1. Lecture 7-<br />Medical Microbiology<br />MBBS-Phase II- ims- MSU<br />Date: Monday; 18/10/2010<br />Antibiotics and antimicrobial chemotherapy<br />Chemotherapy is the treatment of infectious diseases by administration of drugs (antibiotics) which are lethal or inhibitory to the causative organisms.<br />History<br />Rapid development in antimicrobial chemotherapy began in 1935 with the discovery of sulphonamides by Domagk. In 1928, (1)Alexander Fleming noted that the product of a mould Penicillium notatum can inhibit the growth of staphylococci and other organisms. The active part of this product was successfully purified by Florey et al., in 1940, and named penicillin. This was followed by the isolation of several antibiotics from the filtrate of Streptomyces e.g. streptomycin, tetracyclines, chloramphenicol, … etc. <br />An antibiotic is an antimicrobial substance produced by a living microorganism and is active in high dilutions. In recent years, many of these antibiotics have been chemically synthesized. The term is used for any antimicrobial chemotherapeutic agent whether naturally produced or synthetic. <br />Types of action of antimicrobial chemotherapeutics<br />From their behavior toward bacterial populations antibacterial agents are divided into two classes:<br /> <br />Bactericidal drugs: these have a rapid lethal action against the pathogenic agents e.g. penicillins, cephalosporins, and aminoglycosides. <br />Bacteriostatic drugs: these merely inhibit the division of the pathogenic agents i.e. growth of organisms e.g. sulphonamides, tetracyclines and chloramphenicol. <br />ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ<br />(1)Originally noticed by a French medical student, Ernest Duchesne, in 1896 and rediscovered by Alexander Fleming, a Scottish bacteriologist in London, discovered penicillin by mistake when he was trying to study staphylococci bacteria in 1928. He was running experiments with the bacteria in his laboratory at London's St. Mary's Hospital, and set a laboratory dish containing the bacteria near an open window. Upon returning to the experiment, he found that some mold blown in through the open window onto the dish, contaminating the bacteria. Instead of throwing away his spoiled experiment, Fleming looked closely at it under his microscope. Surprisingly, he saw not only the mold growing on the staphylococci bacteria, but a clear zone around the mold. The Penicillium mold, the precursor to penicillin, was dissolving the deadly staphylococci bacteria.<br />Range of Action of Antimicrobial Chemotherapeutics: <br />Antibiotics fall into three main categories: <br />Active mainly against gram-positive organisms e.g. penicillin, erythromycin and lincomysin. <br />Active mainly against gram-negative organisms e.g. polymyxin and nalidixic acid. <br />Active against both gram-positive and gram-negative organisms (broad-spectrum activity) e.g. tetracyclines, chloramphenicol, and ampicillin. <br />Broad Spectrum Antibiotics<br />Broad-spectrum antibiotics are antibiotics which are designed to work against a broad spectrum of bacteria, rather than narrow-spectrum antibiotics, which are only effective against a smaller range of bacteria. These medications are classically used in cases in which a doctor is not sure about the identity of a disease-causing organism and wants to provide a patient with medication which will rapidly attack the infection, rather than waiting for culture results and prescribing a narrow-spectrum antibiotic which is more targeted in effect.<br />Some examples of broad-spectrum antibiotics include; <br />penicillin, <br />cephalosporin, <br />tetracycline, <br />ciprofloxacin, <br />levofloxacin. <br />These drugs work on both gram-negative and gram-positive organisms. When a patient appears to have a bacterial infection, a broad-spectrum antibiotic is the most likely to provide effective treatment without knowing which organism is behind the infection. For example, when a patient comes to a doctor with bronchitis, the doctor may prescribe a general antibiotic medication to treat the infection without taking a culture.<br />If an infection persists or it appears unusual in nature, cultures will be done. In a culture, a sample from the patient is collected and cultured on suitable media in the laboratory to find out which organism is responsible for the infection. Furthermore, a culture can also be used to test antibiotics in case an organism is antibiotic-resistant. In this case, the culture is used to find the drug which will be most effective so that the patient does not have to try several unsuccessful broad-spectrum antibiotics before finding one which works.