Antibiotic Resistance Lecture PDF

Antimicrobial Drugs: Mechanism of Action Antibiotic Resistance  Antibiotics- Naturally occurring and synthetically derived organic compounds that inhibit or destroy selective bacteria, generally at low concentrations. Probiotics- Live, nonpathogenic bacteria, which excludes...

Antimicrobial Drugs: Mechanism of Action Antibiotic Resistance  Antibiotics- Naturally occurring and synthetically derived organic compounds that inhibit or destroy selective bacteria, generally at low concentrations. Probiotics- Live, nonpathogenic bacteria, which excludes the pathogen from binding sites on the mucosa (colonization resistance).  The history of antibiotics 1928, Sir Alexander Fleming In 1928, while working on influenza virus, he observed that mold had developed accidently on a staphylococcus culture plate and that the mold had created a bacteria-free circle around itself. He was inspired to further experiment and he found that a mold culture of Penicillium fungi prevented growth of staphylococci, even when diluted 800 times. In 1939 Ernst Chain began, with Sir Howard Florey, a systematic study of antibacterial substances produced by micro-organisms. This led to his best known work, the reinvestigation of penicillin and to the discovery of its chemotherapeutic action. Fleming, Florey and Chain jointly received the Nobel Prize in Medicine in 1945. In 1943, Selman Waksman discovered that soil Streptomyces produce antibiotics. Nobel prize in 1952 for discovery of Streptomycin. He discovered over twenty antibiotics (a word which he coined) and introduced procedures that have led to the development of many others. CLASSIFICATION OF ANTIBACTERIAL AGENTS Antimicrobials are classified in several ways, including: Spectrum of activity Effect on bacteria Spectrum of activity Broad-spectrum antibiotics active against several types of microorganisms, e.g., tetracyclines are active against many gram - negative rods, chlamydiae, mycoplasmas, and rickettsiae. Narrow-spectrum antibiotics active against one or very few types, e.g., vancomycin is primarily used against certain gram-positive cocci namely, staphylococci & enterococci. Effect on bacteria Bactericidal = kill bacteria Bacteriostatic = slow or interfere with growth of bacteria (inhibits their growth but does not kill them) (1) the bacteria can grow again when the drug is withdrawn, and (2) host defense mechanisms, such as phagocytosis, are required to kill the bacteria. Bactericidal and bacteriostatic activity of antimicrobial drugs  4 Major Targets for Antibiotic Action ▪ Cell wall ▪ Cell membrane ▪ Ribosomes (protein synthesis) ▪ Nucleic acid Mechanism of Action of Important Antibacterial and Antifungal Drugs Mechanism of Action Drugs Inhibition of cell wall synthesis Penicillins, cephalosporins, imipenem aztreonam(beta-lactams), vancomycin, Antifungals Inhibition of protein synthesis 50S Chloramphenicol, erythromycin, clindamycin, linezolid, 30S Tetracyclines and aminoglycosides Inhibition of nucleic acid synthesis Sulfonamides, trimethoprim, Quinolones,(e.g.ciprofloxacin) Rifampin Alteration of cell membrane function Polymyxin, daptomycin & Antifungals(Amphotericin B, nystatin) Other mechanisms of action Isoniazid, metronidazole, Antifungals Antibiotics Acting on Cell Wall The Action of Penicillin Penicillins (and cephalosporins) act by inhibiting transpeptidases, the enzymes that catalyze the final cross-linking step in the synthesis of peptidoglycan. MECHANISMS OF ACTION Two additional factors are involved in the action of penicillin: Penicillin binds to a variety of receptors in the bacterial cell membrane and cell wall, called penicillin-binding proteins (PBPs) Autolytic enzymes called murein hydrolases (murein is a synonym for peptidoglycan) are activated in penicillin-treated cells and degrade the peptidoglycan Some bacteria (e.g., strains of S. aureus) are tolerant to the action of penicillin, because these autolytic enzymes are not activated. Penicillin-treated cells die by rupture as a result of the influx of water into the high-osmotic-pressure interior of the bacterial cell. Penicillin kills cells only when they are growing Disadvantages of Penicillins Limited effect against Gram (-) strains Hydrolysis by gastric acid- no oral use Inactivation by beta lactamase enzymes Hypersensitivity (1-10%), Anaphylaxis (0-5%) Other Beta-lactams Cephalosporins, carbapenems & Aztreonam are beta-lactam drugs that act in the same manner as penicillins; i.e., they are bactericidal agents that inhibit the cross-linking of peptidoglycan. Cephalosporin s Cephalosporins are beta-lactam drugs that act in the same manner as penicillins. The structures are different: Cephalosporins have a 6-membered ring adjacent to the beta-lactam ring, while penicillins have a 5-membered ring. Bactericidal Products of the mould cephalosporium Five generations. 