Microbiology Lecture 5: Antimicrobial Chemotherapy - October 2022 PDF

Lecture 5 Microbiology: Antimicrobial Chemotherapy, drug resistance and its prevention  Content Antimicrobial Chemotherapies and their targets Drug resistance, drug-bacteria relationship, clinical implications, and prevention  Hi...

Lecture 5 Microbiology: Antimicrobial Chemotherapy, drug resistance and its prevention  Content Antimicrobial Chemotherapies and their targets Drug resistance, drug-bacteria relationship, clinical implications, and prevention  History Use of drugs since 17th century (eg, quinine for malaria and emetine for amebiasis) Chemotherapy as a science started in the beginning of the 20th century The first planned chemotherapeutic regimen against syphilis was developed by German physician Gerhard Domagk, Nobel Prize 1939 and scientist Paul Erlich 1935 – Discovery of sulphonamides (die prontosil rubrum) start of the Discovery of Era of Chemotherapy Penicillin, Nobel 1952 – Selman Waksman discovery Prize 1945 of Streptomycin (Aspergillus) Selman Waksman, Nobel Prize 1952  Selective toxicity - The ability to kill or inhibit the growth of a microorganism without harming the host cells. Often, selective toxicity is relative rather than absolute; this implies that a drug in a concentration tolerated by the host may damage an infecting microorganism. Examples: some antibiotics acts on bacterial cell wall synthesis – the organelle that does not exist in eukaryotic cells. some agents act on 70S ribosomes and not in 80S ribosomes.  ANTIMICROBIAL AGENTS ANTIBIOTICS: Natural compounds produced by microorganism which inhibit the growth of other microorganism Antibiotics – antimicrobials of microbial origin , most of which are produced by fungi or bacteria of the genus Streptomycetes. Antimicrobials – substances used for treatment of infectious diseases, it implies the agent, that is not an antibiotic in strict sense, of originating a bacteria or fungus, but is still used in treatment of infections.  ANTIMICROBIAL AGENTS CHEMOTHERAPEUTIC AGENTS: Chemically synthesized antibiotics compounds similar to natural antibiotics. Semi-synthetic antibiotics – chemically modified natural compounds of bacterial origin. Synthetic antibiotics – do not have natural analogues at all.  Activity BACTERICIDAL: antimicrobial activity that is lethal for bacteria/kills bacteria. BACTERIOSTATIC: antimicrobial activity that inhibits growth but does not kill the organism. MIC –minimal inhibitory concentration Minimal inhibitory concentration (MIC)- a laboratory term that defines the lowest concentration (μg/ml) able to inhibit growth of the micro-organism in vitro.  TERMS Resistant/nonsusceptible – term applied when organisms are not inhibited by clinically achievable concentrations of antimicrobial agent. Sensitive/susceptible - term applied to microorganims indicating that they will be inhibited by concentrations of the antimicrobial that can be achieved clinically.  Spectrum of activity Spectrum – an expression of the categories of microorganisms against which an antimicrobial is typically active. Broad spectrum : Gram positive & Gram negative bacteria Narrow spectrum: selected microrganisms Limited spectrum: effective against a single organism or disease  Spectrum of activity Most of the microorganisms that produce antibiotics are resistant to the action of their own antibiotic, although the organisms are affected by other antibiotics, and their antibiotic may be effective against closely-related strains.  