Antibiotics- Classification, Mode of actions, various generations of Antibiotics

Antimicrobial chemotherapy is the use of drugs to control  microorganisms for the prevention and treatment of disease.  The most successful drugs interfere with vital processes that differ between the pathogen and host, thereby seriously damaging the target microorganism while harming its host as little as possible.

Ideally, chemotherapeutic agents used to treat infectious disease destroy pathogenic microorganisms or inhibit their growth at concentrations low enough to avoid undesirable damage to the host. Most of these agents are antibiotics [Greek anti, against, and bios, life], microbial products or their derivatives that can kill susceptible microorganisms or inhibit their growth. Drugs such as the sulfonamides are examples of synthetic chemotherapeutic agents and are not microbially synthesized.

Chemotherapy

Paul Ehrlich is considered the father of the modern era of chemotherapy. Ehrlich was fascinated with dyes that specifically bind to and stain microbial cells-that would selectively destroy pathogens without harming human cells—a “magic bullet.” By 1904 Ehrlich found the dye trypan red active against the trypanosome that causes African sleeping sickness and arsphenamine (commercially, Salvarsan) active against the syphilis spirochete. In 1927, Gerhard Domagk discovered Prontosil Red, (the source of sulfonamides, or sulfa drugs) against pathogenic Streptococci and Staphylococci. Domagk received the 1939 Nobel Prize in Physiology or Medicine for his discovery of sulfonamides, or sulfa drugs. In the 1920s, Alexander Fleming, a Scottish physician, found that human tears contained a naturally occurring antibacterial substance that he termed “lysozyme.” The first true antibiotic Penicillin was later discovered by Fleming.

In 1896, by a 21-year-old French medical student named Ernest Duchesne had actually discovered penicillin but it was Fleming’s discovery which made it famous.  

Penicillium mold colony secretes penicillin that kills Staphylococcus aureus

 Fleming, Florey, and Chain received the Nobel Prize in 1945 for the discovery and production of penicillin. The discovery of penicillin stimulated the search for other antibiotics.

Selman Waksman announced in 1944 and his associates found a new antibiotic, streptomycin, produced by the actinomycete Streptomyces griseus. Streptomycin was the first drug that could successfully treat tuberculosis. Waksman received the Nobel Prize in 1952

Search for other antibiotic-producing soil microorganisms led to the isolation of chloramphenicol, neomycin and tetracycline by 1953. The discovery of chemotherapeutic agents and the development of newer, more powerful drugs have transformed modern medicine and greatly alleviated human suffering.

Characteristics of Antimicrobial Drugs

To be successful a chemotherapeutic agent must have selective toxicity: it must kill or inhibit the microbial pathogen while damaging the host as little as possible.

The degree of selective toxicity is expressed as

(1) the therapeutic dose, the drug level required for clinical treatment of a particular infection

(2) the toxic dose, the drug level at which the agent becomes too toxic for the host.

The therapeutic index is the ratio of the toxic dose to the therapeutic dose. The larger the therapeutic index, the better the chemotherapeutic agent (all other things being equal).

A drug that disrupts a microbial function not found in eucaryotic animal cells often has a greater selective toxicity and a higher therapeutic index. For example, penicillin inhibits bacterial cell wall peptidoglycan synthesis but has little effect on host cells because they lack cell walls; therefore penicillin’s therapeutic index is high. A drug which inhibits the same process in host cells or damages the host in other ways may have a low therapeutic index. The undesirable effects on the host, or side effects, are of many kinds and may involve almost any organ system. Because side effects can be severe, chemotherapeutic agents should be administered with great care.

