Antimicrobial Chemotherapy to Control Microorganisms بسم الله الرحمن الرحيم Antimicrobial Chemotherapy to Control Microorganisms 2013-2014 Diagnostic Medical Microbiology-Laboratory Manual

Antimicrobial Control Agents Usually, microbial controls are used to avoid contamination of pure cultures, prevent infection, or treat existing diseases. A microbiocidal effect kills microorganisms. A microbiostatic effect prevents the reproduction of microorganisms. Antiseptics are chemicals used on living tissues to inhibit the growth of microorganisms. Disinfectants are chemicals used on nonliving surfaces to inhibit the growth of microorganisms. Chemotherapeutic agents are chemicals used to destroy or inhibit the growth of microorganisms in living tissues.22

Antimicrobial Chemotherapeutic Agents Antimicrobial Chemotherapy : is the use of chemicals to inhibit or kill microorganisms in the host. Selective Toxicity: This means that the agent used must inhibit or kill the microorganism without seriously harming the host. Based on their origin, there are 2 general classes of antimicrobial chemotherapeutic agents: Antibiotics: substances produced as metabolic products of one microorganism which inhibit or kill other microorganisms. Antimicrobial Chemotherapeutic Chemicals: chemicals synthesized in the laboratory which can be used therapeutically on microorganisms.

Antimicrobial agents are : Cidal in action: they kill microorganisms. Static in action : they inhibit microbial growth long enough for the body's own defenses to remove the organisms. Antimicrobial agents also vary in their spectrum: Broad spectrum : Drugs which are effective against a variety of both gram-positive and gram-negative bacteria. Narrow spectrum : Drugs which are effective against just gram-positive bacteria, just gram negative bacteria, or only a few species are termed. If a choice is available, a narrow spectrum is preferable since it will cause less destruction to the body's normal flora. In fact, indiscriminate use of broad spectrum antibiotics can lead to super-infection by opportunistic microorganisms.

Several tests may be used to tell a physician which antimicrobial agent is most likely to combat a specific pathogen: 1. Tube dilution tests: A series of culture tubes are prepared, each containing a liquid medium and a different concentration of a chemotherapeutic agent. The tubes are then inoculated with the test organism and incubated for 18-24 hours at 37 oC . After incubation, the tubes are examined for turbidity (growth). Minimum Inhibitory Concentration (MIC): Is the lowest concentration of chemotherapeutic agent capable of preventing growth of the test organism. Minimum Bactericidal Concentration (MBC): Is the lowest concentration of the chemotherapeutic agent that allows less than 0.1% of the inoculum to survive , also called “ Minimum Lethal Concentration (MLC)”

2. The agar diffusion test (Bauer-Kirby test): In this test, the in vitro response of bacteria to a standardized antibiotic-containing disc has been correlated with the clinical response of patients given that drug. In the development of this method, a single high-potency disc of each chosen chemotherapeutic agent was used. Zones of growth inhibition surrounding each type of disc were correlated with the minimum inhibitory concentrations of each antimicrobial agent (as determined by the tube dilution test). The MIC for each agent was then compared to the usually-attained blood level in the patient with adequate dosage. Categories of "Resistant,“ "Intermediate," and "Sensitive" were then established. 3. Epsilometer test: It’s a new technique for direct detection of MIC, a graduated increasing concentration of the antibiotic is fixed along a rectangular plastic test strip which is applied to the surface of an inoculated agar plate, after over night incubation a tear drop shaped inhibition zone is seen. The zone edge intersect the graded test strip at the MIC of the antimicrobial.

Disk-Diffusion Test

E Test

Effects of Combinations of Drugs Additive (indifferent) effect: the activity of two drugs in combination is equal to the sum (or a partial sum) of their independent activity when studied separately. Synergistic effect: the activity of two drugs in combination is greater to the sum of their independent activity when studied separately. Antagonistic effect: the activity of two drugs in combination is less to the sum (or a partial sum) of their independent activity when studied separately.

Additive (indifferent) effect

Synergistic effect

Antagonistic effect

Haw To Choose Antimicrobial Drugs? Narrow spectrum OR broad spectrum. Old age OR child. Male OR female. Pregnant OR lactating women. In patient OR out patient. Type of microorganisms. Site of infection.

The Action of Antimicrobial Drugs

Antimicrobial agents that inhibit peptidoglycan synthesis. Examples of commonly used antimicrobial chemotherapeutic agents arranged according to their mode of action: Antimicrobial agents that inhibit peptidoglycan synthesis. Inhibition of peptidoglycan synthesis in actively-dividing bacteria results in osmotic lysis. Penicillins (produced by the mold Penicillium) There are several classes of Penicillins: 1. Natural Penicillins: Are highly effective against gram-positive bacteria (and a very few gram negative bacteria) but are inactivated by the bacterial enzyme penicillinase. Examples include Penicillin G and V.

