4 - Antibiotic Sensitivity Testing.pdf

PHARM232 Antibiotic Sensitivity Testing 9/25/2024 Outline Ideal antimicrobial agents Broad versus narrow-spectrum drugs Antibiotics by mechanism Side effects Methods that directly measure antimicrobial activity 9/25/2024 Learning...

PHARM232 Antibiotic Sensitivity Testing 9/25/2024 Outline Ideal antimicrobial agents Broad versus narrow-spectrum drugs Antibiotics by mechanism Side effects Methods that directly measure antimicrobial activity 9/25/2024 Learning Objectives Understand some examples of the clinical considerations in prescribing antimicrobial drugs. Understand the categories of antibiotic drugs and their mechanisms of action Understand how to perform and interpret the Kirby-Bauer procedure (disk- diffusion) for the evaluation of antimicrobial activity of chemotherapeutic agents. Contrast the method and results of the disk diffusion antimicrobial susceptibility test with the methods that detect the minimal inhibitory concentration. Understand the factors considered for standardization of antimicrobial susceptibility testing. Define the susceptibility testing interpretive categories. State the advantages and the disadvantages of the different methods that directly measure antimicrobial activity (refer to the assigned reading) 9/25/2024 What are the Attributes of an Ideal Antimicrobial Agent? Selectively Toxic Soluble in bodily fluids Non-allergenic Reasonable half-life Long shelf life Inexpensive 9/25/2024 Selective Toxicity An ideal antimicrobial agent exhibits selective toxicity, which means that the drug is harmful to a pathogen without being harmful to the host. 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 Targets Receptors not found in host Selctive Toxicity Targets Biochemical pathways not essential for host ‘dosis sola facit venenum’…'only the dose makes the poison’ - Paracelsus 9/25/2024 Side Effects Toxicity Drugs may be toxic to kidneys, liver, or nerves (e.g., aminoglycosides- accumulation of these agents within both renal cortex and the perilymph of the inner ear) Considerations needed when prescribing drugs to pregnant women Tetracycline - form complexes with calcium that can be incorporated into bones and developing teeth, causing malformation of the skull and stained weakened tooth enamel to the fetus) Disruption of normal microbiota May result in secondary infections 9/25/2024 Range of Effectiveness of Antibiotics Broad Spectrum Works on a wide range of bacteria and pathogens More likely to disrupt your micriobiota or off target effects Narrow Specrum Increased selection, reduce non-selective toxicity More information is needed to use them 9/25/2024 Antibiotics Mechanisms of Action The mechanisms of antibiotics typically inhibit the sythesis of or disruption of : The Cell Wall Protein Nucleic Acid The Cell Membrane Metabolites 9/25/2024 Inhibitors of Cell Wall Synthesis Beta-Lactams: Penicillins Examples: Penicillin, Ampicillin, Amoxicillin Cephalosporins Examples: Cephalexin, Cefaclor, Cefotaxime Other Cell Wall Inhibitors https://tmedweb.tulane.edu/pharmwiki/doku.php/betalactam_phar m Examples: Vancomycin Beta-lactams Peptidoglycan Side chains Penicillin-biding peptidase Peptidoglycan 9/25/2024 Inhibitors of Protein Synthesis Anti-50S Examples: Chloramphenicol Inhibits peptide chain elongation during protein synthesis Bind 23S rRNA subunit Anti-30S Examples: Aminoglycosides, tetracycline Interfere directly and causing misreading of mRNA 9/25/2024 Inhibitors of Nucleic Acid Synthesis DNA Examples: Quinolines and fluoroqunilones Inhibit DNA gyrase and topoisomerase; blocking DNA replication RNA Examples: Rifampin Interferes with bacterial DNA-dependent RNA polymerase 9/25/2024 https://www.biomol.com/resources/biomol-blog/how-do- antibiotics-affect-nucleic-acid-synthesis Disruptors of the Cell Membrane Example: Polymyxin B Binds to plasma membrane and increases its permeability, while disrupting its structure https://pubs.rsc.org/en/content/articlelanding/2021/fd/d1fd00036e 9/25/2024 Antimetabolite Antibiotics Inhibitors of folic acid synthesis Important for many functions including growth and RNA/DNA synthesis Examples: Sulfonamides and trimethroprim Inhibitors of mycolic acid synthesis Specific for mycobacteria as it is the major lipid component of their cell walls Example: Isoniazid https://www.researchgate.net/publication/348841080_Permeation_of_Silver_Sulfadiazine_Into 9/25/2024 _TEMPO-Oxidized_Bacterial_Cellulose_as_an_Antibacterial_Agent/figures?lo=1 Goals of Antibiotics Bacteriocidal Kills bacteria Can be bacteriostatic at low concentrations Bacteriostatic Stops bacterial growth If removed bacteria can continue to grow Requires the hosts immune system to kill the bacteria 9/25/2024 Measurement of Antimicrobial Activity Measures of antimicrobial activity are accomplished by using several methods such as: Conventional susceptibility testing: Disk Diffusion Broth Dilution Agar Dilution Automated antimicrobial susceptibility testing Note: We are focusing on methods applied for aerobic and facultative anaerobic bacteria 9/25/2024 Important Terms for Measurement Minimum Inhibitory Concentration (MIC) the lowest antimicrobial concentration that completely inhibits visible growth of an organism Minimum Bactericidal Concentration (MBC) the lowest antimicrobial