic acids (DNA or RNA). Antiviral agents in this category specifically interfere with the activities of enzymes (e.g. polymerases) that direct viral DNA or RNA synthesis; and their inclusion or incorporation into viral nucleic acids automatically inhibit or obstruct the biosynthesis of DNA or RNA in the virion. A wide variety of antiviral agents are nucleoside analogues or inhibitors of viral nucleic acid synthesis. Examples of antiviral agents that are nucleoside analogues include acyclovir, zidovudine or azidothymidine (AZT), cidofovir, ribavirin, lamivudine, and zalcitabine or dideoxycytidine amongst others. AZT, a reverse-transcriptase (RT) inhibitor is used to manage HIV/AIDS patients. The drug (i.e. zidovudine) interferes with RT in retroviruses (e.g. HIV) in order to inhibit their activity in viruses; and it is selectively toxic in action because the human cell lacks reverse transcriptase enzymes. RT is an RNA-dependent-DNA-polymerase enzyme that is mainly responsible for the synthesis of viral DNA from viral RNA (which serves as a template for this purpose) in retroviruses (particularly HIV).

Other viral agents: Nelfinavir, saquinavir, indinavir and ritonavir are viral protease synthesis inhibitors. Antiviral agents that are in this category (i.e. protease inhibitors) are novel antiviral agents; and they inhibit viral assemblage and release from infected host cells which is the primary function of protease enzymes in viruses. Viral particles easily develop

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resistance to protease inhibitors, thus they should be used in combination with other antiviral agents (e.g. AZT) especially in HIV/AIDS patients.

PRODUCTION AND SYNTHESIS OF ANTIBIOTICS AND ANTIMICROBIAL AGENTS

Antibiotics can be produced naturally from microbes, or by synthetic and semi-synthetic processes. THE MAJOR SOURCES OF ANTIBIOTICS ARE:

Microorganisms (e.g., Streptomyces species and Penicillium species). Chemical synthesis. For example, from compounds that possesses antimicrobial activity. Semi-synthesis. For example, a part of the antibiotic is produced via fermentation using a

specific microbe, and the product is further chemically modified into the final antibiotic. Herbal plants. Some plants possess bioactive compounds that have antimicrobial efficacy; and

with the current rise in microbial resistance to some available antimicrobial agents, efforts are now geared towards sourcing novel antimicrobial agents from herbal plants and natural products.

The production and synthesis of antibiotics and antimicrobial agents is an aspect of industrial microbiology that is geared towards transforming and harnessing the metabolic potentials of antibiotic-producing bacteria to produce antimicrobial agents (e.g., antibiotics) that are of clinical or medical importance to man, plants, animals and the environment. Microorganisms are metabolic entities that secrete both primary and secondary metabolites, but not all microorganisms have the potential to secrete substances or metabolites that have the ability to inhibit the growth or kill other susceptible bacteria or microbes. Antibiotics and antimicrobial agents are majorly secreted by specific species and strains of bacteria and fungi. Antibiotics may be produced wholly by fermentation; but today, antibiotics are largely produced by semi-synthetic processes – in which a product obtained by fermentation is chemically modified by the chemical introduction of side chains. Antibiotics are examples of secondary metabolites. Antibiotics are mainly produced through the process of fermentation (which is the large scale cultivation of microorganisms aerobically or anaerobically) that allows for the large scale cultivation of microorganisms which convert raw materials into antibiotics or antimicrobial agents. After the large scale production by fermentation, the antibiotics or antimicrobial agents produced, are recovered, concentrated and then purified. The next stage is a chemical conversion stage, which is common for the production of semi-synthetic antibiotics; and it is at this stage that the active pharmaceutical ingredient (API) or bulk antibiotic is produced. Once produced, the bulk antibiotic (API) is further formulated into the required dosage forms – which include tablets, solutions, vials for injection, ointments and syrups. Tablets, vials for injection, syrups, ointments and solutions are the examples of dosage forms of antibiotics after production through the process of fermentation. The stage of antibiotic production from the fermentation to the production of bulk antibiotic (API) is called primary manufacture. The stage from the API to the formulated (dosage) forms is called the secondary manufacture. The fermenter is the vessel in which the fermentation process takes place.

REQUIREMENTS AND PRODUCTION OF PENICILLIN

1. Strain of organism: Penicillium chrysogenum and Penicillium notatum. Some fungi including Cephalosporium and Aspergillus can also be used.

2. Fermentation process: A fed-batch type of fermentation – in which nutrients are intermittently supplied aseptically for microbial growth, is used in the production of penicillin. The steps in the fermentation stage include:

Inoculum preparation. At this stage, a pure inoculum of the organism is produced in sufficient and fast-growing (logarithmic) phase in order to establish a high population density of the organism at a short time. This phase of rapid growth is called trophophase; and it can last for about 24 to 48 hours.