<br />One problem with broad-spectrum antibiotics which began to grow in the late 20th century was the emergence of antibiotic resistance in bacteria. Almost as soon as humans started developing antibiotics, bacteria started swapping genes which they could use to survive antibiotic therapy. In some cases, organisms developed resistance to multiple broad-spectrum antibiotics, making treatment of infections involving these organisms very challenging (complicated). More advanced classes of antibiotics were developed in response, but bacteria also adapted to address these. A broad-spectrum antibiotic is only useful as long as it kills most bacteria and organisms which can quickly adapt to resist antibiotics present a significant challenge.<br />Mechanism of Action of Antimicrobial Chemotherapeutics <br />An ideal antimicrobial agent should have selective toxicity i.e. it can kill or inhibit the growth of a microorganism in concentrations that are not harmful to the cells of the host. Disinfectants e.g. phenol and antiseptics e.g. alcohol and iodine, destroy bacteria but they are highly toxic to tissue cells and are unsuitable for use as chemotherapeutic agents. <br />Thus, the mechanism of action of a chemotherapeutic must depend on the inhibition of a metabolic channel or a structure that is present in the microbe but not in the host cell. Several mechanisms are known: <br />Inhibition of cell wall synthesis: Due to its unique structure and function, the bacterial cell wall is an ideal point of attack by selective toxic agents. Some antibiotics e.g. penicillin, cephalosporins and vancomycin, interfere with cell wall synthesis and cause bacteriolysis. <br />Inhibition of cytoplasmic membrane function: Some antibiotics cause disruption of the cytoplasmic membrane and leakage of cellular proteins and nucleotides leading to cell death. Polymyxins, amphotericin B, and nystatin are examples. <br />Inhibition of protein synthesis: Many antimicrobial chemotherapeutics block protein synthesis by acting on the 30s or 50s subunits of the bacterial ribosome. Examples are chloramphenicol, tetracycline, erythromycin and the aminoglycosides e.g. tobramycin, gentamycin and streptomycin. <br />Inhibition of nucleic acid synthesis: These can act on any of the steps of DNA or RNA replication e.g. quinolones, trimethoprim, rifampicin, nalidixic acid, novobiocin and metronidazole. <br />Competitive inhibition: in which the chemotherapeutic agent competes with an essential metabolite for the same enzyme e.g. p-aminobenzoic acid (PABA) is an essential metabolite for many organisms. They use it as a precursor in folic acid synthesis which is essential for nucleic acid synthesis. Sulphonamides are structural analogues to PABA so they enter into the reaction in place of PABA and compete for the active center of the enzyme thus inhibiting folic acid synthesis. <br />Mechanisms of Resistance to Antimicrobial Agents<br />In the treatment of infectious diseases, one of the serious problems commonly faced with, is the development of bacterial resistance to the antibiotic used. The mechanisms by which the organism develops resistance may be one of the following: <br />The organism produces enzymes that destroy the drug e.g. production of:<br /> β-lactamase - that destroys penicillin – by penicillin resistant staphylococci<br />Acetyltransferase produced by gram negative bacilli destroys chloramphenicol. <br />The organism changes its permeability to the drug, by modification of protein in the outer cell membranes, thus impairing its active transport into the cell e.g. resistance to polymyxins. <br />The organism develops an altered receptor site for the drug e.g. resistance to aminoglycosides is associated with alteration of a specific protein in the 30s subunit of the bacterial ribosome that serves as a binding site in susceptible organisms. <br />The organism develops an altered metabolic pathway that bypasses the reaction inhibited by the drug e.g. sulphonamide-resistant bacteria acquire the ability to use preformed folic acid with no need for extracellular PABA. <br />Origin of Resistance to Antimicrobial Agents <br />These mechanisms may be of non genetic or genetic origin: <br />Non genetic Drug Resistance:<br />Metabolic inactivity: Most antimicrobial agents act effectively only on replicating cells. Non multiplying organisms are phenotypically resistant to drugs. Tubercle bacilli survive for several years in tissues and their resistance to antituberculous drugs is due in part to their metabolic inactivity (dormancy).