1st active against G+ Cocci, 2, 3, 4 more against G- Activity of Selected Cephalosporins Cephalosporin s Few allergic reactions Some are oral, but most are given parentally. Wide distribution after absorption E.gs Cefuroxime, cefotaxime and ceftriaxone Broadspectrum, active against most Gram+, some Gram– activity Carbapenem s Carbapenems are β-lactam drugs that are structurally different from penicillins and cephalosporins. Wide bactericidal activity against many gram – positive (e.g., streptococci and staphylococci), gram – negative (Pseudomonas, Haemophilus, Escherichia coli), and anaerobic bacteria (Bacteroides and Clostridium). Eg. Imipenem, Meropenem etc. Antimicrobial agents affecting Bacterial Protein Synthesis Normal protein synthesis in Bacteria Transcription Translation Transcription and translation Transcription occurs in the cell nucleus, where the DNA is held. The DNA is "unzipped" by the enzyme helicase, leaving the single nucleotide chain open to be copied. RNA polymerase reads the DNA strand , while it synthesizes a single strand of messenger RNA. The single strand of mRNA leaves the nucleus and migrates into the cytoplasm. The synthesis of proteins is known as translation. Translation occurs in the cytoplasm, where the ribosomes are located. Ribosomes are made of a small and large subunit that surround the mRNA. In translation, messenger RNA (mRNA) is decoded to produce a specific polypeptide. This uses an mRNA sequence as a template to guide the synthesis of a chain of amino acids that form a protein. Affecting Bacterial Protein Synthesis Aminoglycosides and tetracyclines act at the level of the 30S ribosomal subunit. Chloramphenicol, erythromycins,and clindamycin act at the level of the 50S ribosomal subunit.  Aminoglycoside ▪ Aminoglycosides inhibit s bacterial protein synthesis by binding to the 30S subunit, which blocks the initiation complex and cause a misreading of the genetic code. This subsequently leads to the interruption of normal bacterial protein synthesis. ▪ No peptide bonds are formed, and no polysomes are made. ▪ Aminoglycosides are a family of drugs that includes: Gentamicin Kanamycin Neomycin Streptomycin Amikacin Aminoglycoside s Poor oral absorption No oral forms, only IV TOXICITY cautions Nephro oto Bactericidal action against G- rods like Pseudomonas, E.coli, Klebsiella Poor entry into Cerebrospinal fluid - CSF  Tetracycline They inhibit protein synthesis by binding to s the 30S ribosomal subunit and by blocking tRNA from entering the acceptor site on the ribosome The tetracyclines are a family of drugs; Doxycycline is used most often. Broad spectrum, originally derived from Streptomyces. More recent compounds are semi-synthetic or synthetic. Bacteriostatic action against gram +ve/-ve and some protozoa. Absorbed orally Tetracycline, Vibramycin, Minocycline  Erythromyci n ▪ Erythromycin inhibits bacterial protein synthesis by blocking the release of the tRNA after it has delivered its amino acid to the growing polypeptide. ▪ Erythromycin is a member of the macrolide family of drugs that includes azithromycin and clarithromycin. Clindamyci n Bacteriostatic drug against anaerobes Important side effect of clindamycin is pseudomembranouscolitis (suppression of the normal flora of the bowel by the drug and overgrowth of a drug-resistant strain of Clostridium difficile) causing severe bloody diarrhea.  Chloramphenico l at same site as erythromycin. Binds to the 50S subunit Chloramphenicol inhibits bacterial protein synthesis by blocking peptidyl transferase, the enzyme that adds the new amino acid to the growing polypeptide. Active against many gram +ve & gram-ves Side effects: - bone marrow toxicity - “gray baby” syndrome Antimicrobial agents affecting Bacterial Protein Synthesis Erythromycin blocking the release of the tRNA INHIBITION OF NUCLEIC ACID SYNTHESIS 1.Inhibition of precursor synthesis e.g Sulphonamides & Trimethoprim inhibit synthesis of tetrahydrofolic acid which is required for synthesis of nucleic acid precursors A, G, T Sulphonamides & Trimethoprim Bacteriostatic, good against gram –ve’s Sulphonamides & Trimethoprim are two drugs but are used together for synergistic effect. E.g Septran Side Effects= Anemia, thrombocytopenia, Photosensitivity (Avoid tanning , Avoid sunlight). INHIBITION OF NUCLEIC ACID SYNTHESIS 2. Inhibition of DNA synthesis by inhibiting DNA gyrase an enzyme which unwinds