Antibiotic Sensitivity Testing PDF

ANTIBIOTIC SENSITIVITY TESTING (ANTIMICROBIAL SUSCEPTIBILITY TESTING) AZLIN SHAM RAMBELY/DR MAIMUNAH MUSTAKM DEPARTMENT OF MEDICAL LABORATORY TECHNOLOGY FACULTY OF HEALTH SCIENCES UNIVERSITI TEKNOLOGI MARA (UITM) LEARNING OUTCOMES Upon completion of this course, student...

ANTIBIOTIC SENSITIVITY TESTING (ANTIMICROBIAL SUSCEPTIBILITY TESTING) AZLIN SHAM RAMBELY/DR MAIMUNAH MUSTAKM DEPARTMENT OF MEDICAL LABORATORY TECHNOLOGY FACULTY OF HEALTH SCIENCES UNIVERSITI TEKNOLOGI MARA (UITM) LEARNING OUTCOMES Upon completion of this course, students will: Understand mechanisms of action of antimicrobial agents Understand theory, principles and limitations of susceptibility testing and interpretations of breakpoints.  ANTIMICROBIAL CHEMOTHERAPY CHEMOTHERAPY the use of chemical substances to kill pathohenic organisms without injuring the host (Paul Ehrlich) CHEMOTHERAPEUTIC AGENT any chemical substance used in medical practice drugs in microbiology antimicrobial agents ANTIMICROBIAL AGENTS special group of chemotherapeutic agents used to treat diseases caused by microbes HISTORY AND DEVELOPMENT OF ANTIMICROBIAL DRUGS Paul Ehrlich “Magic bullet” ü Chemicals with selective toxicity ORIGIN: Selective stains DRUG: Arsphenamine (1910) “606” Salvarsan NOBEL: 1908 HISTORY AND DEVELOPMENT OF ANTIMICROBIAL DRUGS Alexander Fleming ü Microbes make antibiotics ORIGIN: moldy culture plate DRUG: Penicillin (1928) NOBEL: 1945  DISCOVERY OF ANTIBIOTICS - ALEXANDER FLEMING discovered penicillin while working with Staphylococcus no Staph. colonies growing near a mold contaminant colonies appeared to be melting Bactericidal - kills identified mold as Penicillium (producing bactercidal bacteria substance that was effective against wide range of microbes) unable to purified compound & abandoned his study  DISCOVERY OF ANTIBIOTICS Gerhard Domagk üDrugs are changed in the body ORIGIN: Prontosil (only selective in vivo) DRUG: Sulfonamide (1935) NOBEL: 1939 Ernest Fourneau- sulfanilamide portion of the prontosil molecule  DISCOVERY OF ANTIBIOTICS Ernst Chain & Howard Florey purifiried penicillin 1941: tested on human subject with life threating Staphylococcus aureus infection treatment effective initially supply of penicillin ran out before disease under control (patients dies of infection) WWII- mass production of penicillin (treatment of wounded soldrs & war workers)  DISCOVERY OF ANTIBIOTICS Selman Waksman 1943: isolated streptomycin from soil bacterium (Streptomyces griseus) comes up with definition of antibiotics ORIGIN: Penicillin development DRUG: Streptomycin (1943) NOBEL: 1952  DEFINITIONS OF ANTIBIOTICS OLD: An antibiotic is a chemical substance produced by various species of microorganisms that is capable in small concentrations of inhibiting the growth of other microorganisms NEW: An antibiotic is a product produced by a microorganism or a similar substance produced wholly or partially by chemical synthesis, which in low concentrations, inhibits the growth of other microorganisms  DEVELOPMENT OF NEW GENERATION DRUGS 1960: alteration of drug structure gave them new properties Penicillin G altered to create ampicillin Broadened spectrum of antimicrobial killing FEATURES OF ANTIMICROBIAL DRUGS Most modern antibiotics come from organisms living in the soil - includes : bacterial species (Streptomyces & Bacillus) and fungi (Penicillium & Cephalosporium) To commercially produce antibiotics: 1. strain is inoculated into broth medium 2. incubated until maximum antibiotic concentration is reached 3. drug is extracted from broth medium 4. antibiotic extensively purified 5. in some cases, drugs are chemically altered to impart new characteristics - semi-synthetic  FEATURES OF ANTIMICROBIAL DRUGS Antimicrobial action: Drugs may kill or inhibit bacterial growth vinhibit = bacteriostatic vkill = bacteriocidal bacteriostatic drugs rely on host immunity to eliminate pathogen bacteriocidal drugs are useful when host defenses cannot be relied upon to control pathogen  FEATURES OF ANTIMICROBIAL DRUGS Spectrum of activity: antimicrobials vary with respect to range of