<br />Loss of target structure: L-forms of bacteria are penicillin resistant, having lost their cell wall which is the structural target site of the drug. <br />Genetic Drug Resistance <br />Plasmid mediated resistance <br />Resistance (R) factors are a class of plasmids that mediate resistance to one or more antimicrobial agent. Plasmids frequently carry genes that code for the production of enzymes that inactivate or destroy antimicrobial agents e.g. p-Iactamase which destroys the p-Iactam ring in penicillin and cephalosporins. Plasmids may result in epidemic resistance among bacteria by moving from one to the other by conjugation, transduction, or transformation. <br />Transposon-mediated resistance <br />Many transposons(2) carry genes that code for drug resistance. As they move between plasmids and chromosomes they can transfer this property to bacteria. The process is called transposition. <br />Chromosomal drug resistance <br />This develops as a result of spontaneous mutation in a gene that controls susceptibility to an antimicrobial agent. The most common result of chromosomal mutation is alteration of the receptors for a drug. For example, streptomycin resistance can result from a mutation in the chromosomal gene that controls the receptor for streptomycin located in the 30s bacterial ribosome. <br />ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ<br />(2)Transposons are sequences of DNA that can move or transpose themselves to new positions within the genome of a single cell. The mechanism of transposition can be either quot;
copy and pastequot;
or quot;
cut and pastequot;
. Transposition can create phenotypically significant mutations and alter the cell's genome size. Barbara McClintock's discovery of these jumping genes early in her career earned her a Nobel prize in 1983. <br />Complications of Antibacterial Chemotherapy <br />Development of drug resistance <br />This is one of the most serious complications of chemotherapy. The emergence of resistant mutants is encouraged by:<br />Inadequate dosage, <br />Prolonged treatment, <br />The presence of a closed focus of infection and <br />The abuse of antibiotics without in vitro susceptibility testing<br />The problem is more serious when resistant strains develop in the community, e.g. in hospitals it is common to find that about 90% of strains of Staph. aureus are resistant to penicillin. <br />Drug toxicity <br />Many of the antibacterial drugs have toxic side effects. This can be due to;<br />Over-dosage, <br />prolonged use or narrow margin of selective toxicity e.g. streptomycin affects the 8th cranial nerve leading to deafness, chloramphenicol may cause depression of the bone marrow, the aminoglycosides (e.g. garamycin, netilmycin, tobramycin) are nephrotoxic. Tetracyclines inhibit growth and development of bones and teeth in the developing fetus and infants. <br />Super-infection<br />Superinfection may occur by pre-existing resistant strains present in the environment e.g. penicillin resistant Staph. aureus in hospital infections. <br />Another type of superinfection is due to suppression of normal flora by the antibiotic used and their replacement with drug-resistant. organisms which cause disease e.g.: <br />Overgrowth of Candida in the vagina causing vaginitis or in the mouth causing oral thrush. <br />Prolonged oral chemotherapy leading to suppression of intestinal flora and overgrowth of staphylococci causing staphylococcal enterocolitis or C. difficile which causes pseudomembranous colitis. <br />Overgrowth of gram-negative organisms naturally drug-resistant e.g. pseudomonas, proteus or enterobacter, may account for respiratory tract superinfection. <br />Hypersensitivity<br />The drug may act as a hapten, binds to tissue proteins, and stimulates an exaggerated immune response leading to tissue damage i.e. hypersensitivity. Any type of hypersensitivity reaction can occur with several antibiotics. The most serious is anaphylactic shock, this may occur with penicillin or cephalosporins. Milder