Chapter 13.4-13.5 PDF

Chapter 13.4-13.5 How do we eliminate pathogens from the body? new cant use disinfectant - toxic to humans ◦ They lack specificity in their action and can affect animals as well as microbial cells. We use anitbiotics ◦ Made in nature ◦ Chemical compounds synthesized by one microbe t...

Chapter 13.4-13.5 How do we eliminate pathogens from the body? new cant use disinfectant - toxic to humans ◦ They lack specificity in their action and can affect animals as well as microbial cells. We use anitbiotics ◦ Made in nature ◦ Chemical compounds synthesized by one microbe to selectively kill other microbes Many are made biologically, other are synthesized chemically The generic terms for these are chemotherapeutic agents Antibiotics Exhibits Selective Toxicity selective toxicity is possible because key aspects of bacterial pathogen’s physiology are different from those of eukaryotes. ◦ They target peptidoglycan that is lack by eukaryotes ‣ Prevent peptidoglycan synthesis ◦ Ribosomes are different for eukaryotes and prokaryotes ‣ Tetracycline - binds to bacterial 30S ribosomal subunits ◦ They inhibit bacterial growth but they are invisible to host cells becauase they dont interact with them, ◦ Eukaryotic pathogens have similar components to their host (humans) which it makes it difficult for treatment 0 antibiotics toxicities are unrelated to their antimicrobial modes of actions ◦ Drugs that target 50S ribosomal subunits - can interfere with the development of blood cells in bone marrow ‣ Result to failure to produce red blood cells antibiotics (antibacterial) are selectively toxic to bacteria without toxicty to eukaryotic organisms ◦ Magic bullet concept ◦ They still have unintended Targets onlybacterian and not humans host side effects ‣ Age dependent A Cirpofloxacin - can cause defects in human bone growth plates and should nor be administered for pregananr and children ‣ Cell development In ‣ Metabolism dependent The liver metabolizes and detoxifies foreign chemicals, including antibioticsma and can become damaged by the toxic metabolic products. Kidneys which excretes drugs and metabolites, cna also damage some antibiotics. ‣ Allergy Drug does not directly impact mammalian physiology Extreme reaction towards penicillin - this could lead to treatment that is worst than the infection itself They have to use alternative medications how likelymicroorganism inhibited Students must be able to distinguish between drug susceptibility and drug sensitivity. ◦ A microbe is susceptible to the drug’s action, but a human cna develop an allergic sensitivity to the drug. bydrug Spectrum of Activity not single chemotherapeutic agent affects all microbes Ibody's reaction to drug Spectrum activity - the types of organisms that they can affect ◦ Antibacterial, antiprotozoal, antiviral ◦ Antibacterial (bacteria) - within this group it must still be narrowed The types of organisms the antibiotic affect: antifungal, antibacterial, antiprotozoal… Within a group → Antibacterial agent → Narrow or broad spectrum? ◦ Penicillin → Gram (+) = Narrow spectrum ◦ Ampicillin → Gram (+) + Gram (-) = Broad spectrum ‣ They have added amino group that helps the drug penetrate the gram negative outer membrane ◦ Isoniazid → Very narrow spectrum. ‣ Only useful for mycobacterium tuberculosis -> tuberculosis Bacteriostatic and Bactericidal antibiotics patients typically believe that all antibiotics kill their intended targets - this is completely wrong ◦ Many just prevents their growth and let the immune system kill the microbe Therefore they can also be classified if they can kill or not the microbe ◦ bactericidal antibiotics kill the pathogens o ◦ Bacteriostatic antibiotics prevents bacterial growth -> immune response able to get rid of the pathogen. o Minimum inhibitory concentration - how do we compare antibiotics? in vitro tests for antibiotic effectiveness Minimal inhibitory concentration (MIC) ◦ Defined as the lowest concentration of the drug that will prevent the growth of an organism. ◦ This will differ depending on the bacterial species ‣ The reason includes the ease with which the drug penetrates each cell and the affinity of the drug for its molecular target. ◦ How do we measure this? ‣ An antibiotic is serially diluted along a row of test tubes containing nutrient broth ‣ After dilution, the organism to be tested is inoculated at identical low population densities into each tubes, and