Media addition: Media for penicillin production comprise corn steep liquor (nitrogen source), glucose or lactose (carbohydrate source), buffer (calcium carbonate or phosphates), and other precursor compounds.

Fermenter: The fermentation process takes place inside a closed, vertical, cylindrical stainless steel vessel known as fermenter. Fermenters measures between 50,000 to 500,000 liters in capacity. The fermenter is fitted with channels for aeration (oxygen supply), media/nutrient addition, product (antibiotic) recovery and temperature control.

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Fermenters are now automated to monitor pH, O2 level, penicillin content, temperature level and nutrient level.

pH: The pH is maintained at 6.8 and 7.4. Acid or base is automatically added as necessary in order to maintain this pH level.

Oxygen supply: Penicillin production requires large amount of O2 (because the fungus is a highly aerobic organism). O2 is supplied as filter-sterilized air from a compressor.

Temperature control: Penicillin production generates a lot of metabolic heat, and this must be controlled via cooling. Penicillin is sensitive to heat or temperature.

Addition of defoaming agent: Microbial cultures form foams when subjected to vigorous aeration and stirring. Penicillin production also causes foam formation; and thus deforming agents are added to control this factor of foam formation.

Addition of phenylacetic acid (PAA): PAA is the source of the side chain for the particular penicillin (e.g., benzylpenicillin) to be produced.

Termination of fermentation: The fermentation process is stopped when there is no further conversion of the raw material into antibiotics.

3. Extraction or recovery of antibiotic: After fermentation, the penicillin is usually in solution extracellularly, and in combination with other metabolites and media components. However, some techniques including filtration, ultrafiltration, and centrifugation are used aseptically to separate the fungal cells from the broth or liquid medium. To recover the penicillin from the broth, techniques such as solvent extraction, ion-exchange chromatography and precipitation techniques are often used. Depending on the usage, the penicillin is produced as a salt for clinical usage or as an input for the production of another antibiotic by a semi-synthetic process.

4. Packaging, distribution and usage: The extracted antibiotic (penicillin) is further processed or treated to remove pyrogens (fever-causing substances) by dry-heat sterilization technique. The antibiotic (penicillin) produced is:

packaged in sterile vials as freeze-dried powder or suspension (that will be reconstituted before final usage); or

processed into film-coated tablets for oral usage. Finally, a searching test is conducted on a good number of random samples of the produced and packaged antibiotic in order to ensure that they meet the strict quality control requirements for antibiotic potency, freedom from pyrogens, sterility and purity. Only then can the antibiotic be released for public and clinical usage or consumption. A sufficient number of product samples from each batch of the product are subjected to sterility testing in order to give “an acceptable degree of confidence” in the ultimate results obtained from the sterility test; and thus certify the batch of the product free from contaminating microorganisms.

QUALITY CONTROL OF PHARMACEUTICAL PRODUCTS The quality control of pharmaceutical products (e.g., antibiotics, vaccines, interferons, water for injection) is mainly intended to provide assurances of the probable efficacy and safety of every batch of the finished product. Quality control (QC) is defined as a monitoring system that is used for detecting and correcting analytical errors by establishing performance limits. It is mainly geared towards finding a problem that is associated with a given product and managing it so that the product can be certified safe for consumption. QC is a procedure or set of procedures intended to ensure that a manufactured product including pharmaceutical and medical products or performed services adheres to a defined set of quality criteria or meets the laid down requirements of the products. QC is similar to, but not identical with, quality assurance (QA). Quality assurance (QA) is a planned and systematic process used for evaluating and monitoring the quality and appropriateness or suitability of a product or given service. QA is defined as the total process whereby the quality of laboratory reports can be guaranteed. Thus, QA is summarily the right result, at the right time, on the right clinical/environmental specimen, from the right patient, with the result interpretation based on the correct reference data, and at the right place. QA encompasses all the processes used to evaluate and monitor the quality of services rendered in a healthcare facility, hospital laboratory, pharmaceutical companies and other allied industries. QA is the summation of all the processes ensured during the production of a product in order to ensure that the final product is of the desired quality.

