An antimicrobial is an agent that kills microorganisms or stops their growth Antimicrobial medicines can be grouped according 10 the microorganisms they act primarily against, For example. antibiotics are used against bacteria and antifungals are used against fungi. They can also be classified according to their function. Agents that kill microbes are called microbicidal, while those that merely inhibit their growth are called biostatic. The ideal antimicrobial agent should he nontoxic to the host (selective toxicity), non-allergenic, soluble in body Muids, able to be maintained at therapeutic levels. have a low probability of eliciting resistance, long shelf life. and low cost. ‘The main classes of antimicrobial agents are: Disinfectants (“nonselective antimicrobials" such as bleach), which kill a wide range of ing surfaces to prevent the spread of illness. microbes on non Antiseptics (which are applied to living tissue and help reduce infection during surgery) Antibiotics: substance produced by a microorganism, [or a ilar product produced wholly (synthetic) or partiglly (semi-synthetic) by chemical synthesis] that is capable. in low concentrations, of inhibiting the growth of or killing other microorganisms, Most are produced by either fungi (¢.g.. penicillin, cephalosporins), Bacillus species (e. polymyxin, bacitracin), or Streptomyces species (streptomycin, tetracycline. erythromycin, kanamycin, neomycin. nystatin) Antibact agents can be further subdivided into bactericidal agents. which kill bacteria, and bacteriostatic agents, which slow down or stall bacterial growth. Preservatives Immunological agents Antibiotic classes and their mechanisms of action: There are several major ways antibioties kill microbes or inhibit their growth Antibiotics generally target basic bacterial structures/functions necessary for lie andlor replication. ‘ Different mechanisms of action include: 1) Inhibition of cell wall synthesis 2) Inhibition of protein synthesis 4) Inhibi essential metabolites. jon of folate metabolism--necessary for production of DNA and other BLP iption of the cell membrane and production of tree radicals that damage DNA.‘An ‘antibiotic elass? refers to a group of antibiotics with a very similar chemical jotic class structure. Because of their similar chemical structure members of an ant have the same basic mechanism of action. Generally, within a class, there is the same core nucleus critical to function, while differing side chains modify the drug's toxicity. icillins and quinolones are different spectrum, pharmacokinetics, etc. For example, pe antibiotic classes. Ampicilfin and piperacillin are two different kinds of penicillins (both inhibit cell wall synthesis but have different antibiotic spectrums) while levofloxacin inds of quinolones (both alter nucleic acid metabolism, and ciprofloxacin are different tut are dosed once daily and twice daly, respectively). Below. the different antibioticclasses are grouped = by -— their = mechanism = of -—_ action: Cytoplasm + Nitroimidazoles (e.g. Metronidazole) produces Chromosome oxygen free radicals which —_* Diaminopyramidines (e.g. Trimethoprim) damage proteins and DNA interferes with folic acid synthesis + Lipopeptides (e.g. + Quinolones (e.9. Ciprofloxacin, Daptomycin) depolarises Levofloxacin) inhibits ONA Coiling cell membranes Inside cell _» Rifampicin inhibits RNA polymerase + Nitrofurantoin actual mechanism is unknown but causes direct damage to Cell Membrane + Polymyxin (e.g Colistin) binds to phospholipids disrupting the cell membrane Ribosome * Macrolides and Lincosamides (e.g, Erythromycin, Clarithromycin, Azithromycin, Clindamycin) prevents protein elongation and inhibits ribosome formation Cell Wail + Aminoglycosides (e.g. Gentamicin, + Beta-Lactams (e.9. Amikacin, Tobramycin) interfere with Penicillins, Cephalosporins, translation and protein formation * Carbapenems) inhibit cell + Tetracyclines and Glycyicyclines (e.g. wall formation Doxycycline, Tigecycline) prevents + Glycopeptides protein synthesis. (Vancomycin, Teicoplanin) * Oxazolidinones (e.g. Linezolid) prevents Prevent peptidoglycan ribosome formation cross-linkage + Fusidic Acid blocks elongation factor G, Preventing protein formation + Chloramphenicol inhibits Protein synthesis + Nitrofurantoin actual mechanism is unknown but interferes with translation Inhibit cell wall synthesis: + Penicillins* Cephalosporins + Monobactams (aztreonam) + Carbapenams + Glycopeptides (vancomycin) Inhibit Protein synthesis: + Aminoglycosides * Tetracyclines + Glycyleycline (tigecycline) 5 + Macrolides 4 + Lincosamides (clindamycin) + Streptogramins (quintapristin/dalfopristin) * Oxazolidinones (linezolid) * Phenicols (chloramphenicol) Alter nucleie acid metabolis + Rifamycins (rifampin) * Quinolones Inhibit folate metabolism: + Trimethoprim + Sulfonamides Antibiotics that inhibit cell wall synthesis: Beta-lactam antibiotics (penicillins, cephalosporins, monobactams, carbepenams): beta-lactam antibiotics refer to several classes of antibiotics (listed above) that all share the same beta-lactam ring consisting of 3 carbon atoms and | nitrogen atom. The beta-lactam ring is the square at the cemer of the penicillin nucleus Baie TX o o oF 7OH The different classes of beta-lactams differ from each other by structures (such as side chains/rings) apart from the beta-lactam ring. Beta-lactam antibiotics all inhibit cell wall synthesis by blocking the action of transpeptidases (aka penicillin binding proteins, PBPs) which are membrane bound and produce peptidoglycan, the major cell wallcomponent. These antibiotics cause dividing bacteria to lyse and die as shown in the diagram below. ° Drug molecules © 5 . renne) Sf oe Gram-negative Gram-positive w. : 6 ‘acterium ace 86 eo . Scotian 8 NS aa + ten a ser on z= \ Biscenase Peinasmicrpece {© . a “N Pestapycas — = = Bacal moreno — o Meniwesrg rote MN SP | Lysis ecu gms EB Vancomycin (a glycopeptide antibiotic) Vancomy: also 's cell wall synthesis by interfering with the groduction of peptidoglycan (but it is not a beta-lactam ic) It does this by binding to the D-Ala-D-Ala Vancomycin-susceptible staphylococci “e Inhibit n of cell-wall synthesis, Emo) Z a OAe 30) > ‘Tripeptide containing intermediates ‘cell-wall synthesis terminals of peptidoglycan precursors on the outer surface of the cell membrane. As a result, the precursors cannot incorporate into the peptidoglycan matrix. This process causes dividing cells to lyse and die. With a rare exception, vancomycin is only active against Gram positive bacteria, Bacitracin blocks the secretion of NAG and NAM from cytoplasm. 5Protein Synthesis Inhibitors ‘The protein synthesis inhibitors interfere with different aspects of translation, Some classes act on the 30S ribosome while others act on the 50S ribosome as demonstrated below. Most protein synthesis inhibitors cause a reversible inhibition of protein synthesis and many are bacteriostatic (prevent bacterial growth but don’t kill them). An exception are aminoglycosides which bind irreversibly to the 30S ribosome and are generally bactericidal (cause cell death). Macrolides (erythromycin, azithromycin, clarithromycin, _dirithromycin, troleandomycin, etc.) bind reversibly to the 50S subunit. They can inhibit elongation of the protein by the peptidyltransferase, the enzyme that forms peptide bonds between the amino acids. . Aminoglycosides (tetracycline) blocks bacterial translation by binding irreversibly to the 30S subunit and distortins in such a way that the anticodons of the charged tRNAS cannot align properly with the codons of the mRNA. Growing polypeptide an O Binds to 508 portion and Inhibits formation of {2) Three-dimensional detall of the Protein synthesis site showing the 4508 and 505 subunit portions of the ‘708 prokarytic ribosome Messenger BNA Chimrsorsdagrorm Rc os ena a meg tanta saeco {£1 Bioram inciating the dierent points ot which chloramphenicol cyclins, and streptomycin exert thelr activi Antibiotics that affect bacterial nucleic acid metabolism: Rifamycins Rifamycins include several antibiotics including rifampin (aka rifampicin). They inhibit MRNA