each tubes are usually incubated overnight. Growth of the organism is seen as turbidity Tubes with the highest concentration of the drug were clear = no grwoth The tube containing the MIC is the tube with the lowest concentration of drug that shows no growth ◦ MIC does not tell whether the drug is Bacteriostatic or Bactericidal ◦ It is great for estimating a single drugs effectiveness against a bacterial pathogen isolated from a patients ◦ They are not particle for more than 20 drugs need to be screened. ‣ They take time ‣ The time can be reduced via strip test = avoids the need of dilutions It’s contains a gradient of antibiotic, is placed over a fresh lawn of bacteria spread on an agar plate. While the bacteria are trying to grow, the drug diffuses out of teh strip and into the medium Drug diffusing from the more concentrated areas of the strip than will achieve and maintain MIC farther away from the strip than will drug diffusing from less concentrated areas of the strip This will result in an elliptical zone of inhibiton - antibiotic has stopped bacterial growth ◦ The MIC is the point at which the elliptical zone intersects with the strip Disk Diffusion Assay Kirby-Bauer assay ‣ 100 more bacterial isolates in one day so individual MIC is not practical ‣ Uses a series of round filter paper disks impregnated with different antibiotics ‣ A dispenser delivers up to 12 disks simultaneously to teh surface of an agar plaste covered by a bacterial lawn. ‣ Standardized medium is mueller-Hinton agar ‣ Each disk is marked to indicate the drug used ‣ During incubation, the drugs diffuse away from the disks into surrounding agar and inhibit growth of the lawn to different distances ◦ Zone of inhibition is measures around filter paper disks impregnated with 12 antibodies ‣ The sone of inhibition vary in width depending on: the antibiotic used Concentration of the drug in the disk larger zone of inhibitionBetter drug The susceptibility of the organism to the drug ‣ Teh diameter of the zone around an antibiotic disk correlates to the MIC of that antibiotic against the organism tested. ◦ Following parameters have been standardized ‣ Size of agar plate ‣ Depth of the medium ‣ Composition of the medium ‣ Number of organisms psread on the agar plate ‣ Size of the disks ‣ Concentrations of antibiotics in the disks ‣ Incubation temperature ◦ Changing one of these parameters will alter the zone of inhibition and give incorrect assessment ◦ Will not tell you whether the drug is bactericidal, because there is no way of trekking whether they are viable or dead Minimal bactericidal concentration (MBC) Requires further plating to determine if any cells survived. Dilution test is performed and the antibiotic will be removed Any cells remaining in the growth tubes are first forced to the bottom of the tube by centrifugations Antibiotic medium is removed, and cells at the bottom of the tube are suspended in fresh medium If the cells grow they are not killed The lowest concentration of drug that does not show growth represents the MBC almost any antibiotic can be bactericidal (MBC) at high concentration (MIC). However, a drug is deemed to have a bactericidal mode of action if its MBC is no more than four times its MIC. Determining whether an antibiotic is clinically useful Results of an MIC determination or a disk diffusion test must be evaluated to determine whether a drug is clinically useful -> high concentration of frug may stop growth in labs but not work in patients -> for patients = the drugs concentration in tissue must remain higher than the MIC at all times Half life = how long drug stay in tissue, depends on how quickly the antibiotics is removed from the body via the secretion by the kidneys or destruction on the liver. clinical considerations ◦ Drugs concentration in tissue (based on its half-life) > MIC at all times during the treatment ◦ Higher doses than MIC or multiple doses to keep the levels > than lab MIC ◦ Usefulness also depends on whether side effects appear at concentrations needed to affect the pathogen. ‣ As long as the concentration of the drug in the tissue or blood remains higher than MIC (without side effects appearing) -> drug will be effective ‣ The doctor can give higher does -> more side effects ‣ Or by doing a second dose before 1st dose goes down the MIC That’s why some patients are asked to take medication two times or once a day. The chemotherapeutic index When