Some bacteria and fungi are able to naturally produce many of the commonly employed antibiotics. In contrast, several important chemotherapeutic agents, such as sulfonamides, trimethoprim, chloramephenicol, ciprofloxacin, isoniazid, and dapsone, are synthetic—that is, manufactured by chemical procedures. Semisynthetic antibiotics are natural antibiotics that have been structurally modified by the addition of chemical groups which make them less susceptible to inactivation by pathogens (e.g., ampicillin, carbenicillin, and methicillin). Many semisynthetic drugs have a broader spectrum of antibiotic activity than their parent molecule. e.g., ampicillin, amoxycillin

 Microbial Sources of Some Antibiotics

Microorganism	Antibiotic
Actinomyces
Streptomyces spp.	Amphotericin B Chloramphenicol (also synthetic) Kanamycin Neomycin Nystatin Rifampin Streptomycin Tetracyclines Vancomycin
Bacteria
Micromonospora spp.	Gentamicin
Bacillus spp.	Bacitracin Polymyxins
Fungi
Penicillium spp.	Griseofulvin Penicillin
Cephalosporium spp.	Cephalosporins

 

Drugs have different range of effectiveness. Many are narrow-spectrum drugs— effective only against a limited variety of pathogens. Broad-spectrum drugs act against many different kinds of pathogens.

Drugs may also be classified based on the general microbial group they act against: antibacterial, antifungal, antiprotozoan, and antiviral. Some agents can be used against more than one group; for example, sulfonamides are active against bacteria and some protozoa.

Chemotherapeutic agents, like disinfectants, can be either cidal or static. Static agents reversibly inhibit growth; if the agent is removed, the microorganisms will recover and grow again. A cidal agent kills the target pathogen. The effect of an agent also varies with the target species: an agent may be cidal for one species and static for another. A static agent may not be effective if the host’s resistance is too low.

The minimal inhibition concentration (MIC) is the lowest concentration of a drug that prevents growth of a particular pathogen. The minimal lethal concentration (MLC) is the lowest drug concentration that kills the pathogen. A cidal drug generally kills pathogens at levels only two to four times the MIC, whereas a static agent kills at much higher concentrations.

 

ANTIBACTERIAL DRUGS

Antibiotics damage pathogens in several ways. Examples of antibiotics with the process inhibited, are the following: chloramphenicol (bacterial protein synthesis), cycloserine (peptidoglycan synthesis), nalidixic acid and novobiocin (bacterial DNA synthesis), rifampin (bacterial RNA synthesis), cycloheximide (eucaryotic protein synthesis), polyoxin D (fungal cell wall chitin synthesis), and cerulenin (fatty acid synthesis.

A few antibacterial drugs are described here, with emphasis on their mechanisms of action.

1. Inhibitors of Cell Wall Synthesis
2. Inhibitors of Protein Synthesis
3. Inhibitors of Nucleic acid Synthesis
4. Antimetabolites

Inhibitors of Cell Wall Synthesis 

The most selective antibiotics are those that interfere with bacterial cell wall synthesis. Drugs like penicillins, cephalosporins, vancomycin, and bacitracin have a high therapeutic index because they target structures not found in eukaryotic cells.

Penicillins

Most penicillins (e.g., penicillin G or benzylpenicillin) are derivatives of 6-aminopenicillanic acid and differ from one another with respect to the side chain attached to its amino group.  β-lactam ring,  is essential for bioactivity. Many penicillin-resistant bacteria produce penicillinase (β -lactamase), an enzyme that inactivates the antibiotic by hydrolyzing a bond in the β lactam ring.

Penicillin structure resemble the terminal D-alanyl-D-alanine found on the peptide side chain of the peptidoglycan subunit. This structural similarity blocks the enzyme catalyzing the transpeptidation reaction that forms the peptidoglycan cross-links. Thus formation of a complete cell wall is blocked, leading to osmotic lysis. Hence, penicillins act only on growing bacteria that are synthesizing new peptidoglycan.

Penicillins also bind to several periplasmic proteins (penicillin-binding proteins, or PBPs) and may also destroy bacteria by activating their own autolytic enzymes. Penicillin may stimulate special proteins called bacterial holins to form holes or lesions in the plasma membrane, leading directly to membrane leakage and death. 

The two naturally occurring penicillins, penicillin G and penicillin V, are narrow-spectrum drugs. Penicillin G is effective against gonococci, meningococci, and several gram-positive pathogens such as streptococci and staphylococci. However, it is destroyed by stomach acid and it must be administered by injection (parenterally). Penicillin V is similar to penicillin G in spectrum of activity, but can be given orally because it is more resistant to acid.

