General Chemotherapy

 

Chemotherapy

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Chemotherapy is the branch of Pharmacology which deals with the use of chemicals/drugs (called chemotherapeutic agents like anti-bacterials, anti-fungals, anti-neoplastic and anti-virals etc.) that are selectively toxic to the foreign cells (pathogens) with minimal or no impact over the normal host cells.

Antimicrobials: These are substances having natural, semi-synthetic or synthetic origin and are effective against a wide range of micro-organisms (like bacteria, protozoa etc.). Antibacterials, antivirals and antiprotozoals are examples of antimicrobial drugs.

Antibacterials: Their origin may be natural, semi-synthetic or synthetic but their activity is only against bacteria.

Antibiotics: Their origin is always natural (derived from bacteria or fungi) or semi-synthetic but their activity may be against a wide range of micro-organisms (like bacteria, protozoa etc.).

Bacteriostatic: It is that antibacterial agent which inhibits the growth of susceptible bacteria e.g. Tetracyclines, Amphenicols, Macrolides and Lincosamides.

Bactericidal: It is that antibacterial agent which causes destruction/lysis of susceptible bacteria e.g. Penicillins, Cephalosporins, Aminogycosides, Quinolones, Vancomycin and Bacitracin.

Antimicrobial spectrum: It is the range of micro-organisms covered by a particular chemotherapeutic agent. It is of three types.

(a) Narrow spectrum: It is used for those antibacterial agents that are effective only against gram positive or gram negative bacteria. For example Erythromycin is effective against gram positive while Streptomycin covers only gram negative bacteria.

(b) Broad spectrum: Broad spectrum indicates the activity of concerned antibacterial against a wide range of bacteria, covering gram positive as well as gram negative, e.g. Oxytetracycline and Chloramphenicol.

(c) Extended spectrum: It is subdivided into two types. Extended gram positive spectrum indicates that respective agent is originally active against gram positive bacteria but it also covers sufficient number of gram negative bacteria (but not all) e.g. Amoxycillin. Extended gram negative spectrum shows that concerned drug is basically effective against gram negative but it also covers sufficient number of gram positive bacteria e.g. Nalidixic acid.

Potency: It may be defined as the antimicrobial activity contained in milligram of a chemotherapeutic agent. It is usually expressed on the basis of minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and minimum antibiotic concentration (MAC).

(a) Minimum inhibitory concentration (MIC): It is the lowest concentration of an antimicrobial drug that prevents visible growth of bacteria when grown against sequentially varying drug concentrations in vitro.

(b) Minimum bactericidal concentration (MBC): It is the lowest concentration of an antimicrobial drug that kills/destroys the bacterial cells.

(c) Minimum antibiotic concentration (MAC): It is the concentration of an antimicrobial drug that reduces the growth of an organism (pathogen) in vitro by a factor of 10 (one log).

Post antibiotic effect: It is the persistence of antimicrobial action for longer periods (few hours) after brief exposure to or in absence of detectable concentration of an antimicrobial drug. It can affect the dosing interval for some antimicrobials (Aminoglycosides are given at 12-24 hours intervals, although their half lives are much shorter).

 

Generalized structure of a bacterial cell

 

Bacteria are prokaryotic, unicellular micro-organisms belonging to class Schizomycetes. They exist in three main morphological forms, i.e., cocci (spherical), bacilli (rod-shaped) and spirilla (filamentous). On the basis of staining properties they can be divided into gram positive, gram negative and acid-fast bacteria. A typical bacterial cell consists of lipopolysaccharide capsule (glycocalyx), cell wall, cell membrane, cytoplasm (containing genetic material and ribosomes) and appendages like flagella or pili. Certain bacterial species are capable to produce spores or endospore for acquiring a dormant state. Capsule is meant for protecting bacteria from dessication and phagocytosis and in-vivo it aids in the adherence of pathogenic bacteria to host surfaces. Cell wall is a tough and rigid framework that protects bacteria from mechanical damage and osmotic lysis.  The prokaryotic ribosomes (e.g., bacterial ribosomes) have a sedimentation value of 70S (where “S” stands for Swedberg unit) with subunits 50S (larger subunit) and 30S (smaller subunit) while eukaryotic ribosomes (e.g., mammalian ribosomes) have a sedimentation value of 80S with subunits 60S (larger subunit) and 40S (smaller subunit).

 

Structure of gram positive bacterial cell wall: The major portion of gram positive bacterial cell wall consists of peptidoglycan or murein that supports the lipoprotein membrane. Peptidoglycan is composed of linear strands of two alternatively amino-sugars (i.e., N-acetylglucosamine and N-acetyl muramic acid) linked with each other by means of β (1-4) glycosidic linkage. Another important component is polymer of glycerol phosphate or ribitol phosphate known as teichoic acid, a major cell wall surface antigen of gram positive bacteria. The gram positive bacterial cell wall contains very little lipids.