Sources of natural antibiotics Moulds: Pencillinum sp. – Penicillin Bacteria: Streptomyces sp. – tetracyclin, erythromycin, chloramphenicol, etc. Bacillus sp. - polymyxin and bacitracin These organisms all have in common that they live in soil and they form some sort of a spore or resting structure. Antibiotics are secondary metabolites and they are produced at the same time that the cells begin their sporulation processes. Selection of antibiotics/drugs  Therapeutic index (TI) Effective dose ED50 –a dose that will produce an effect that is half of the maximal response. Toxic dose TD 50 – dose that will cause toxic effect in 50% of tested animals LD50 - dose that will kill 50 percent of animal tested TI - is a ratio of the LD50 to the ED50 of a drug. It gives an estimate of the relative safety of a drug. The ratio of the toxic dose (to the patient) to the therapeutic dose (to eliminate the infection). TI = toxic dose/therapeutic dose The larger the index, the safer is the drug (antibiotic) for human use. Examples: Penicillin: High (10-100) Aminoglycosides: Low (1.0-10.0) Polymyxin B : the lowest (0.1-1.0)  MECHANISMS OF ACTION OF ANTIMICROBIALS 1.Inhibition of cell wall synthesis 2. Inhibition of cell membrane function 3. Inhibition of protein synthesis (ie, inhibition of translation and transcription of genetic material) 4. Inhibition of nucleic acid synthesis 5. Anti-metabolite OR competitive antagonism. Targets of antibiotics  Cell wall inhibition The cell wall contains a complex polymer consisting of polysaccharides and a highly cross-linked polypeptide. The polysaccharides contain the amino sugars N-acetylglucosamine and N-acetylmuramic acid. N-acetylmuramic acid is found only in bacteria. N-acetylglucosamine is present in extracellular matrix of animal cells.  LACTAM ANTIBIOTICS: All β-lactam drugs are selective inhibitors of bacterial cell wall synthesis and therefore active against growing bacteria. This inhibition is only one of several different activities of these drugs Contain : Beta- Lactam ring & organic acid. Natural & Semi-synthetic Bactericidal Bind to Penicillin binding protein (PBP), interfere with trans-peptidation reaction Toxicity: mainly: Hypersensitivity Anaphylaxis, Diarrhea, etc.  Binding of the drug to cell receptors - penicillin-binding proteins [PBPs] 3 1 4 2 Cell wall inhibition mechanism Two major enzymes: transpeptidase and D-alanyl carboxypeptidase (PENICILLIN BINDING PROTEIN) contribute to "cross-linking"... vital step in completing the cell wall. The Beta-Lactam Ring binds at the active site of the transpeptidase enzyme by mimicking the D-alanyl-D-alanine residues that would normally bind to this site (The pink objects are the Beta-Lactam Rings).  Cell wall inhibition Bacteria constantly remodel their peptidoglycan cell walls, simultaneously building and breaking down portions of the cell wall as they grow and divide. After a β-lactam drug has attached to one or more receptors, the transpeptidation reaction is inhibited, and peptidoglycan synthesis is blocked. β-Lactam antibiotics inhibit the formation of peptidoglycan cross-links in the bacterial cell wall; this is achieved through binding of the four-membered β-lactam ring of penicillin to the enzyme DD-transpeptidase. Consequently, DD-transpeptidase cannot catalyze formation of these cross-links, and an imbalance between cell wall production and degradation develops, causing the cell to rapidly die. Spheroplasts are created from gram-negative bacteria, protoplasts – from gram- positives.  PBPs There are at least six different PBPs (molecular weight [MW], 40–120 kilodaltons [kD]), some of which are transpeptidation enzymes. Different receptors different affinities for a drug different effect. E.g. attachment of penicillin to one PBP abnormal elongation of the cell E.G. attachment to another PBP defect in the periphery of the cell wall cell lysis. PBPs are under chromosomal control mutations alteration of affinity for β-lactam drugs i.e. drug resistance  ANTIMICROBIALS THAT INHIBIT CELL WALL SYNTHESIS 1- Beta –Lactam antimicrobial agents: Penicillins Cephalosporins e.g. cephamycin Carbapenems e.g. imipenem & meropenem Monobactams e.g. aztreonam Beta-lactamase inhibitors Penicillins, cephalosporins, 2- Vancomycin (Teicoplanin) carbepenems and monobactams differ in terms of the structure fused to the beta-lactam ring.  