concentration required to kill a particular bacterium 9/25/2024 Determining Minimum Bactericidal and Inhibitory Concentrations Drug 32 μg/ml 16 μg/ml 8 μg/ml 4 μg/ml 2 μg/ml 1 μg/ml Concentration No Drugs No Colonies No Colonies Colonies MBC MIC 9/25/2024 Standardization The potential influence of environmental factors on antibiotic activity should be minimized To control such factors, the conditions for susceptibility testing are standardizedin the following reasons: Optimized growth conditions → Inhibition of growth is due to antimicrobial agent Optimized integrity of antimicrobial → failure to inhibit growth is the organisms’ resistance mechanisms, not environmental inactivation Reproducibility and consistency in the resistance profile 9/25/2024 The standardized components of antimicrobial susceptibility testing Time → 16 h Concentrations of antimicrobial → 2-fold dilution series Total volume → 2 mL for macrodilution; 100 μL for microdilution Initial inoculum concentration → 0.5 McFarland scale or 0.08-0.12 at 625 nm == 1 x 105 CFU/mL https://microbenotes.com/mcfarland-standards/ Chemical solution of barium chloride and sulfuric acid at varying amounts leads to difference in turbidity 9/25/2024 Limitations of standardization The in vivo environment cannot be reproduced, where the actual interaction between the bacteria and antibiotic will take place Some other factors may play a role in patient outcome and are not taken or cannot be taken into account by testing, such as: Antibiotic diffusion into tissues and host cells Serum protein binding of antimicrobial agents Status of patient immune system Virulence of the infecting bacterium 9/25/2024 Broth Dilution Testing The range of concentrations examined for each antibiotic is a series of doubling dilutions The concentration range examined often varies from one drug to the other depending on specific criteria, such as: the safest therapeutic concentration possible in a patient’s serum. the level of drug required to reliably detect a particular resistance mechanism. In this case, the test concentration for a drug may vary depending on the organism and its associated resistances. The lowest concentration of antibiotic that inhibits the in vitro growth of the bacteria is recorded as the MIC. The MICs are then translated into one of the following categories: susceptible, intermediate, or resistant. The interpretive criteria for these categories are based on studies that correlate the MIC with serum-achievable levels for each antimicrobial agent, particular resistance mechanisms, and therapeutic successful outcomes. 9/25/2024 Broth Dilution Testing (Cont.) Two main types macrodilutions (2 mL) and microdilutions (100 μL) Advantages: Quantatative method (MIC) Straightforward and technically simple (easy reproducibility between people) Opportunity for automation (especially with micro dilutions) Flexible Disadvantages: Time consuming and tedious Media usage (especially for macrodilution testing) Possiblity of error (preparing antibiotic solutions, innoulum concentrations, etc) 9/25/2024 Broth Macrodilution Testing 9/25/2024 Broth Microdilution Testing 9/25/2024 Agar Dilution Testing It is similar to the previous technique, but here the serial dilutions of the antibiotic are added to melted solid media, which are then poured and left to cool and solidify. This procedure involves the use of a series of agar plates, each containing a different concentration of antibiotic. Then a fixed number of organisms (standardized as before) is inoculated on the surface of agar plates, and examined by colony count after incubation. 9/25/2024 Agar Dilution Testing (Cont.) Advantages More gradual changes Disadvantages Labor intensive Expensive Space inefficient 9/25/2024 Disc Diffusion Method (Kirby-Bauer procedure) Bacterial suspension should be standardized Concentration of antibiotics in the disk should be standardized (provided from the manufacturer) Incubation time and temperature should be standardized Standardized agar medium (The most frequently used media is Mueller- Hinton agar) 9/25/2024 Disc Diffusion Method Protocol 1. Make bacterial lawn Dip swab into broth culture. Remove excess liquid Swab entire agar surface – 3 directions and around the rim Should be no “bare spots” or “brush strokes” on agar 9/25/2024 Disc Diffusion Method Protocol 2. Dispense the antibiotic disks 3. Press GENTLY with forceps for disks to adhere. Alcohol-flame forceps to sterilize. 4. Label plates on the edges. 5. Invert plates and incubate. 9/25/2024 Zone of inhibition measurements If the zones of adjacent antibiotic disks overlap, the zone diameter can be determined by measuring the radius of the zone. Measure from the center of the antibiotic disk to a point on the circumference of the zone where a distinct edge is present. Multiply this measurement by 2 to determine the diameter of the zone of inhibition. 