organisms controlled: vNarrow spectrum - work on narrrow range of organisms - Gram-positive only OR Gram-negative only vBroad spectrum - work on broad range of organisms - Gram-positive AND Gram-negative - disadvantage: disruption of normal flora  ANTIBIOTIC @ ANTIMICROBIAL AGENT The ideal antimicrobial agent should: - kill @ inhibit the growth of pathogens - cause no damage to the host - cause no allergic reaction in the host - be stable when stored in solid liquid @ liquid form - remain in specific tissue in the body long enough to be effective - kill the pathogens before they mutate & become resistant to it  Antimicrobial drug must inhibit @ destroy the pathogen without damaging the host Mechanisms of antibiotic action involve the blockage of a wide variety of How anti- metabolic activities: microbial - energy metabolism agents - the function of bacterial membrane work? - the synthesis of protein - the metabolism of nucleic acid - the synthesis of peptidoglycan The blockage of some of these function is lethal to the cell wall @ limit cell growth  How anti- microbial - agents work? INHIBITION OF CELL WALL SYNTHESIS bacteria- rigid external cel wall inhibiting cell wall synthesis - damages bacterial cells penicillin & cephalosporin contain β-lactam ring, which attaches to the enzymes that cross-link peptidoglycans & prevent cell wall synthesis INHIBITION OF PROTEIN SYNTHESIS structure of prokaryotic ribosome acts as target for many antimicrobials - differences in prokaryotic & eukaryotic ribosomes responsible for selective toxicity DETERMINING SUSCEPTIBILITY OF BACTERIAL TO ANTIMICROBIAL AGENTS AST One of the most important function of a diagnostic microbiology lab The physician need help from the lab to find the most effectiveness antibiotic using the AST AST When reporting, lab must indicate the response to various antibiotic (sensitive @ resistance), so that if the patient is allergic to any of them @ some of them prove to be ineffectiveness, an alternative choice can be made The report of the AST will be available in 18-24 hrs  PURPOSE AST is used to determine the susceptibility of bacteria to various antibiotics This standardized test is used to measure the effectiveness of a variety of antibiotics on a specific organism in order to prescribe the most suitable antibiotic therapy  METHOD (i) disk diffusion (ii) tube dilution Disk diffusion: Is based on the inhibition of bacterial growth measured under standard conditions (standardization of inoculum using McFarland standard)  The most widely used method: Kirby-Bauer DISK DIFFUSION Stokes Comparative Ericsson DISK DUFFUSION METHOD Kirby-Bauer disc diffusion routinely used to qualitatively determine susceptibility standard concentration of strain uniformly spread on standard media discs impregnated with specific concentration of antibiotic clear zone of inhibition around disc reflects placed on plate & incubated susceptibility - based on size of zone , organism can be described as susceptible or resistant  STANDARDIZATION OF INOCULUM Reproducibility on the disk diffusion test largely depends on the size of the inoculum used The zone of the inhibition decreases with increasing size of the inoculum, because the antimicrobial agent has to react with a greater no. of bacteria → it is important to use McFarland standard which is compared with the turbidity of the inoculum  MCFARLAND 0.5 TURBIDITY STANDARD Contains Barium with the optical density of the suspension is 0.5. AST by the disk-diffusion (Kirby-Bauer) method is standardized for bacterial inoculums with this optical density. Bacteria are diluted in sterile saline until the optical density matches the McFarland standard, then used to streak a lawn of bacteria to be used for Kirby-Bauer  PRINCIPLE The bacteria to be tested was spread evenly all over the culture plate (Muller-Hinton agar) Then a series of disks contain with known KIRBY-BAUER concentration of an antibiotics are placed on METHOD an agar plate (each white disk represents a different antibiotic eg: ampicillin, tetracycline, gentamicin)  The plate is incubated for 19-23 hrs at 37˚C, during this period, the