The semisynthetic penicillins,  have a broader spectrum of activity. Ampicillin can be administered orally and is effective against gram-negative bacteria such as Haemophilus, Salmonella, and Shigella. Carbenicillin and Ticarcillin are potent against Pseudomonas and Proteus.

Many bacteria have become resistant to natural penicillins and many of the semisynthetic analogs. Specific semisynthetic penicillins that are not destroyed by β -lactamases such as, methicillin, nafcillin, and oxacillin are used to combat antibiotic-resistant pathogens such as methicillin-resistant bacteria.

Although penicillins are the least toxic of the antibiotics, about 1 to 5% of the adults may be allergic to them. Occasionally, a person will die of a violent allergic response; therefore, patients should be questioned about penicillin allergies before treatment is begun.

Cephalosporins

Cephalosporins are a family of antibiotics originally isolated in 1948 from the fungus Cephalosporium acremonium. They contain a β -lactam structure that is very similar to that of the penicillins. Cephalosporins also inhibit the transpeptidation reaction during peptidoglycan synthesis. 

They are broad-spectrum drugs frequently given to patients with penicillin allergies (although about 10% of patients allergic to penicillin are also allergic to cephalosporins).

Cephalosporins are broadly categorized into generations (groups of drugs that are sequentially developed) based on their spectrum of activity.

First generation cephalosporins are more effective against Gram-positive organisms, such as staphylococci and streptococci, and minimal coverage against Gram-negative bacteria. Examples include cefazolin, cephalothin, cephradine, cefadroxil, and cephalexin.   

Second-generation drugs, developed after the first generation, have improved effects on gram negative bacteria with some anaerobe coverage. These cephalosporins are more effective against Gram-negative bacteria, but less effective against Gram-positive bacteria. Has better cell penetration. Increased resistance to beta lactamases. Examples include cefuroxime and cefprozil.

Third-generation drugs are  effective against gram-negative pathogens, and some reach the central nervous system. This is important because many antimicrobial agents do not cross the blood-brain barrier.  Examples include cefotaxime, ceftazidime, cefdinir, ceftriaxone, cefoperazone, and cefixime.  

Fourth-generation cephalosporins are broad spectrum with excellent gram-positive and gram-negative coverage and, like their third-generation predecessors, inhibit the growth of the difficult opportunistic pathogen Pseudomonas aeruginosa. These cephalosporins have an improved gram-positive spectrum while retaining the expanded gram-negative activity of the third generation. Wider spectrum of activity and improved resistance to beta lactamases. Examples include cefepime and cefpirome.  

Fifth generation These cephalosporins include ceftaroline and ceftobiprole. These are approved for treating critical infections, such as hospital-acquired pneumonia.

Vancomycin 

Vancomycin is a glycopeptide antibiotic produced by Streptomyces oreintalis. It is a cup-shaped molecule composed of a peptide linked to a disaccharide. 

Vancomycin’s peptide portion blocks the transpeptidation reaction by binding specifically to the D alanine-D-alanine terminal sequence on the pentapeptide portion of peptidoglycan. The antibiotic is bactericidal for Staphylococcus and some members of the genera Clostridium, Bacillus, Streptococcus, and Enterococcus. 

It is given both orally and intravenously and is important in the treatment of antibiotic resistant staphylococcal and enterococcal infections.

Vancomycin has been considered the “drug of last resort” in cases of antibiotic resistant S. aureus. However, vancomycin-resistant strains of Enterococcus and resistant Staphylococcus aureus (VRSA) are prevalent now

Teicoplanin 

Teicoplanin is a glycopeptide antibiotic from the actinomycete Actinoplanes teichomyceticus that is similar in structure and mechanism of action to vancomycin, but has fewer side effects. It is active against Staphylococci, Enterococci, Streptococci, Clostridia, Listeria, and many other gram-positive pathogens.

Protein Synthesis Inhibitors

Many antibiotics inhibit protein synthesis by binding with the prokaryotic ribosome. Because these drugs discriminate between prokaryotic and eucaryotic ribosomes, their therapeutic index is fairly high, but not as high as that of cell wall inhibitors. Some drugs bind to the 30S (small) ribosomal subunit, while others attach to the 50S (large) subunit. 