Penicillines: Pencillin G – the oldest penicillin, remains active against Gram positive organisms, Gram negative cocci, some spirochetes including Treponema pallidium – the cause of syphilis. Should be administered via injections. Semi-synthetic penicillins: Penicillin V – the acid stable modification of Penicillin G - Should be administered orally. Some penicillins are inactivated by staphylococcal pencilillinases/ β-lactamases. Methicillin, nafcicillin, oxacillin – have narrow spectrum, but are resistant to β-lactamases produced by S. aureus. Amino-penicillins: ampicillin, amoxacillin – have broad action against Gram negative bacteria, as they penetrate through outer membrane of some Gram negative bacteria (Enterobacteria, but not Pseudomonas sp.). Ureidopenicillins/Acylaminopenicillins: piperacillin, ticarcillin, mezlocillin- have narrow activity than penicillin G, however combat Pseudomonas sp.  Lactamases The α-lactamases open the β-lactam ring of penicillins and cephalosporins and abolish their antimicrobial activity. β-Lactamases are characteristic for Gram-positive and Gram-negative bacteria. Some β-lactamases are plasmid mediated (eg, penicillinase of Staphylococcus aureus), others - chromosomally mediated (eg, many species of Gram-negative bacteria). Plasmid mediated lactamases may be transferred within different species.  Extended spectrum α-lactamases (ESBLs) There is one group of β-lactamases that is Currently throughout much of the world, the CTX-M occasionally found in certain species of enzymes have become more prevalent. These Gram-negative bacilli such as, Klebsiella enzymes are more active against cefotaxime and pneumoniae. These enzymes are termed ceftriaxone than ceftazidime and seem to be extended spectrum α-lactamases (ESBLs) inhibited more readily by tazobactam than the other because they confer upon the bacteria the β-lactamase inhibitors. Of most concern is the additional ability to hydrolyze the β-lactam emergence of K. pneumoniae carbapenemases rings of cefotaxime, ceftazidime, or aztreonam. (KPC), which are ESBL type enzymes that confer resistance to third- and fourth generation The classification of β-lactamases is complex, cephalosporins and carbapenems. This resistance based on the genetics, biochemical properties, mechanism is plasmid mediated and has spread and substrate affinity for a β-lactamase nosocomially among many hospitals throughout the inhibitor (clavulanic acid). United States and other countries  CEPHALOSPORINS: penicillinase resistant First Generation: Cephradine Ceohalexine (active against Gram positive organisms and some Enterobacteriacea) Second generation: Cefuroxime Cephamycin (cefoxitin, cefaclor) (improved coverage of Enterobacteriaceae) Third generation: Ceftriaxone, Cefotaxime, Ceftazidime, Ceftazidime (expanded spectrum, increasing potency against Gram negative bacteria, lower MIC) Fourth generation: Cefepim (extended spectrum against Enterobacteriaceae, Ps. aerugionsa, Neisseria, H.Influenzae) Fifth generation: Ceftarolin (active against Meticillin Resistant S. aureus – MRSA)  Carbapenems Carbapenems have the broadest spectrum of all β-lactam antibiotics. This is due to the combination of easy penetration of Gram-negative and Gram-positive bacterial cells and high level of resistance to β-lactamases.  Monobactrams Monobactram (e.g. Aztreonam) activity is strictly limited to Gram-negative aerobes: Pseudomonas, Enterobacteria, Serratia, Haemophilus. This antibiotic should be used among the patients severely allergic to penicillines.  