2. Semisynthetic Penicillins : Are effective against gram-positive bacteria but are not inactivated by penicillinase. Example Methicillin. 3. Semisynthetic broad spectrum Penicillins : Are effective against a variety of gram-positive and gram-negative bacteria but are inactivated by penicillinase. Example Ampicillin. 4.Semisynthetic broad spectrum penicillins combined with beta lactamase inhibitors such as clavulanic acid and sulbactam : Although the clavulanic acid and sulbactam have no antimicrobial action of their own, they inhibits penicillinase thus protecting the penicillin from degradation. Example Augmentin (Amoxicillin plus Clavulanic acid). Ampicillin acts as a competitive inhibitor of the enzyme transpeptidase, which is needed by bacteria to make their cell walls, It inhibits the third and final stage of bacterial cell wall synthesis binary fission, which ultimately leads to cell lysis. used to treat urinary tract infections, otitis media, uncomplicated community-acquired pneumonia, Haemophilus influenzae, salmonellosis and Listeria meningitis. Penicillin G is Intramascular therapy (Im). Penicillin V is orally therapy that is not effected by the acids of stomach. Methicillin is insensitive to penicillinase enzyme secreted by many penicillin-resistant bacteria. The presence of the ortho-dimethoxyphenyl group directly attached to the side chain carbonyl group of the penicillin nucleus facilitates the β-lactamase resistance. Bactoprenol phosphate transports NAM and NAG across the cell membrane in the synthesis of peptidoglycan.

B. Cephalosporins (produced by the mold Cephalosporium): Cephalosporins are effective against a variety of gram positive and gram-negative bacteria and are resistant to penicillinase (although some may be inactivated by other beta-lactamase enzymes similar to penicillinase). Four "generations" of cephalosporins have been developed over the years in an attempt to counter bacterial resistance : 1. First generation cephalosporins : Cephalexin (Keflex). 2. Second generation cephalosporins : Cefuroxime (Zinnat) 3. Third generation cephalosporins : Ceftriaxone (Claforan) 4. Fourth generation cephalosporins : Cefepime (Maxipime). brand name Augmentin (amoxicillin with potassium clavulanate). Clavulanic acid the similarity in chemical structure allows the molecule to act as a competitive inhibitor of beta-lactamases secreted by certain bacteria to confer resistance to beta-lactam antibiotics Cephalosporium acremonium Cephalosporins First  Generation     Cefazolin  Cephalexin Cephalothin  Cephaprin  Cephradine Cefadroxil Second Generation  Cefamandole  Cefonicid  Ceforanide Cefotetan Cefoxitin  Cefuroxime Cefaclor Third Generation Cefoperaxone  Cefotaxime Ceftazidime Ceftibuten Ceftizoxime eftriaxone Cefdinir Fourth Generation  Cefepime

d. Vancomycin (produced by the bacterium Streptomyces): c. Monobactems: They work only against Gram-negative bacteria, beta lactam antibiotics resistant to beta lactamase. The only commercially available monobactam antibiotic is Aztreonam. d. Vancomycin (produced by the bacterium Streptomyces): Vancomycin and teichoplanin are glycopeptides that are effective against gram-positive bacteria. Vancomycin is drug of "last resort", used only after treatment with other antibiotics had failed. e. Bacitracin (produced by the bacterium Bacillus): Bacitracin is used topically against gram-positive bacteria. f. Ethambutol (a synthetic chemical): Ethambutol is bacteriostatic against actively growing M.tuberculosis bacilli, it works by obstructing the formation of cell wall, That Inhibits incorporation of mycolic acid. Monobactems They work only against Gram-negative bacteria, The only commercially available monobactam antibiotic is aztreonam. Vancomycin is drug of "last resort", used only after treatment with other antibiotics had failed. Bacitracin a toxic and difficult-to-use antibiotic, bacitracin doesn't work well orally. However, it is very effective topically. Its action is on gram positive cell walls. Glycopeptide antibiotics are a class of antibiotic drugs. The class is composed of glycosylated cyclic or polycyclic nonribosomal peptides. Ethambutol is bacteriostatic against actively growing TB bacilli, it works by obstructing the formation of cell wall. Mycolic acids attach to the 5'-hydroxyl groups of D-arabinose residues of arabinogalactan and form mycolyl-arabinogalactan-peptidoglycan complex in the cell wall. It disrupts arabinogalactan synthesis by inhibiting the enzyme arabinosyl transferase. Disruption of the arabinogalactan synthesis inhibits the formation of this complex and leads to increased permeability of the cell wall. Bactoprenol phosphate transports NAM and NAG across the cell membrane in the synthesis of peptidoglycan. Bacitracin inhibits this process