synthesis (transcription) by binding to the bacterial DNA dependent RNA Polymerase, Resistance may occur as a result ofa single-step mutation wi that encodes the f- subupit of the RNA polymerase. in the gene Because resistance can easilyOccur, rifampin is almost always used in combination with other antibiotics. One exception is for [Link] prophylaxis. Quinolones Quinolones inhibit DNA synthesis and cause cell death. They do this by inhibiting the ‘opoisomerases responsible for supercoiling DNA (DNA gyrase) or relaxing the supercoiled DNA (topoisomerase IV). Ciprofloxacin is a quinolone that became a household name during the anthrax scare. Damaging the plasma membrane (Nystatin, amphotericin B. miconazole and ketoconazole) 8. Polyenes drugs incroporate into cell membranes causing membrane integrity damage (Porin formation) resulting cellular lysis. Amphoteri in B side effects in humans binds to cholesterol which is ar to fungal ergosterol b. Azoles (fluconazole) ‘ind Allyamines (turbinafine) antifungal drugs Inibits the synthesis of ergosterol which is necessary for fungal membrane structure (much like cholesterol is necessary for human membrane structure) Inhibitors of folate metabolism Both trimethoprim and sulfonamides inhibit folate metabolism. When folate metabolism is inhibited, formation of DNA precursors (e.g. purines) is reduced and ultimately, DNA synthesis is inhibited. Sulfonamides and trimethoprim act in different steps in folate metabolism and are often used together. Sulfonamides inhibit tetrahydropteroie acid synthetase which inhibits PABA“Wdihydrofolic acid. Trimethoprim inhibits the conversion of dihydrofolic acid to tetrahydrofolic acid by inhi reductase, " ing. dihydrofolate Determination of Antibiotic Efficacy Susceptibility can be determined in several ways, including broth di disk diffusion ion, E-test, and 4m broth dilution, tubes with liquid media antl‘inereasing concentrations of antibiotic are innoculated with the organism. After 24 hours, the tubes are observed. The first tube (the tube with the lowest concentration of antibiotic) that has no visible growth represents the MIC, In the figure below, the MIC=8 jig/ml, If the organism is Klebsiella ‘pneumoniae andthe iotic i i i antibiotic is cefepime (a cephalosporin), the organism would be considered susceptible to cefepime (the NCCLS guideline says that and MIC <8 jg/ml is susceptible.) 64 2 0 peyml Anubiote 64 32 16 test (Epsilometer test): The e-test can also determinethe MIC. The e-stiP contains amtibiotics in a gradient from a low to high concentration. The organism 1s inoculated onto a plate of solid media, the e-strip is placed on the media, and the plate is incubated for 24 hours. Antibiotie-an the e-strip will diffuse into the solid media (also on & gradient). The plate is observed and there will be an elliptical-shaped inhibition of growth. The lowest point on the e-strip that corresponds fo an absence of growth equals the MIC. In this example, the MIC appears to be 4 mg/L. Disk Diffusion: This classic method of determining antibiotic susceptibility does not yield an MIC value—it just tells you if the organism is susceptible, intermediate. oF 8resistant. In this method the organism is inoculated onto a plate of solid media. One or more antibiotic disks are placed onto the plate and the antibiotic will diffuse into the ‘media. The plates are incubated for 24 hours and then observed. For each antibiotic disk, the zone of inhibition around it is measured. Depending on the diameter of the Zone of inhibition, the organism will be classified as sensitive, intermediate, or resistant (NCCLS guidelines are used), Obviously. if there is no zone of inhibition, the organism is resistant to the antibiotic. As stated above, in order for a pathogen to be sensitive to an antibiotic, the antibitoric concentration at the site of infection must be at or preferably above the MIC. Some anti jotics are concentration dependent agents-the higher the concentration of antibiotic above the MIC, the more killing occurs. Other anti jotics are time-dependent