β -Lactamase inhibitors β -Lactamase inhibitors - β -Lactams with little or no antibacterial activity Irreversibly bind to β -lactamase enzyme rendering them inactive Penicillin activity is enhanced in the presence of β - Lactamase inhibitors AUGMENTIN possesses the distinctive properties of a broad-spectrum antibiotic and a β-lactamase inhibitor. Clavulanic acid, Sulbactam, Tazobactam – these compounds are known as “suicide inhibitors” because they must first be hydrolized by β -Lactamase before becoming effective inactivators of this enzyme. They are highly effective against staphylococcal penicillinases and broad spectrum β -lactamases. eg. amoxicillin/clavulanic acid, ticarcillin /clavulanic acid and piperacillin /tazobactam.  Glycopeptide antimicrobials - are a type of antibiotic that inhibits bacterial cell wall formation by inhibiting peptidoglycan synthesis VANCOMYCIN and TEICOPLANIN – bind directly to amino acid side chains. Vancomycin inhibits cell wall synthesis and may be used to kill penicillinase-producing staphylococci. Used primarily against methicillin-resistant S. aureus (MRSA). Bacteriocidal action May be used orally  INHIBITION/ALTERATION OF CELL MEMBRANE FUNCTION Cytoplasmic membrane provides: selective permeability barrier carries out active transport functions controls the internal composition of the cell. Disruption of functional integrity of the cytoplasmic membrane leads: loss of macromolecules and ions from the cell cell damage cell death Structure of cytoplasmic membrane of bacteria and fungi ≠ animal cells Selective chemotherapy  INHIBITION/ALTERATION OF CELL MEMBRANE FUNCTION (2) Detergents, which contain lipophilic and hydrophilic groups, disrupt cytoplasmic membranes and kill the cell. Antibiotics polymyxins, consists of detergent-like cyclic peptides that selectively damage membranes containing phosphatidylethanolamine. Inhibitors of protein synthesis  ANTIBIOTICS THAT INHIBIT PROTIEN SYNTHESIS AMINOGLYCOSIDES - act on 30S ribosomal subunit TETRACYCLINE S – act on 30S ribosomal subunit CHLORAMPHENICOL – act on 50 Sub Unit of 23 r RNA MACROLIDES – act on 50 Sub Unit of 23 r RNA AMINOGLYCOSIDES - inhibit protein synthesis by binding, with high affinity, to the A-site on the 16S ribosomal RNA of the 30S ribosome Aminoglycoside must be transported into cell by oxidative metabolism/oxidative phosphorilation. Bactericidal action Acts only on Gram positive and Gram negative bacteria, including Pseudomonas, Streptococci & anaerobes are naturally resistant Slow development of resistance Examples: Gentamicin, Amikacin, Neomycin Given by injection Nephrotoxic & Ototoxic - dose related  TETRACYCLINES Tetracicline block tRNA attachment Broad spectrum Bacteriostatic activity Oral absorption Active against intracellular organisms eg. Mycoplasma, Rickettsia, Chlamydia,Brucella also for V. cholera & Nocardia Classes: Short acting: Tetracycline Long acting: Minocycline, Doxycycline ( CSF penetration). New tetracycline : Tigycycline (active against MRSA,MSSA, some Gram negative bacteria and anaerobes. Side effects : Teeth discoloration, GIT disturbance  CHLORAMPHENICOL Blocks peptidyl transferase Broad spectrum Bactericidal activity Affects bone marrow cells and cause a plastic anemia Used for severe infections not responding to treatment, also for Rickettsial diseases  MACROLIDES: Erythromycin & Clindamycin Bacteriostatic activity Active against Legionella, Camylobacter, Gram negative and positive infections for patients allergic to Penicillins and Cephalosporins. Clindamycin acts on anaerobes as well Cause GIT disturbance, Pseudomembraneous colitis. New Macrolides: Azithromycin & Clarithromycin  INHIBITORS OF NUCLEIC ACID SYNTHESIS Rifampicin Quinolones AND Fluoroquinolones Nalidixic acid: recommended for treatment of urinary infections Ciprofloxacin - Inhibits DNA gyrase