Antimicrobial agents that alter the cytoplasmic membrane. Alteration of the cytoplasmic membrane of microorganisms results in leakage of cellular materials : a. Polymyxin B (produced by the bacterium Bacillus): Polymyxin B is used in severe Pseudomonas infections. b. Amphotericin B (produced by the bacterium Streptomyces): Amphotericin B is used for systemic fungal infections. c. Nystatin (produced by the bacterium Streptomyces): Nystatin is used mainly for Candida yeast infections. d. Imidazoles (produced by the bacterium Streptomyces): The imidazoles are antifungal antibiotics used for yeast infections, dermatophytic infections, and systemic fungal infections. Example Miconazole. Polymyxin B is an antibiotic primarily used for resistant gram negative infections. Bactericidal for gram-negative; little to no effect on gram-positive since cell wall is too thick to prmit access to membrane. Amphotericin B is produced by Streptomyces nodosus

Antimicrobial agents that inhibit protein synthesis These agents prevent bacteria from synthesizing structural proteins and enzymes. a. Agents that block transcription (prevent the synthesis of mRNA from DNA): Rifampins (produced by the bacterium Streptomyces): Rifampins are effective against some gram-positive and gram-negative bacteria and Mycobacterium tuberculosis. They inhibit the enzyme RNA polymerase. b. Agents that block translation (alter bacterial ribosomes to prevent mRNA from being translated into proteins): 1. Agents that bind irreversibly to the 30s ribosomal subunit and cause a misreading of the mRNA (the aminoglycosides produced by the bacterium Streptomyces). Examples include Streptomycin & Amikacin. Ethambutol is bacteriostatic against actively growing TB bacilli, it works by obstructing the formation of cell wall. Mycolic acids attach to the 5'-hydroxyl groups of D-arabinose residues of arabinogalactan and form mycolyl-arabinogalactan-peptidoglycan complex in the cell wall. It disrupts arabinogalactan synthesis by inhibiting the enzyme arabinosyl transferase. Disruption of the arabinogalactan synthesis inhibits the formation of this complex and leads to increased permeability of the cell wall. Ethambutol Inhibits incorporation of mycolic acid The ribosome then contains three RNA binding sites, designated A, P and E. The A site binds an aminoacyl-tRNA (a tRNA bound to an amino acid); the P site binds a peptidyl-tRNA (a tRNA bound to the peptide being synthesized); and the E site binds a free tRNA before it exits the ribosome.

c. Antimicrobial agents that interfere with DNA synthesis: 2. Agents that bind reversibly to the 30s ribosomal subunit and interfere with the binding of charged tRNA to the bacterial ribosome. Examples include Tetracycline & Doxycycline ( produced by the bacterium Streptomyces). They are effective against a variety of gram-positive and gram-negative bacteria. 3. Agents that bind reversibly to the 50s ribosomal subunit and prevent the release of uncharged tRNA from the bacterial ribosome. Erythromycin is used against gram positive bacteria and some gram-negative bacteria. c. Antimicrobial agents that interfere with DNA synthesis: Quinolones (synthetic chemicals): The quinolones block bacterial DNA replication by inhibiting the DNA gyrase, the enzyme needed by bacteria to produce their circular DNA. They are broad spectrum and examples include Norfloxacin, *Nalidixic acid & Ciprofloxacin. Nalidixic acid : antibiotic used only to treat UTI caused by Gram negative bacteria. d. Co-trimoxazole (synthetic chemicals): Co-trimoxazole is a combination of sulfamethoxazole and trimethoprim,which blocks the bacterial synthesis of folic acid needed to make DNA bases. DNA gyrase It is not present in humans. This makes gyrase a good target for antibiotics. Two classes of antibiotics that inhibit gyrase are: The aminocoumarins (including novobiocin). Aminocoumarins work by competitive inhibition of energy transduction of DNA gyrase by binding to the ATPase active site . The quinolones (including nalidixic acid and ciprofloxacin). Quinolones bind these enzymes and prevent them from decatenating replicating DNA. Quinolone-resistant bacteria frequently harbor mutated topoisomerases that resist quinolone binding.