DNA strands for replication. Quinolones inhibit DNA synthesis in bacteria by blocking DNA gyrase the enzyme that unwinds DNA strands so that they can be replicated. Quinolones are a family of drugs that includes ciprofloxacin, ofloxacin, and levofloxacin. Fluoro-Quinolone s 5 generations Newer forms (ciprofloxacin, ofloxacin and levofloxacin) have wider activity. Used for RTI, UTI, GIT, Skeletal & soft tissue infections Contraindicated in pregnancy & young child -it damages growing bone and cartilage  INHIBITION OF NUCLEIC ACID SYNTHESIS 3. Inhibition of RNA synthesis Rifampin inhibits RNA synthesis in bacteria by blocking the RNA polymerase that synthesizes mRNA. Rifampin is typically used in combination with other drugs because there is a high rate of mutation of the RNA polymerase gene, which results in rapid resistance to the drug. The action of antimicrobial drugs Chemoprophylaxi s Antimicrobial drugs are used to prevent infectious diseases as well as to treat them. Chemoprophylactic drugs are given primarily in three circumstances: to prevent surgical wound infections, to prevent opportunistic infections in immunocompromised patients, in people with normal immunity who have been exposed to certain pathogens.  Probiotics Probiotics are live, nonpathogenic bacteria that may be effective in the treatment or prevention of certain human diseases. Oral administration of live Lactobacillus rhamnosus strain GG significantly reduces the number of cases of nosocomial diarrhea in young children. The yeast Saccharomyces boulardii reduces the risk of antibiotic-associated diarrhea caused by C. difficile. Adverse effects are few Side Effects Toxic effects. These effects arise from direct cell and tissue damage in the macroorganism. Blood concentrations of some substances must therefore be monitored during therapy if there is a risk of cumulation due to inefficient elimination (examples: aminoglycosides, vancomycin). Allergic reactions -The Pathological Immune Response (example: penicillin allergy). Biological side effects. Example: change in or elimination of normal flora,interfering with its function as a beneficial colonizer. Antibiotic Resistance Definitio n Resistance. Relative or complete lack of effect of antibiotic against a previously susceptible microbe Natural (intrinsic) resistance. Resistance characteristic of a bacterial species, genus, or family. Acquired resistance. Strains of sensitive taxa can acquire resistance by way of changes in their genetic material. Intrinsic Resistance Intrinsic resistance is the innate ability of a bacterial species to resist activity of a particular antimicrobial agent through its inherent structural or functional characteristics, which allow tolerance of a particular drug or antimicrobial class. This can also be called “insensitivity” since it occurs in organisms that have never been susceptible to that particular drug. Such natural insensitivity can be due to: lack of affinity of the drug for the bacterial target inaccessibility of the drug into the bacterial cell exclusion of the drug by chromosomally encoded active exporters innate production of enzymes that inactivate the drug Biofilms  MECHANISMS OF ⚫ RESISTANCE Enzymatic inhibition ⚫ Bacteria produce enzymes that inactivate the drug (e.g., β-lactamases can inactivate penicillins and cephalosporins by cleaving the β-lactam ring of the drug) ⚫ Alteration of target sites Altered ribosomal target sites Altered cell wall precursor targets Altered target enzymes ⚫ Alteration of bacterial membranes Outer membrane permeability Inner membrane permeability Level of resistance high-level resistance - refers to resistance that cannot be overcome by increasing the dose of the antibiotic. A different antibiotic, usually from another class of drugs, is used. Resistance mediated by enzymes such as β-lactamases often result in high- level resistance, as all the drug is destroyed. Low-level resistance -refers to resistance that can be overcome by increasing the dose of the antibiotic. Resistance mediated by mutations in the gene encoding a drug target is often low level, as the altered target can still bind some of the drug but with reduced strength.  Molecular genetics of antibiotic resistance ⚫ The genetic basis of drug resistance, mediated by genetic change in bacteria, is most important in the development of drug resistance in bacteria. ⚫ This is of three types as follows: ⚫ (a) chromosome-mediated resistance, ⚫ (b) plasmid-mediated resistance, and ⚫ (c) transposons-mediated resistance. GENETIC BASIS OF RESISTANCE Chromosome-Mediated Resistance Chromosomal resistance is due to a mutation in the gene that codes for either the target of the drug or the transport system in the membrane that controls the entry of drugs into cells. Plasmid-Mediated Resistance (1) It occurs in many different species, especially gram-negative rods. (2) Plasmids frequently mediate resistance to multiple drugs. (3) Plasmids have a high rate of transfer from one cell to another, usually by conjugation.  