and topoisomerase blocks DNA supercoiling Recommended for treatment urinary tract infections Metronidazole  RIFAMPICIN: Semi-synthetic, bactericidal, Inhibits RNA synthesis, acts on Gram positive bacteria and selected Gram negative bacteria. Reserved for Tuberculosis Resistance develops quickly Used in combination Causes discoloration of body fluids & hepatotoxicity  QUINOLONES : Synthetic, bactericidal, inhibit DNA Gyrase and /or topoisomerase, blocks DNA supercoiling Generations: First generation: nalidexic acid – locally acting Second generation: fluoroquinolones eg. ciprofloxacin, norfloxacin, ofloxacin,levofloxacin Third generation: sparfloxacin, gatifloxacin Fourth generation: moxifloxacin, trovafloxacin Side effects: on cartilage & heart  Metronidazole Nitroimidazole active on anaerobic bacteria, and parasite Caused DNA breakage Used for B.fragilis, Trichomonas vaginalis, amoebiasis and giardiasis.  Folate inhibitors Folic acid is derived from para-aminobenzoic acid (PABA), glutamate and a pteridine unit. It is essential coenzyme for the transport of one-carbon compounds into synthesis of purins, thymidine, and some aminoacids. Thus, folic acid is indirectly essential for synthesis of nucleic acids and proteins. Sulfonamids are structural analogues of PABA and compete with it in initial stage of foliate synthesis. ANTIMETABOLITES (foliate inhibitors): Trimethoprim-Sulfamethoxazole (TMP-SMX) Combination of TMP-SMX called : Bactrim / Septrin Block sequential steps in folic acid synthesis Used to treat :Nocardia, Chlamydia, Protozoa & P.cranii Urinary tract infections, Lower Respiratory Tract Infections, Otitis Media, Sinusitis, infectious diarrhea. Side effects: GIT, hepatitis, bone marrow depression, hypersensitivity  ANTI TUBERCULOUS AGENTS First line: ISONIAZIDE (INH) RIFAMPICIN ETHAMBUTOL PYRAZINAMIDE Second line: STREPTOMYCIN PASA CYCLOSERINE CAPREOMYCIN  ISONIAZIDE (INH) Bactericidal Affects mycobacteria at different sites of lung tissues Used for the treatment & prophylaxis of tuberculosis Cause peripheral neuritis (pyridoxine (vitamin B6) pyridoxine  Ethambutol BACTERICIDAL CONCENTRATED IN PHAGOLYSOSOME OF ALVEOLI OPTIC NEURITIS Pyrazinamide ACID ENVIRONMENT OF MACROPHAGES HEPATITIS & ARTHRALGIA ANTIBIOTIC RESISTANCE IN BACTERIA INDISCRIMINATE USE OF ANTIMICROBIALS SELECTIVE ADVANTAGE OF ANTIBIOTICS TYPES OF RESISTANCE: PRIMARY: Innate eg. Streptococcus & anaerobes are resistant to gentamicin.  ANTIBIOTIC RESISTANCE IN BACTERIA (Continue) Acquired resistance : 1- MUTATION: MTB RESISTANT TO SRTEPTOMYCIN 2- GENE TRANSFER : plasmid mediated or through transposons Cross resistance : Resistance to one group confer resistance to other drug of the same group. eg. Resistance to erythromycin and clindamycin Dissociate resistance: resistance to gentamicin does not confer resistance to tobramicin.  MECHANISMS OF RESISTANCE 1- Permeability changed 2- modification of site of action, eg. MUTATION 3- inactivation by enzymes. eg. Beta- Lactamase & aminoglycoside inactivating enzymes 4- passing blocked metabolic reaction eg. PABA folic acid, plasmid mediated  Resistance mechanisms (1) 1. Microorganisms produce enzymes that destroy the active drug. Examples: Staphylococci resistant to penicillin G produce a β-lactamase that destroys the drug. Other β-lactamases are produced by Gram-negative rods. Gram-negative bacteria resistant to aminoglycosides (by virtue of a plasmid) produce adenylating, phosphorylating, or acetylating enzymes that destroy the drug. 2. Microorganisms change their permeability to the drug. Examples: Tetracyclines accumulate in susceptible bacteria but not in resistant bacteria. Resistance to polymyxins is also associated with a change in permeability to the drugs. Streptococci have a natural permeability barrier to aminoglycosides. This can be