The Action of Antimicrobial Drugs Adverse effects of chloramphenicol : Aplastic anemia Bone marrow suppression Leukemia

Microbial Resistance To Antimicrobial Chemotherapeutic Agents A common problem in antimicrobial chemotherapy is the development of resistant strains of bacteria. A variety of mutations can lead to antibiotic resistance. Most bacteria become resistant to antimicrobial agents by one or more of the following mechanisms: Enzymatic destruction of drug, e.g., penicillinase and other beta-lactamases. Prevention of penetration of drug, e.g., producing an altered cytoplasmic membrane. Alteration of drug's target site, e.g., altered ribosomal subunit. Rapid ejection of the drug. Developing an alternate metabolic pathway. Increasing the production of a certain bacterial enzyme. Resistance genes are often on plasmids or transposons that can be transferred between bacteria. Transposons are sequences of DNA that can move around to different positions within the genome of a single cell, a process called transposition. In the process, they can cause mutations and change the amount of DNA in the genome. Transposons were also once called jumping genes.

efflux is a mechanism responsible for extrusion of toxic substances and antibiotics outside the cell; this is considered to be a vital part of xenobiotic metabolism. This mechanism is important as it can contribute to bacterial antibiotic resistance to pump out unwanted toxic substances through specific efflux pumps Efflux pumps are proteinaceous transporters localized in the cytoplasmic membrane of all kinds of cells. They are active transporters, meaning that they require a source of chemical energy to perform their function.

Antimicrobial susceptibility testing by the disk diffusion method (Kirby-Bauer) & Antibiotic profil Purpose of Procedure To test isolated bacteria for its susceptibility to antimicrobial agents Specimen Requirements In general a pure growth of the isolate. Equipment Sterile cotton swabs. Reagents Required Mueller Hinton Agar plates. Appropriate antibiotic discs or dispenser. McFarland Standard (0.5). Pure culture of the test organism.

The basic steps for the Bauer-Kirby method of antimicrobial susceptibility testing are: Prepare a standard turbidity inoculum ((0.5) McFarland Standard ) of the test bacterium so that a certain density of bacteria will be put on the plate. Inoculate a (90 mm diameter, 4 mm agar depth of Mueller-Hinton agar plate) with the standardized inoculum so as to cover the entire agar surface with bacteria. Place standardized antibiotic-containing discs on the plate. Incubate the plate at 37 oC for 24 hours. Measure the diameter of any resulting zones of inhibition in millimeters. Determine if the bacterium is susceptible, intermediate, or resistant to each antimicrobial agent using a *standardized table. *Standarized table according to manufactory pamphlet.

Mueller Hinton Agar INGREDIENTS Beef Extract. Digest of Casein ( peptone ). Starch. Agar ....... 1.7 %. Final pH is ~7.3 at 25°C. Pour cooled Mueller Hinton agar into sterile Petri dishes on a level, horizontal surface to give a uniform depth of about 4 mm and cool to room temperature.

0.5 McFarland standard McFarland standards are used as a reference to adjust the turbidity of bacterial suspensions so that the number of bacteria will be within a given range. Were mixing specified amounts of barium chloride and sulfuric acid together. Mixing the two compounds forms a barium sulfate precipitate, which causes turbidity in the solution. A 0.5 McFarland standard is prepared by mixing 0.05 mL of 1.175% barium chloride dihydrate (BaCl2•2H2O), with 9.95 mL of 1% sulfuric acid (H2SO4). Turbidity of 0.5 McFarland standard is approximately equal 1.5X10^8 CFU/mL of bacterial suspension.

Procedure: Adjust the concentration of the inoculum by comparing the turbidity of Normal Saline tube to that of a 0.5 McFarland standard. Note: If the turbidity of the inoculum is greater than that of 0.5 McFarland standard, dilute with sterile normal saline. If the turbidity of the standard is greater than the inoculum, add more of the test organism. 2. Dip a sterile cotton swab into the adjusted inoculum tube and drain excess fluid by pressing the swab against the walls of the test tube.

3. Hold Muller-Hinton plate half or partially open and streak the plate using the wet cotton swab covering all the area even at the sides. 4. Place the plates aside for about 10 – 15 minutes at room temerature. Allow the inoculum to dry.

5. Using a sterile forceps or needle, apply a set of suitable antibiotic disks. Five to six disks for each plate, or 8 disks if you use the automatic dispenser. 6. Let the plates stand for 10 - 15 minutes at 4oC, then incubate in inverted position at 37 oC for 18-24 hours.

7. Using a ruler or caliper, measure the zone of inhibition around each antimicrobial disk and record it. 8. Consult the special chart provided by the manufacturer of the antimicrobial disks and interpret results as Sensitive, Resistant, or Intermediate.

END LECTURE Any Questions ?