Several different steps in protein synthesis can be affected: aminoacyl-tRNA binding, peptide bond formation, mRNAreading, and translocation.

Aminoglycosides

Aminoglycoside antibiotics contain a cyclohexane ring and amino sugars. Streptomycin, kanamycin, neomycin, and tobramycin are synthesized by different Streptomyces species, whereas gentamicin comes from another actinomycete, Micromonospora purpurea. 

Aminoglycosides bind to the 30S (small) ribosomal subunit and interfere with protein synthesis by directly inhibiting the synthesis process and also by causing misreading of the mRNA. 

These antibiotics are bactericidal and are most effective against gram-negative pathogens. Streptomycin is effective in treating tuberculosis and but widespread drug resistance is a problem. Gentamicin is used to treat Proteus, Escherichia, Klebsiella, and Seratia infections. 

Aminoglycosides can be quite toxic, however, and can cause deafness, renal damage, loss of balance, nausea, and allergic responses.

Tetracyclines

The tetracyclines are a family of antibiotics with a common four ring structure to which a variety of side chains are attached. Oxytetracycline and chlortetracycline are produced naturally by Streptomyces species while others are semisynthetic drugs. 

These antibiotics are similar to the aminoglycosides and combine with the 30S (small) subunit of the ribosome. This inhibits the binding of aminoacyl-tRNA molecules to the A site of the ribosome. 

Because their action is only bacteriostatic, the effectiveness of treatment depends on active host immune system. 

Tetracyclines are broad-spectrum antibiotics that are active against gram-negative, as well as gram-positive bacteria, Rickettsias, Chlamydiae, and Mycoplasmas. High doses may result in nausea, diarrhea, yellowing of teeth in children, and damage to the liver and kidneys.

Macrolides

The macrolide antibiotics contain 12- to 22-carbon lactone rings linked to one or more sugars. 

Erythromycin is usually bacteriostatic and binds to the 23S rRNA of the 50S (large) ribosomal subunit to inhibit peptide chain elongation during protein synthesis. Erythromycin is a relatively broad-spectrum antibiotic effective against gram-positive bacteria, mycoplasmas, and a few gram-negative bacteria. It is used with patients who are allergic to penicillins and in the treatment of whooping cough, diphtheria, diarrhea caused by Campylobacter, and pneumonia from Legionella or Mycoplasma infections. 

Clindamycin is effective against a variety of bacteria including staphylococci, and anaerobes such as Bacteroides. Azithromycin, is particularly effective against Chlamydia trachomatis.

Chloramphenicol

Chloramphenicol was first produced from cultures of Streptomyces venezuelae but is now synthesized chemically. Like erythromycin, this antibiotic binds to 23S rRNA on the 50S ribosomal subunit to inhibit the peptidyl transferase reaction. It has a very broad spectrum of activity but, is quite toxic. Allergic responses or neurotoxic reactions may be seen. The most common side effect is depression of bone marrow function, leading to aplastic anemia and a decreased number of white blood cells. So, this antibiotic is used only in life-threatening situations when no other drug is adequate.

Metabolic Antagonists

Several  drugs act as antimetabolites—they antagonize, or block, the functioning of metabolic pathways by competitively inhibiting the use of metabolites by key enzymes. These drugs can act as structural analogs, molecules that are structurally similar to naturally occurring metabolic intermediates. These analogs compete with intermediates in metabolic processes because of their similarity, and prevent normal cellular metabolism. They are bacteriostatic and broad spectrum.

Sulfonamides or Sulfa Drugs 

The first antimetabolites to be used successfully as chemotherapeutic agents were the sulfonamides, discovered by G. Domagk. Sulfonamides, or sulfa drugs, are structurally related to sulfanilamide, an analog of p-aminobenzoic acid, or PABA.

PABA is used in the synthesis of the cofactor folic acid (folate). When sulfanilamide or another sulfonamide enters a bacterial cell, it competes with PABA for the active site of Dihydropteroate synthase, an enzyme involved in folic acid synthesis, causing a decline in folate concentration. Folic acid is a precursor of purines and pyrimidines, the bases used in the construction of DNA, RNA, and other important cell constituents so this decline is harmful to the bacterium. The resulting inhibition of purine and pyrimidine synthesis leads to inhibition of protein synthesis and DNA replication, thus the pathogen dies.