manifestations may be urticaria, purpural eruptions, skin rash, diarrhoea, vomiting and' jaundice. <br />Chemoprophylaxis<br />Chemoprophylaxis is the use of antimicrobial agents to prevent rather than to treat infectious diseases. The following are principal conditions for which prophylactic antibiotics are positively indicated: <br />The use of benzathine penicillin G injections every 3-4 weeks to prevent reinfection with Strept. pyogenes in rheumatic patients. <br />A single large dose of amoxycillin given immediately prior to dental procedures is recommended for patients with congenital or rheumatic heart disease to prevent endocarditis. <br />The oral administration of rifampicin 600 mg twice a day for 2 days to exposed persons during epidemics of meningococcal meningitis. <br />Oral administration of tetracycline to prevent cholera caused by Vibrio cholerae. <br />Chemoprophylaxis in surgery: little is known about its effectiveness. <br />However, conditions in which chemoprophylaxis is indicated are: <br />Large bowel surgery. <br />Major orthopedic and cardiac surgery. <br />Amputation of an ischaemic limb. <br />Clinical Use of Antibiotics:<br />The objective of antibiotic therapy is to cure the patient with minimal complications. At the same time, it is important to discourage the emergence of drug-resistant organisms. The following principles should be observed: <br />Antibiotics should not be given for trivial infections.<br />They should be used for prophylaxis only in special circumstances.<br />Treatment should be based on a clear clinical and bacteriological diagnosis. Suitable specimens should be sent to the laboratory before treatment is begun. However, treatment can be started after taking the sample; but should be modified later according to results of antibiotic sensitivity testing in vitro. <br />Antibiotics for systemic treatment should be given in full therapeutic doses for adequate period. <br />Combined therapy with two or more antibiotics is required in some conditions e.g. : <br />Serious resistant infections e.g. infective endocarditis or meningitis. <br />In treatment of tuberculosis 2 or 3 drugs are given in combination to delay emergence of resistant mutants. Also to decrease toxic effects of the drugs by lowering the dose of each. <br />Severe mixed infections e.g. peritonitis following perforation of the colon. <br />Antimicrobial Chemotherapeutics<br />Killing pathogens, but not us!<br />Definitions<br />Chemotherapeutics<br />Antimicrobials<br />Antibiotics<br />Antifungals<br />Strategies<br />Look for metabolic targets not found in human cells<br />Bacteria – some<br />Fungi – fewer<br />Protozoa – even less<br />Viral – very few<br />Targets in Bacteria<br />Cell Wall <br />Protein synthesis<br />Cytoplasmic Membrane<br />General metabolic pathways<br />Nucleic Acid synthesis<br />Inhibition of Attachment<br />Targets of Antimicrobials<br />Cell Wall <br />Penicillin<br />Cephalasporin<br />Vancomycin<br />Protein synthesis<br />Streptomycin<br />Tetracycline<br />Erythromycin<br />Cytoplasmic Membrane<br />Amphotericin B & Fluconazole<br />Polymyxins<br />General metabolic pathways<br />Sulfanilamide<br />Protease Inhibitors<br />Nucleic Acid synthesis<br />Rifampin - RNA<br />Ciprofloxacin – helicase<br />AZT & Acyclovir<br />Inhibition of Attachment<br />Antivirals - pleconaril<br />Spectrum of Activity<br />Determining Efficacy of Antibiotics<br />Kirby Bauer Test<br />MIC<br />Broth Dilution<br />Etest<br />MBC<br />Effect of Route of Administration<br />Side Effects<br />Kidney Damage<br />Nerve Damage<br />Allergies<br />Immunities<br />Interactions with Calcium<br />Disruption of Normal Microbiota<br />Hairy Tongues<br />Metronidazole<br />Developing Resistance<br />Mutation and Selection<br />Horizontal Gene Transfer<br />Mechanisms<br />Alteration of target<br />Membrane & Cell wall permeability <br />Enzymes<br />Changes in metabolic pathway<br />Cross Resistance<br />Countering Drug Resistance<br />Completing the regimen<br />Synergism<br />Using Narrow spectrum drugs<br />Limiting use<br />Developing new drugs<br />Multi-drug treatments<br />Are bacteria winning the war against antibiotics?<br />Antiprotozoans & Antihelminths<br />Quinolones – antimalarial<br />Mebendazole – Pinworms and protozoans<br />Ivermectin – flaccid paralysis<br />Cultures for some microorganisms that produce antibiotics <br />