treating an infection, which antibiotic to prescribe ◦ Therapeutic does: Minimum dose per kg of body weight that stops growth of pathogens ◦ Toxic dose: maximum dose tolerated by the patient ◦ Ratio of the toxic dose to therapeutic does is called the chemotherapeutic index ‣ The higher the chemotherapeutic index, the safer the drug ‣ Ideally, the therapeutic dose us much lower than the toxic dose Synergism and antagonism ◦ Combinations of antibiotics can be either synergistic or antagonistic ◦ Synergistic - drugs have greater effectiveness when used together (combined effects is greater than additive effect) ‣ Example: aminoglycoside (inhibits protein synthesis) + vancomycin ( a cell wall synthesis inhibitor) Used for serious enterococcus infections of the blood (sepsis) and of the heart valve (endocarditis) Neither drug is very effective, but when combined weakens the enterococcus cell wall, thereby facilitating the entry of the amino glycoside into the bacterial cell. ◦ Antagonistic drugs - interfere with each other and decrease effectiveness ‣ Occurs when Bacteriostatic agent is combined with bactericidal agent ‣ Example: penicillin (bactericidal) + macrolides Penicillin - kills growing cells that are actively making peptidoglycan but has little effect on non growing cells, Macrolides - Bacteriostatic and merely prevent growth. One relys on stopping growth while the other relys on the growth of bacteria. 13.5 Antimicrobial Mechanisms of Action There are three ways of classifying antibacterial agents: 1. Bactericidal or Bacteriostatic 2. target site (5) 3. Chemical structure no targetsite in Eukaryotes How do antibiotics work? Antibiotics that target the cell wall NAG and NAM are made by the cell and linked together into long peptidoglycan chains assembled at the cell wall. NAG and NAM (short disaccharides ) with short peptide side chains are made in the cytoplasm and then shuttled across the membrane by a lipid carrier molecule (step1-4) The dissacharide units are assembled outside the cytoplasmic membrane to form long strands of peptidoglycan (step 5) Adjacent strands are then snapped together by cross-linking the short peptide side chains that spring from individual NAM molecules (step 6) Cross linking produces a rigid, cage like macro molecular structure essential for maintaining cell shape Bacteria small sub 30 40 largesubunit 50 60 70 80 Antibiotics that target the cell wall beta-lactam antibiotics ◦ Enzymes that attach the disaccharide units to preexisting peptidoglycan and produce the peptide cross-links are collectively called penicillin-binding proteins (PBPs) ‣ Because penicillin and related antibiotics binds to them. ‣ Penicillin is naturally produced antibiotic that contains a critical beta lactam ring structure that chemically resembles a piece of the peptidoglycan peptide side chain ◦ Because penicillin looks like peptidoglycan side stain, the drug will bind to enzymes that mediate peptide cross linking and inhibit their activity. ‣ Lack of peptide cross-bridging is not good for bacteria that is trying to grow larger before they divide ◦ Intact cell wall acts a a rigid “exoskeleton” that restrains the hydrostatic pressure form within the cell. ‣ Penicillins action compromises the cell wall Growing cell will eventually die/burst from internal pressure Penicillin is bactericidal drug. ◦ Narrow spectrum because it kills gram positive organisms better than gram negative bacteria. ◦ Gram negative bacteria’s outer membrane blocks penicillin’s entry. Microbial Resistance to cell wall-inhibiting antibiotics Antibiotics - magic bullets Microbes constantly evolve to become resistant to new antibiotics bacterial resistance was discovered and was recognized to have caused occasional treatment failures they develop resistance to penicillin in two basic ways ◦ Inheriting a gene encoding (beta-lactamase enzymes) ‣ Which cleave the critical beta-lactam ring structure of this class of antibiotics ‣ This enzyme is transported out of the cell and into surrounding medium (gram positive) or the periplasm (gram negative) Where they will destroy penicillin before the drug even reaches the cell ‣ They are still susceptible to certainl modified penicillins and cephalosporins that are no longer susceptible to the enzyme ◦ Second form of penicillin resistance is when a key penicillin-binding protein is altered by mutation. ‣ Altered protein can still synthesize cell wall peptidoglycan but