partly overcome by the simultaneous presence of a cell wall-active drug such as a penicillin. Resistance to amikacin and to some other aminoglycosides may depend on a lack of permeability to the drugs caused by an outer membrane change that impairs active transport into the cell  Resistance mechanisms (2) 3. Microorganisms develop an altered structural target for the drug. Examples: Erythromycin-resistant organisms have an altered receptor on the 50S subunit of the ribosome, resulting from methylation of a 23S ribosomal RNA. Resistance to some penicillins and cephalosporins may be a function of the loss or alteration of PBPs. Penicillin resistance in Streptococcus pneumoniae and enterococci is attributable to altered PBPs. 4. Microorganisms develop an altered metabolic pathway that bypasses the reaction inhibited by the drug. Example: Some sulfonamide-resistant bacteria do not require extracellular PABA but, similar to mammalian cells, can use preformed folic acid.  Resistance mechanisms (3) 5. Microorganisms develop an altered enzyme that can still perform its metabolic function but is much less affected by the drug. Example: In trimethoprim-resistant bacteria, the dihydrofolic acid reductase is inhibited far less efficiently than in trimethoprim-susceptible bacteria. 6. Microorganisms can develop efflux pumps that transport the antibiotics out of the cell. Many Gram-positive and especially Gram-negative organisms have developed this mechanism for tetracyclines (common), macrolides, fluoroquinolones, and even β-lactam agents.  Origin of drug resistance (1) Nongenetic Origin of Drug Resistance Examples: Mycobacteria often survive in tissues for many years after infection yet are restrained by the host’s defenses and do not multiply. Such “persisting” organisms are resistant to treatment and cannot be eradicated by drugs. Yet if they start to multiply (eg, after suppression of cellular immunity in the patient), they are fully susceptible to the same drugs. Aminoglycosides such as gentamicin are not effective in treating Salmonella enteric fevers because the salmonellae are intracellular and the aminoglycosides do not enter the cell.  Origin of drug resistance (2) Genetic Origin of Drug Resistance A. Chromosomal Resistance This develops as a result of spontaneous mutation in a locus that controls susceptibility to a given antimicrobial drug. The presence of the antimicrobial drug serves as a selecting mechanism to suppress susceptible organisms and favor the growth of drug-resistant mutants. Spontaneous mutation occurs with a frequency of 10^12–10^7 and thus is an infrequent cause of the emergence of clinical drug resistance in a given patient. However, chromosomal mutants resistant to rifampin occur with high frequency (~10^7–10^5 ). Consequently, treatment of bacterial infections with rifampin as the sole drug often fails. B. Extrachromosomal Resistance Bacteria often contain extrachromosomal genetic elements called plasmids. Some plasmids carry genes for resistance to one—and often several—antimicrobial drugs. Plasmid genes for antimicrobial resistance often control the formation of enzymes capable of destroying the antimicrobial drugs. Thus, plasmids determine resistance to penicillins and cephalosporins by carrying genes for the formation of β lacatamases. Genetic material and plasmids can be transferred by transduction, transformation, and conjugation  PRINCIPLES OF ANTIMICROBIAL THERAPY INDICATION CHOICE OF DRUG ROUTE DOSAGE DURATION DISTRIBUTION EXCRETION TOXICITY COMBINATION PROPHYLAXIS: SHORT TERM: MENINGITIS : LONG TERM: TB, UTI, RHEUMATIC FEVER CRITERIA FOR IDEAL ANTIMICROBIAL: SELECTIVE TOXICITY NO HYPERSENSITIVITY PENETERATE TISSUES QUICKLY RESISTANCE NOT DEVELOP QUICKLY NO EFFECT ON NORMAL FLORA BROAD SPECTRUM DILUTION TESTS DIFFUSION TESTS Reading