agents, For these agents, little is gained as the concentration increases above the MIC. For these agents, it is the duration of time that the concentration of antibiotic remains above the MIC that influences the extent of bacterial killing, Resistance to Antimicrobials ‘A. Microbial susceptibility and resistance Resistance to an antibiotic means that a microorganism, that was formerly susceptible to the action of that antibiotic is mo longer affected by it. Antibiotic resistance can sometimes be transferred among bacteria on extra chromosomal DNA molecules known as plasmids. Resistance may be due to changes in the sensitivity of affected enzymes. Plasmids changes in the selective permeability of cell walls and membranes, increased production of a competitive substrate, or enzymatic alteration of the drug itself. Unnecessary exposure to antibioties has brought about a significant increase in antibiotic-resistant microbes, A variety of mutations can lead to antibiotic resistance. Resistance genes are often on plasmids or transposons that can be transferred between bacteria Reasons for antibiotic registance 1. Misuse of antibioties selects for resistance mutants. Misuse includes: a. Using outdated, weakened antibiotics b. Using anti jotics for the common cold and other inappropriate conditions ©. Use of antbi ics in animal feed . 4, Failure to complete the prescribed regiment ¢, Using someone else's leftover prescriptionMechanisms of antibiotic resistance 1. Enzymatic destruction of drug B-lactam ring ° ' Z8\_/CHy I 7S\_/CHs R-C-NH-CH7-CH R-G-NH-CH-CH CQ, ea 1 SCH tt 1 SCH {8—N— HCOOH Penicillinase o-c NCH COOH 0 on dt “i Bus Penicillin uw Penicilloic acid — 2. Inducing changes in the cell membrane prevention of penetration of drug into cell 3. Alteration of drug's target site 4, Rapid ejection of the drug; pumping the drug out of the cell x ” 5, Altering the cell's metabolic pathway 4. Blocking wo Antibiotic entry + 2. inactivating” enzymes enzymes _ target molecule i= 3. Alteration 4, Efflux of of target antibiotic molecule rf Sex fliProduction of a Protein (A) that Interferes Before the Action of Antibloue fetams (@.g. ESBLs. AmpC) 2 Macrolides and Lincosamiges + Aminogtycosides (e.9. aminoglycoside modifying enzymes) + Nitroimidazoles (2.9. Catal 2 Tetracyelines and se) Chioramphenicel (e.9. Acety! tennaferases) inhibitors STetracyclines (proteins knock Tetracyclines off ribosome) = Nitrofurantoln (inhibition of activating enzyme) Reduced Entry of the Antibiotic Into the Celt Reduced Cell Membrane Permeability BBttactectems, Diaminopyramidines ond Chierampnenicel Gram-negative Cel! Membrane Blocks Enery, ‘ Siretrotides, Lincdkamises, Giycopeptides and Lipopeptides Loss of Porta Guinolones, Retarding Drug Resistance 1. Using sufficiently high concentrations of a drug for Mutation or Change In Active Site Prevents Binding of the Antibiotic At the Ribosome SMacrolides, Lincosarmid Aminogiveosides, Oraromdinones, FusidK ACO 2nd At the Cell wat Mpatatnctams and Giycopeptides At the Chromosome Abliminapyrameaines. Quinolones excessive Target Site SSiannnooyramiaines and Giycopeptias No Target Site Nechann in Gram-positive Dacter'e Efflux Pumps Remove the Antibiotic from the Bacteria before its Action Sera lactams, Oaminopyramiaines. Placrondes, Lincosamiges, a sufficient time to kill all sensitive cells and inhibit others long enough for the body's defenses to destroy them 2, Using antimicrobials in combination, promoting synergism. the interplay between drugs that results in efficacy that exceeds the efficacy of either drug alone 3, Limiting the use of antimicrobials to necessary cases, preseribing and uncontrolled use 4, Developing new variatigns of existing drugs avoiding indiscriminateAntiseptics: microbicidal agents harmless enough to be applied to the skin and mucous membrane; should not be taken internasify. Examples: mercurials, silver nitrate, iodine solution, alcohols, detergents. Disinfectants: Agents that Kill microorganisms, but not necessarily their spores,not safe for application to living tissues; they are used on inanimate objects such as tables, floors, utensils, etc. Examples: chlorine, hypochlorites, chlorine’ compounds, lye, copper sulfate, quaternary ammonium compotinds. 