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QC is a major good manufacturing practice (GMP) carried out in production pharmaceutical companies to avoid microbial contamination of finished products (e.g., antibiotics). Good manufacturing practice (GMP) is simply defined as those general rules that govern the manufacture and/or production of a safe, efficacious and microbial-free pharmaceutical product. GMP are practices and the systems required to be adapted in pharmaceutical manufacturing, quality control, and quality system covering the manufacture and testing of pharmaceuticals or drugs including active pharmaceutical ingredients, diagnostics, foods, pharmaceutical products, and medical devices. GMPs are guidance that outlines the aspects of production and testing that can impact the quality of a product so that the finished product is deemed fit for consumption and free from microbial or chemical contamination. Quality control covers all the aspects of quality assurance (QA) which concerns itself with the control of errors in the performance of tests and verification of test results. In quality control processes, every available and necessary test including microbial tests to be carried out are conducted at each stage of the manufacturing process. And QC ensures that finished products are not released into the market for consumption until such products have satisfactorily passed the stipulated QC test that pertains to that particular manufacturing process or plant or product. Several pharmaceutical products including drugs, vaccines and injection water are easily contaminated with pyrogens (fever-inducing agents produced by microbes) during the manufacturing process. This is why it is important that pharmaceutical products pass through a number of purity, freedom from pyrogens, sterility and potency test in order to certify them safe for clinical usage. The outcome or results of the quality control tests must be recorded and authenticated by a quality control specialist or personnel before the finished pharmaceutical product can be certified safe for use. The quality control of pharmaceutical products can be carried out in two (2) stages:

In–process control. This type of control is carried out on the starting materials or raw materials used in the production process. In–process control includes control of raw materials, water, air, personnel, equipment, manufacturing premises and building, and control of packaging material. Any of these materials can serve as an important source of contamination to the production process; and they must be properly controlled to avoid contamination and thus ensure the production of a contamination–free finished product.

Final product control. This type of control involves the testing of the finished product for potency and safety for human or clinical usage or consumption. Safety test, sterility test, pyrogen test, toxicity test, and test for the presence of other contaminating substances are examples of test carried out in the final product control in order to ascertain their safety and potency.

CONCEPT OF GROWTH AND DEATH OF MICROORGANISMS

Growth is simply defined as an irreversible increase in the size of an organism. It is one of the characteristics of living organisms. In microbiological terms, growth is basically the increase in the cell size and cell mass of a bacterial cell and it occur during the development of the organism. Growth in microorganisms unlike other living organisms is usually measured in terms of increase in the number of cells or population of the microbial cells rather than increase in the size of an individual organism. The growth of bacteria occurs when bacterial cells divide into two daughter cells by an asexual reproductive mechanism known as binary fission. Such cells are said to be viable because they are able to reproduce and increase the size of their population. On the other hand, a microbial cell can be said to be “dead” when it can no longer reproduce (i.e., grow and divide) when introduced into a growth medium that support its development. Such a cell is said to be non-viable (i.e., they are not alive and cannot divide or multiply) because they have lost the ability to replicate and multiply. Generally, the growth of microorganisms (e.g., bacteria) follows a particular pattern, and usually includes a lag phase, log (logarithmic) phase, stationary phase and decline or death phase (Figure 10).

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Figure 10: Growth curve of bacteria in a closed system. This figure illustrates the typical growth curve of a bacterial cell with time (hrs) and the number of bacteria (log10) in the X-axis and Y-axis respectively. Microbial populations exhibit this pattern of growth when they are grown in a closed culture system (batch culture). A typical bacterial growth curve as obtainable in this illustration is normally obtained when a fresh growth medium is inoculated with a number of cells, and the cell growth is monitored over a certain period of time. Plotting the cell population against the time gives you the bacterial growth curve obtainable in a closed or batch culture system. This curve is termed a sigmoid curve and it is known as a standard growth curve of bacterial cells. MicroDok.

LAG PHASE: Lag phase represent the stage in the development of a bacterial cell when no growth occurs. It is the period after the initial seeding, culturing or inoculation of a bacterium in a fresh culture/growth medium before observable growth begins (Figure 10). When bacterial cells are initially introduced into a new medium or environment, they usually require some time to synthesize important biosynthetic intermediates (i.e., primary metabolites) required to keep them alive. Lag phase represents a period in the cell division of bacteria in which the respective bacterial cells are maturing or growing but on the other hand, they are not able to divide and increase in their population size. There is enormous synthesis of primary metabolites such as enzymes, nucleic acids and alcohol which are critical to the growth of the bacterial cell.

LOG (LOGARITHMIC) PHASE: Log phase is the period in the growth curve of a microorganism in which the growth of bacteria increases at an exponential rate. This stage of microbial growth is also known as exponential growth phase; and it is characterized by doubling of the bacterial cells (Figure 10). The doubling of the bacterial cells continues at a constant rate unless the growth is inhibited by altering some of the growth conditions such as limiting the supply of nutrients. The growth and doubling of the bacterial cells at the log phase of growth can be inhibited following nutrient depletion and the buildup of toxic wastes. Studies of microbial metabolism and physiology are usually undertaken at the exponential growth phase because the microbial cells are often at their prime or maximum growth in this stage.