R factors Most resistance plasmids have two sets of genes: (1) resistance transfer genes that encode the sex pilus and other proteins that mediate transfer of the plasmid DNA during conjugation, and (2) drug resistance genes that encode the proteins that mediate drug resistance. In addition to producing drug resistance, R factors have two very important properties: (1) They can replicate independently of the bacterial chromosome; therefore, a cell can contain many copies; and (2) they can be transferred not only to cells of the same  Transposon-Mediated Resistance Transposon - resistance genes that are transferred within or between large pieces of either chromosomal DNA or plasmids. Transduction - phage-mediated transfer of resistance genes. Phage which pick up resistance genes that can infect antimicrobial sensitive cells. Transformation - Uptake of resistance transposon by a sensitive bacterium after lysis of a resistant bacteria.  Penicillins & Cephalosporins Penicillins & Cephalosporins Mechanism of Action Inhibition of cell wall synthesis Mechanism of resistance Cleavage by β-lactamases (penicillinases and cephalosporinases) Clavulanic acid and sulbactam are penicillin analogues that bind strongly to β- lactamases and inactivate them. Resistance to penicillin can also be due to changes in the penicillin- binding proteins (PBPs) in the bacterial cell membrane. S. aureus demonstrate tolerance. Failure of activation of the autolytic enzymes, murein hydrolases, which degrade the peptidoglycan. Carbapenem s Carbapenems—Resistance to carbapenems, such as imipenem, is caused by carbapenemases that degrade the β-lactam ring. This enzyme endows the organism with resistance to penicillins and cephalosporins as well. Carbapenemases are produced by many enteric gram-negative rods, especially Klebsiella, Escherichia,and Pseudomonas. Carbapenem-resistant strains of Klebsiella pneumoniae are an important cause of hospital-acquired infections and are resistant to almost all known antibiotics. Tetracyclines and Chloramphenicol Resistance to tetracyclines is the result of failure of the drug to reach an inhibitory concentration inside the bacteria. This is due to plasmid-encoded processes that either reduce the uptake of the drug or enhance its transport out of the cell. Chloramphenicol—Resistance to chloramphenicol is due to a plasmid- encoded acetyltransferase that acetylates the drug, thus inactivating it. Sulfonamide s Resistance is mediated by two mechanisms: (1) a plasmid-encoded transport system that actively exports the drug out of the cell (2) a chromosomal mutation in the gene coding for the target enzyme dihydropteroate synthetase, which reduces the binding affinity of the drug. Trimethoprim and Quinolones Quinolones inhibit DNA synthesis in bacteria by blocking DNA gyrase (topoisomerase)—the enzyme that unwinds DNA strands so that they can be replicated. Quinolones are a family of drugs that includes ciprofloxacin, ofloxacin, and levofloxacin. Resistance to trimethoprim is due primarily to mutations in the chromosomal gene that encodes dihydrofolate reductase. Resistance to quinolones is due to chromosomal mutations that modify the bacterial DNA gyrase. USE OF ANTIBIOTIC COMBINATIONS (1) To treat serious infections before the identity of the organism is known. (2) To achieve a inhibitory effect against certain organisms. (3) To prevent the emergence of resistant organisms. (If bacteria become resistant to one drug, the second drug will kill them, thereby preventing the emergence of resistant strains.)  OVERUSE & MISUSE OF ANTIBIOTICS Some doctors use multiple antibiotics when one would be sufficient, prescribe unnecessarily long courses of antibiotic therapy, use antibiotics in self-limited infections for which they are not needed, and overuse antibiotics for prophylaxis before and after surgery. Antibiotics are used in animal feed to prevent infections and promote growth. This selects for resistant organisms in the animals and may contribute to the pool of resistant organisms in humans. ANTIBIOTIC SENSITIVITY TESTING Minimal Inhibitory Concentration ANTIBIOTIC SENSITIVITY TESTING Disk Diffusion Method References Levinson, Review of Medical Microbiology and Immunology. Chapters 10, 11