Sulfonamides thus have a high therapeutic index. Sulfonamides are selectively toxic for many pathogens because these bacteria manufacture their own fo late and cannot effectively take up this cofactor, whereas humans do not synthesize folate (we must obtain it in our diet). 

The increasing resistance of many bacteria to sulfa drugs limits their effectiveness. 5% of the patients receiving sulfa drugs experience adverse side effects, chiefly allergic responses such as fever, hives, and rashes.

Trimethoprim

Trimethoprim is a synthetic antibiotic that also interferes with the production of folic acid. It binds to dihydrofolate reductase (DHFR), the enzyme responsible for converting dihydrofolic acid to tetrahydrofolic acid, competing against the dihydrofolic acid substrate 

It is a broad-spectrum antibiotic often used to treat respiratory and middle ear infections, urinary tract infections, and traveler’s diarrhea. 

It can be combined with sulfa drugs to increase efficacy of treatment by blocking two key steps in the folic acid pathway.

The inhibition of two successive steps in a single biochemical pthway means that less of each drug is needed in combination than when used alone. This is termed a synergistic drug interaction.

The most common side effects associated with trimethoprim are abdominal pain, abnormal taste, diarrhea, loss of appetite, nausea, swelling of the tongue, and vomiting. Taking trimethoprim with food may reduce some of these side effects. Some patients are allergic to trimethoprim, exhibiting rash and itching. Some patients develop photosensitivity reactions (i.e., rashes due to sun exposure).

Nucleic Acid Synthesis Inhibition 

The antibacterial drugs that inhibit nucleic acid synthesis function by inhibiting DNA polymerase and DNA helicase or RNA polymerase, thus blocking processes of replication or transcription, respectively. These drugs are not as selectively toxic as other antibiotics because procaryotes and eucaryotes do not differ greatly with respect to nucleic acid synthesis.

Quinolones

The quinolones are synthetic drugs that contain the 4-quinolone ring. The quinolones are important antimicrobial agents that inhibit nucleic acid synthesis. The first quinolone, nalidixic acid, was synthesized in 1962. Since that time, generations of fluoroquinolones have been produced. Three of these— ciprofloxacin, norfloxacin, and ofloxacin—are currently used, and more fluoroquinolones are being synthesized and tested.

Quinolones act by inhibiting the bacterial DNA gyrase and topoisomerase II. DNA gyrase introduces negative twist in DNA and helps separate its strands. Inhibition of DNA gyrase disrupts DNA replication and repair, bacterial chromosome separation during division, and other cell processes involving DNA. Fluoroquinolones also inhibit topoisomerase II, another enzyme that untangles DNA during replication.

Quinolones are bactericidal. The quinolones are broad-spectrum antibiotics. They are highly effective against enteric bacteria such as E. coli and Klebsiella pneumoniae. They can be used with Haemophilus, Neiserria, P. aeruginosa, and other gram-negative pathogens. The quinolones also are active against gram-positive bacteria such as S. aureus, Streptococcus pyogenes, and Mycobacterium tuberculosis. 

They are used in treating urinary tract infections, sexually transmitted diseases caused by Neisseria and Chlamydia, gastrointestinal infections, respiratory infections, skin infections, and osteomyelitis (bone infection).

Quinolones are effective when administered orally but can sometimes cause diverse side effects, particularly gastrointestinal upset.