STATIONARY PHASE: Stationary phase is the period in the growth curve of microorganisms

(bacteria) in which the growth of the microbial cells stops. The stationary phase is characterized by a slow growth of bacterial cells due to nutrient depletion and toxic waste buildup in the growth medium. Stationary phase sets in as the growing bacterial cells begin to use up or exhaust all the growth nutrients available to them. Once the stationary phase sets in, the line of the growth curve flattens and assumes a horizontal dimension (Figure 10) because the rate of bacterial growth is equal to the rate of bacteria death. Growth rate is stabilized at this phase as the cell number of the organism is no longer increasing. However, the stationary phase is important because it is at this phase that secondary metabolites (e.g., antibiotics) with immense industrial and economic values are synthesized.

Keys: A = Lag phase B = Log (exponential Phase) C = Stationary phase D = Decline & Death Phase

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DECLINE AND DEATH PHASE: The growth of a bacterial cell begins to decline and recede

once growth nutrients become unavailable and drastically depleted in the growth medium. In some circumstances, the microbial cells may continue to grow and metabolize but such growth is not enduring because the microbial population will eventually die (Figure 10). The decline stage ushers in the death phase which is generally characterized by a period of “no growth”. At the death stage, the rate of bacterial death exceeds the rate of bacterial growth. Growth stops entirely because nutrient has been depleted and toxic wastes have started building up. Bacteria completely loses its ability to reproduce (i.e., grow & divide) at this stage of the growth cycle. However, bacterial cells capable of forming endospores and other protective factors that helps it to survive adverse environmental conditions can survive this stage of the growth cycle until it finds a suitable environment to continue its growth process.

MEDICAL AND NON-MEDICAL USES OF ANTIBIOTICS

Medically, antibiotics are used to treat infectious diseases in humans. But generally, antibiotics and other antimicrobial agents are solely used to achieve a therapeutic goal in either human or animal population. The discovery of antimicrobial agents particularly antibiotics was a transforming moment for clinical medicine because infectious diseases caused by pathogenic microorganisms can be handled with these agents. Uncountable number of lives has been saved from the therapeutic powers of antibiotics over the years and even till now. The bacteriostatic and bactericidal prowess of antibiotics has been exploited by man and medicine in particular, to treat and manage infectious diseases of humans and animals. Antibiotics and other antimicrobial agents are used for both clinical/medical purposes and other non-clinical/medical uses. Medically, antibiotics are used:

for the treatment of various infectious diseases caused by pathogenic bacteria, fungi, protozoa and viruses,

medically during surgery to prevent microbial contamination, in veterinary medicine for treating infectious diseases caused by pathogens in animals.

The non-medical uses of antibiotics involve the use of antibiotics outside of human or veterinary medicine. It also involves the usage of antibiotics or antimicrobial agents for purposes not intended to confer or achieve any form of therapeutic or healing advantage or goal, especially in human population. Some of the non-medical uses of antibiotics involve the use of antibiotics:

in animal feeds, as growth promoters in animals, in animal husbandry, in bee-keeping, in fish farming and aquaculture, in agricultural purposes, in food preservation (e.g., bacteriocins)., Bacteriocin is a proteinaceous toxin or compound

produced by bacteria such as lactobacilli to inhibit the growth of some closely related bacteria. They are used as preservatives in food because bacteriocins possess some antimicrobial activity.

in horticulture (e.g., to tackle plant pathogens), in plant disease control, in ethanol production to prevent bacterial contamination during the fermentation process, in mouthwashes, some creams, and sterilizing agents, in biomedical research (e.g., in cell culture experiments), for domestic purposes. For example, some detergents (e.g., soaps or shampoos) used in the

home contain some compounds (e.g., triclosan) that possess some antimicrobial activity. Triclosan is a synthetic compound that has antibacterial, antifungal and antiviral properties. The use of such compounds can initiate cross resistance of antibiotics in susceptible bacteria, and thus hinder the therapeutic efficacy of the said antibiotics when used clinically for medical purposes.

These non-medical uses of antibiotics have contributed largely to the development and spread of bacterial resistance and other forms of resistance in microbial population and even in human population. The non-medical uses of antibiotics threatens many areas of medicine including surgery and antimicrobial chemotherapy due to the emergence and spread of microbial or bacterial strains that are now recalcitrant

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or resistant to the antimicrobial activity of some available antimicrobials. The irrational use of antibiotics and their widespread usage in non-medical practices as aforesaid have compromised their efficacy and undermined their powers in tackling infectious diseases in human population. Today, antibiotic resistant microbe can be isolated from various environments including sewage, water ways, soil to mention a few. It is important to reduce the use of antibiotics in non-medical practices to the barest minimum. More so, antibiotic stewardship in medical practice is also important in order to contain the emergence and spread of drug resistant microbe both in the hospital and non-hospital environment.

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