will no longer bind antibiotic ‣ Most methicillin resistant bacterua use this strategy = drugs that can affect MRSA is vancomycin Drugs that target the Bacterial Membrane poking holes in a bacterial cytoplasmic membrane is another effective ways to kill bacteria Gramicidin uses this strategy ion channel ◦ They are produced by brevibacillus brevis ◦ Cyclic peptide composed of 15 alternating d and l amino acids ◦ It inserts into the membrane as a dimmers forming a cation channel that enables positive ions to freely cross the membrane and dustruot membrane polarity Polymyxins ◦ Polymyxin B Detergent ◦ Has positively charged polypeptide ring that binds to the outer and inner membranes of bacteria, both of which are negatively charged ◦ They dissolved the inner membrane by distrusting phospholipid interactions, like a detergent ◦ Used topically to treat or prevent infection ◦ Never ingested because it damaged human cell membranes ◦ Used in some bandages for burn patients that are susceptible to gram negative bacteria (P.aeroginosua) Drugs that affect DNA synthesis and integrity Bacteria generally make and maintain their DNA by using enzymes that closely resemble those of mammals. When an identity of an organism is not yet known clinicians gives patients broad spectrum antimicrobial to cover the usuals pathogens that cuases the disease. this is called empirical treatment sometimes there are bacteria that are not covered by the drug Metronidazole anaerobic also know as Flagyl Prodrug - harmless unless activated by reduction ◦ Activated after being metabolized by microbial protein cofactors (ferredoxin) found in anaerobic or micro aero Philip bacteria such as Bacteriodes and fusobacterium ◦ Aerobic microbes are resistant because they lack the electron transport protein needed to reduce metronidazole, ◦ They are activated in anaerobes - they nick the DNA in random, killing the cell. ◦ Humans are unharmed by the drugs = we lack anaerobiosis associated cofactors Quinolones dnagyrase DNA gyrase reverses the positive supercoils that occurs by the unwinding of DNA. -> cell will not die because replication can continue quinolones inhibits DNA gyrase -> positive supercoils accumulate and the pathogen cannot multiply. The DNA gyrase of bacteria is different form mammalian so it doesn’t affect us and our DNA replication Various chemical modification have been done to inctreade antimicrobial spectrum and bloodstream leaf life. ◦ Ciprofloxacin and levofloxacin are quinolones commonly used to treat a variaty of infections. RNA synthesis inhibitors Rifampin Orange Bacterial pathogens must transcribe and translate their genes to grow. we can stop either of those processes and the bacteria will not grow and host immune system can kill them Many antibiotics can stop protein synthesis but only few can stop transcription. ◦ Rifampin (rifamycin family of antibiotics) ‣ selectively binds to bacterial RNA polymerase and prevents newly made mRNA from exiting the enzyme ‣ Teh polymerase stops in its tracks ‣ It is used to treat tuberculosis or meningococcal meningitis ‣ It’s orange-> pee turns orange and breast milks orange as well Protein Synthesis Inhibitors the molecular complexity of translation provides numerous sites that can be attacked by antibiotics Eukaryotes and prokaryotes are different (ribosomes) so we can do selective Can be classified into several groups according to their structure and mechanism of action Most of them are Bacteriostatic Generally works by binding to and interfering with the function of bacterial rRNA, which differs form. Eukaryotic rRNA Look at big picture: If we say anti fungal we do fungi have that humans don’t? Chitin ◦ So they will target chitin ◦ Idk how we gonna know this but I think chapter 11? Drugs that affect intermediary metabolism Sulfonamides Folicacid nucleic acids are known as anti metabolites because they interfere with the syntheisis of metabolic intermediates. Inhibit the synthesis of nucleic acids They prevent this synthesis of folic acids, an important cofactor in the synthesis of nucleic acid precursor Why are they selective to bacteria? ◦ Because mammals don’t synthesize folic acid ◦ We rely on vegetables as source of folic acid 000 ◦ Bacteria make folic acid with the use of PABA, glutamic acid, and pteridine 0 ◦ Sulfanilamidem analog of PABA competes with one of the enzyme in folic acid pathway in bacteria and inhibits the production of folic acid.