 

Structure of gram negative bacterial cell wall: The cell wall of gram negative bacteria is much more complex than that of gram positive bacteria. The teichoic acid found in gram positive bacterial cell wall is absent. The outer membrane is an asymmetrical lipid bilayer containing proteins. Its inner surface resembles that of cytoplasmic membrane while its outer surface is composed of lipo-polysaccharides. The polysaccharide side chains contribute to the complex antigenic specificity of gram negative bacterial cells and are usually called O-polysaccharides because they correspond to the somatic (O) antigens used for serotyping of gram negative bacteria. The other component of lipo-polysaccharide, the lipid A, is responsible for the endotoxic activity of gram negative bacteria.  The outer membrane also contains specialized protein channels called porins through which small molecular weight substances such as sugars and amino acids enter the bacterial cell. The periplasmic space of gram negative bacterial cell wall consists of some lipoprotein molecules, some enzymes and few peptidoglycan chains (1-2).

The enzymes are involved mainly in the active transport of nutrients, metabolites and are also responsible for conferring resistance to some antibiotics (by acting as antibiotic inactivating enzymes, for instance β-lactamse can inactivate β-lactam antibiotics including Penicillins and Cephalosporins).

 

Comparative features of gram positive and gram negative bacterial cell walls
Feature	Gram positive bacterial cell wall	Gram negative bacterial cell wall
Thickness of cell wall	Cell wall is thicker due to presence of more peptidoglycans	Cell wall is thinner due to presence of less peptidoglycans
Teichoic acid	Teichoic acid is present	Teichoic acid is absent
Outer membrane	Outer membrane is absent	Outer membrane is present
Periplasmic space	Periplasmic space is absent	Periplasmic space is present
Porins	Porins are absent	Porins are present

 

Bacterial protein synthesis

 

Bacterial protein synthesis can be divided into three distinct stages, initiation, elongation and termination.

1. Initiation: Bacterial protein synthesis starts with the formation of initiation complex that includes ribosomes, three initiation factors (IF-1, IF-2 and IF-3), mRNA and guanosine triphosphate (GTP). In the initiation process, the inactive 70S ribosome in the presence of IF-1 and IF-3 first dissociates into 50S and 30S subunits. The 30S subunit then occupies a specific site on mRNA. In the presence of initiation factors (IF-1, IF-2 and IF-3) and GTP, a specific tRNA, called the initiator tRNA brings a modified methionine amino acid (known as f-met) to the 30S subunit. The 30S subunit, three initiation factors, mRNA, GTP and f-met-tRNA constitute the initiation complex. The initiation complex then combines with the 50S subunit to form activated 70S ribosome. The 50S ribosomal subunit contains a peptidyl (P) and an aminoacyl (A) site.  This is followed by a process in which GTP is hydrolyzed to GDP and P_i and the initiation factors (IF-1, IF-2 and IF-3) dissociate from the ribosome. This ensures that the initiator tRNA (f-met-tRNA) binds to peptidyl site (the amino acyl site remains free) and positioned at the initiation condon, AUG. The initiator tRNA will be having an anticodon (UAC), complementary to the start codon (AUG) of mRNA. The synthesis of all prokaryotic proteins start with the amino acid methionine, coded by the start codon AUG. The translation takes place in 5^/ to 3^/ direction.

 

2. Elongation: The elongation step is further subdivided into three substages.

(a) Binding of amino acyl-tRNA: A newly amino acyl-tRNA that is complementary to the codon located adjacent to start codon, binds to the free amino-acyl site of 70S ribosomal unit. This step can be blocked by Tetracyclines.

(b) Peptide bond formation (Transpeptidation): In this step, the activated amino acid (f-met) attached to the tRNA in P-site, is transferred to the A-site through the catalyzing action of peptidyl transferase enzyme. This energy independent transpeptidation process results in the attachment of two amino acids to tRNA present at amino acyl-site while the empty tRNA is discharged from the A-site.

(c) Translocation: This energy-dependent step involves the movement of ribosomal complex to the next codon (a specific sequence of three nucleotides) in a 5^/ to 3^/ direction along the length of mRNA. This causes the shifting of dipeptidyl tRNA from A-site to P-site. Thus the A-site once again becomes free and ready for receiving a new amino acyl-tRNA to start a new elongation process. The elongation process continues in the same manner until a stop codon is encountered. The translocation step is blocked by Macrolide antibiotics.

 

3. Termination: The termination of polypeptide chain in signaled by on of the three termination/stop codons in the mRNA. The termination codon is identified and elongation is terminated by the discharge of releasing factors (RF-1, RF-2 and RF-3).