antibiotics diffuses through the agar & may prevent the growth of KIRBY-BAUER the organism METHOD If the bacteria is susceptible (sensitive) to an antibiotic, it will not grow close to the disk (a clear zone will appear around the disk where the growth has been inhibited) If the bacteria is resistant to an antibiotic, it will grow right up to the disk Kirby-Bauer method The diameter of the zone of growth inhibition is measured & scored according to the size of the zone as sensitive, intermediate @ resistant The size of zone of inhibition depends on the sensitivity of the bacteria to the specific antibiotic & the antibiotic ability to diffuse through the agar STOKES METHOD The comparative disk test (stokes’ method) uses both a test organism and a control organism on the same plate The control organism is of defined sensitivity to the antibiotics being tested & this method allows a direct comparison of the diameter of the zone of inhibition between the test and control organism  Control: control bacterial strain (known sensitivity to antibiotics Test: bacterial strain under test A - F: six different antibiotic disks test organism: - sensitive to antibiotics B, C & E - resistant to antibiotics A, D & F  TUBE DILUTION Give quantitative data which is not obtainable with the Kirby-Bauer method AST is expressed in terms of the minimal inhibitory concentration (MIC) and/or the minimal bactericidal concentration (MBC) MINIMUM INHIBITORY CONCENTRATION (MIC) Quantitative test to determine lowest concentration of specific antimicrobial drug needed to prevent growth of specific organism determined by examining strain’s ability to grow in broth containing different concentration of test drug MINIMUM INHIBITORY CONCENTRATION (MIC) MIC is determined by producing serial dilutions with decreasing concentrations of test drugs known concentration of organism is added to each test tube tubes are incubated & examined for growth- growth determined by turbidity of growth medium lowest concentration to prevent growth is MIC  MINIMUM INHIBITORY CONCENTRATION MIC is measured by exposing The lowest concentration of the organism to a range of antimicrobial agent that concentrations of the prevents visible growth of the antimicrobial agent in microorganism after question (usually in doubling overnight incubation dilution series)  After a period of incubation the culture medium is inspected to identify the lowest concentration of antimicrobial agent at which no growth is visible MIC will inhibit growth but not necessary kill The killing concentration is the MBC Bactericidal - kills bacteria Bacteristatic - inhibits bacterial growth  bacteria are inoculated 0.1 0.2 0.4 0.8 1.6 3.1 6.3 12.5 25 50 into a nutrient broth containing a dilution series of antibiotic concentrations (eg: Staph aureus and tetracycline) bacterial growth occurred at a Tetracycline concentration of 0.8 mcg/ml but not at 1.6 mcg/ml. Thus, the MIC is read as 1.6 mcg/ml  MINIMUM BACTERICIDAL CONCENTRATION (MBC) Is determined by sub-culturing each of the No Growth tubes in the MIC test to a solid medium that does not contain antibiotic The antibiotic in the sub-cultured diffuses into the agar, releasing any inhibitory effect it may have had on any living bacteria in the sub-cultured  The MBC is identified as the smallest concentration of antibiotic that prevents any growth of the test bacterium  Tetracycline has no visible turbidity above 1.6 mcg/ml, but plating out the "clear" broth shows growth in 3.1, 6.3, and trace in 12.5 mcg/ml, so while the MIC is 1.6 mcg/ml, 0.1 0.2 0.4 0.8 1.6 3.1 6.3 12.5 25 50 the MBC is 25 mcg/ml. REVIEW  E-TEST modification of disc diffusion method uses strips impregnated with gradient concentration of antibiotic (from highest concention to lowest) test organism will grow & form zone of inhibition - zone is tear-drop shaped - zone will intersect strip at inhibitory concentration E-TEST Determination of MIC by E-test. A – Zone of inhibition; B – Bacterial growth; C – E-test strip; D – the MIC is the point at which the edge of the zone crosses the E - test strip – in this example it is 3 mg/l. REVIEW