Properties of Some Common Antibacterial Drugs

Antibiotic Group		Primary Effect		Mechanism of Action			Members		Spectrum		Common Side Effects
Cell Wall Synthesis Inhibition
Penicillins		Cidal		Inhibit transpeptidation enzymes involved in the cross-linking polysaccharide chains of the bacterial cell wall peptidoglycan. Activate cell wall lytic enzymes			Penicillin G, Penicillin V, Methicillin Ampicillin, Carbenicillin		Narrow (gram-positive) Broad (gram-positive, some gram-negative)		Allergic responses (diarrhea, anemia, hives, nausea, renal toxicity
Cephalosporins		Cidal		Same as above			Cephalothin, cefoxitin, cefaperazone, ceftriaxone		Broad (gram-positive, gram-negative)		Allergic responses, thrombophlebitis, renal injury
Vancomycin		Cidal		Prevents transpeptidation of peptidoglycan subunits by binding to D-Ala-D-Ala amino acids at the end of peptide cross-bridges. Thus it has a different binding site than that of the Penicillins			Vancomycin		Narrow (gram-positive)		Ototoxic (tinnitus and deafness), nephrotoxic, allergic reactions
Protein Synthesis Inhibition
Aminoglycosides		Cidal	Bind to small ribosomal subunit (30S) and interfere with protein synthesis directly inhibiting synthesis and causing misreading of mRNA		Neomycin, kanamycin, gentamicin,     Streptomycin			Broad (gram-negative, mycobacteria   Narrow (aerobic, gram-negative)			Deafness, renal damage, loss of balance, nausea, by allergic responses   Same as above
Tetracyclines		Static	Same as above		Oxytetracycline, chlortetracyclin			Broad (gram-positive and Gram-negative, rickettsia, chlamydia)			Gastrointestinal upset, teeth and discoloration, renal, hepatic injury
Macrolides		Static	Bind to 23S rRNA of large ribosomal subunit (50S) to inhibit peptide chain elongation during protein synthesis		Erythromycin, clindamycin			Broad (aerobic and anaerobic gram-positive, Some gram-negative)			Gastrointestinal upset, hepatic injury, anemia, allergic responses
Chloramphenicol		Static	Same as above		Chloramphenicol			Broad (gram-positive and negative, rickettsia and chlamydia)			Depressed bone marrow function, allergic reactions
Nucleic Acid Synthesis Inhibition
Quinolones and Fluoroquinolones		Cidal		Inhibit DNA gyrase topoisomerase IV, thereby blocking DNA replication and transcription		Norfloxacin, ciprofloxacin, Levofloxacin			Narrow (gram-negatives better than gram-positives) Broad spectrum		Tendonitis, headache, lightheadedness, convulsions, allergic reactions
Rifampin		Cidal		Inhibits bacterial DNA- dependent RNA polymerase		R-Cin, rifacilin, rifamycin, rimactane, rimpin, siticox			Mycobacterium infections and some gram-negative such as Neisseria meningitidis and Haemophilus influenzae		Nausea, vomiting, diarrhea, fatigue, anemia, drowsiness, headache, mouth ulceration, and liver damage
Cell Membrane Disruption
Polymyxin B		Cidal		Binds to plasma membrane and disrupts its structure and permeability properties		Polymyxin B, polymyxin topical ointment			Narrow—gram-negatives only		Can cause severe kidney damage, drowsiness, dizziness
Antimetabolites
Sulfonamides	Static		Inhibits folic acid synthesis by competing with ρ-aminobenzoic acid (PABA)			Silver sulfadiazin, sodium sulfacetamide, and  sulfamethoxazole, sulfanilamide, sulfasalazine, sulfisoxazole				Broad spectrum	Nausea, vomiting, diarrhea; hypersensitivity reactions such as rashes, photosensitivity
Trimethoprim	Static		Blocks folic acid synthesis by inhibiting the enzyme tetrahydrofolate reductase			Trimethoprim (in combination with sulfamethoxazole [1:5])				Broad spectrum	Same as sulfonamides, but less frequent
Dapsone	Static		Thought to interfere with folic acid synthesis			Dapsone				Narrow-mycobacterial infections, principally leprosy	Back, leg, or stomach pains; discolored fingernails, lips, or skin; breathing difficulties fever, loss of appetite, skin rash, fatigue
Isoniazid	Cidal if, bacteria are actively growing, static if bacteria are dormant		Exact mechanism is unclear but it is thought to inhibit lipid synthesis(especially mycolic acid); putative enoyl-reductase inhibitor			Isoniazid				Narrow—mycobacterial infections, principally  tuberculosis	Nausea, vomiting, liver damage, seizures, “pins and needles” in extremities (peripheral neuropathy)