2they are safe for «ig of whether oe in the basis 2 tion of the compound, are distinguished ons the coe for drinking, but «disinfectants and antiseptics fety depends o Nats ia membranes. on, ay as added (0 Me drink. plication 0 muse hypochlorite (CH ant, is hardly For ear Eo hypochlorite) or pxeellentdisinte! vinta "chlorox" (5% hypochlorite), cred in Tat i sn ines an nies we Su Common antiseptics ; Table 2 Common antiseptics and disinfectants Uses Chemical Action ‘og and . ‘ Denatures proteins 2m! ‘Antiseptic used on skin Bthanol (50-70%) solubilizes lipids Denatures proteins M4 jp ntiseptic used on skin Isopropanot (50-70%) solubilizes lipids Reacts with NHz, SH and p;infectant, kills endospores Formaldehyde (8%) coor gous Disinfectant, p ee af Iodine (2% 12 in 70% ractivates proteins | Antiseptic used on skin aleobol Forms hypochlorous acid py sinfect drinking water; general Chlorine (Ch) gas (HCIO), 2 ~— SORE disinfectant oxidizing agent General antiseptic and used in Silver nitrate (AgNO) Precipitates proteins the eyes of newboms Inactivates» proteins by Disinfectant, although Mercurie chloride reacting with sulfide occasioffally used as an groups antiseptic on skin Detergents’ (eg. quatemary 5. Skin antiseptic d ; ie ptics an ‘ammonium compounds) isrupts cell membranes 4; fectants Phenolic compounds (e.g. earboloic ‘Antisepti rs a sptics at - low acid, lysol, bexylresorcino{, Denture. proteins » and ions; disi pexachlérophede) disrupt cell membranes cocaine, eee # : Disinfectant used to sterilize Ethylene oxide gas Alkylating agent heat-sensifive objects such as rubber and plastics Preservatives: static agents used to inhibi i is i u it the growth of microorganisms, most often in foods. IF re at oe be nontoxic. Examples; calcium propionate, sodium benzoate, formaldehyde, ie, lioxide. Table 3 is a list of common preservative and their uses. Table 3. Common food preservatives and their uses 22a Preservative Effective a Cone Uses Propionic acid and enteation propionates 032% Kaa . ae ntifungal agent in breads, cake, Swiss cheeses bic acid and sorbates 0.2% i jelli Ee Antifungal agent in cheeses, jellies, syrups, cakes a o1% Ansitungal ‘agent in margarine, cider, relishes, Denno soft drinks Sodium dis ‘pete liacetate 0.32% Antifungal agent in breads TG ed ae Antimicrobial agent in cheeses, buttermilk, yogurt and pickled foods ‘Antimicrobial agent in dried fruits, grapes, molasses ‘Antibacterial agent in cured meats, fish Prevents microbial spoilage of meats, fish, etc. Sugar pare Prevents microbial spoilage of preserves, jams, syrups, jellies, etc. Prevents microbial spoilage of meats, fish, ete Sulfur dioxide, sulfites 200-300 ppm Sodium nitrite 200 ppm Sodium chloride unknown ‘Wood smoke unknown Pharmaceutical products of microbial origin: Among these are dextrans, amino acids, enzymes, organic acid“and vitamins. They are produced by microbes and are use as pharmaceutical products. Dextrans Dextrans are polysaccharide: genusLeuconostoc (e.g: L. dextranicus and [Link]' to the attention of industrial microbiol gummy masses of dextran clogged pit inks of high but variable molecular weight carried out in large «produced by lactic acid bacteria, in particular members of the ides) following growth on sucrose. These Jlogists because of their polymers of glucose first came ipetines. Dextran is nuisance in sugar refineries where large ‘essentially a glucose polymer consisting of (1A6)-a-li (15000-20000000; Fig. 25.1). Growth of the dextran producer strain is fermenters in media with a low nitrogen but high carbohydrate content. ‘The average ain used. This is important because ra which will depend on their use. lecular weight. ‘The first molecular weight of the dextrans produced will vary with the st must have defined molecular weights, dextrans for clinical use ed for obtaining dextrans of a suitable m ‘Two main methods are employ 23