9/25/2024 Example of Standards for Antimicrobial Disk Diffusion - Zones of Inhibition for Various Antimicrobial Agents Zone of Inhibition Antimicrobial Agent Disc Content Resistant Intermediate Susceptible Ampicilin 10 μg Enterobacteriaceae 17 Staphalococcui 29 Enterococci 17 Other Streptococci 30 Listeria Monocytogenes 20 Bacitracin 10 Units 13 Carbenicillin 10 μg Pseudomonas 17 Other Gram-Negatives 23 Cefoxitin 30 μg 18 9/25/2024 Disk diffusion method advantages and disadvantages Advantages: Simplicity; no special equipment Flexible Reduced labour Easily interpretable Inexpensive Consistent Disadvantages: Diameter instead of concentration → requires interpretation Qualitative Purchased discs means limited antibiotics 9/25/2024 Epsilometer test (E-test) Photograph of E-test susceptibility strip. The minimal inhibitory concentration (MIC) is determined from the point where the zone of inhibition intersects with the numerical scale 9/25/2024 Examples of the E-test interpretation 9/25/2024 Etest Advantages and Disadvantages Advantages Potentially more consistent and accurate Relatively easy Well controlled antibiotic concentrations Disadvantages Higher Costs (~10X cost of discs) Limited number of antibiotics 9/25/2024 Automated Antimicrobial Susceptibility Testing 9/25/2024 Automated Antimicrobial Susceptibility Testing (Cont.) Automatically generates a growth curve based on readings Algorithm-derived MIC Vivtek System Micro plate ‘card’ with preloaded antibiotics Photometric measurements Tests against a astandard to reach appropriate turbitiy MicroScan standard micro-dilution test trays Fluometric measurements BD Phoenix oxidation–reduction detector and a turbidity measurement 9/25/2024 Automated Testing Advantages Less tedious More reproducable Fast (as little as 3 hours) Automated data output Disadvantages Limited types of antimicrobials Limited ability to detect some forms of antimicrobial resistance (β-Lactamases in gram- negative bacteria ) Expensive 9/25/2024 How are breakpoints calculated/selected? In general, all susceptibility testing methods require breakpoints (AKA interpretive criteria), so that the results of the tests can be interpreted and reported as susceptible, intermediate, or resistant. Depending on the testing method, breakpoints are expressed as either a concentration (in mg/L or μg/mL) or a zone diameter (in mm). Hundreds of strains are tested to create reference inhibitory zone-size breakpoints to define susceptible, intermediate, and resistant categories for each antimicrobial agent/bacterial species The inhibition zone sizes are then correlated with MICs recorded by broth or agar dilution. A range of data are used to assist organizations in selecting breakpoints, including in vitro microbiological data, animal and human PK/PD data, and clinical/bacteriological outcome data from clinical studies. 9/25/2024 “Scattergram” of MICs versus zone diameters to determine breakpoint for Zone Diameter Horizontal lines are drawn from the MIC resistant breakpoint and the susceptible MIC breakpoint. Where the horizontal lines intersect the regression line, vertical lines are drawn to delineate the corresponding inhibitory zone size breakpoints in mm. 9/25/2024 Breakpoints Categories Breakpoints are usually set in such a manner to create a third category, i.e., intermediate (susceptibility) This category has multiple purposes, including: “Strains with MICs in the given range is too small to derive robust conclusions” Responses can be variable May be dose dependent Fills in the grey area between resistant and susceptible 9/25/2024  Definitions of Susceptibility testing interpretive categories Category Interpretation Susceptible Indicates that the antimicrobial agent may be an appropriate choice (in this situation). Bacterial resistance is absent or clinically insignificant Intermediate Indicates a number of possibilities, including: -The potential utility of the agent in the body sites where it may be concentrated -The response rates may be lower than those susceptible isolates -Providing a “buffer” between the resistant and susceptible categories to prevent serious interpretive errors Resistant Indicates that the isolates are not inhibited by the usually achievable concentrations of the agent with normal dosage schedules and/or demonstrates that the MICs / Zone diametes that fall in the range where the specific microbial resistance mechanisms are likely and that clinical efficacy against the isolates has not been shown reliably in treatment studies 9/25/2024 Selection of antimicrobial agents for lab testing The antimicrobial agents that are chosen for testing against a particular bacterial isolate are referred to as the antimicrobial battery or panel. A laboratory may use different testing batteries, but the content and application of each battery are based on various criteria such as those mentioned in Table I. 9/25/2024 Antibiogram 9/25/2024 DANGERS OF INDISCRIMINATE USE The indications for administration of antibiotics must sometimes be qualified by the following concerns: 1) Changes in the normal flora of the body, with disease resulting from "superinfection" due to overgrowth of drug-resistant organisms. 2) Direct drug toxicity (eg, renal damage or auditory nerve damage due to aminoglycosides). 3) Development of drug resistance in microbial populations, chiefly through the elimination of drug-sensitive microorganisms from antibiotic-saturated environments (eg, hospitals) and their replacement by drug-resistant microorganisms. 9/25/2024 Always remember! All antibiotic orders must have 3 pieces of information: when to take them how much to take (the right dosage) how many days you should take them 9/25/2024  Recommended Reading From Joseph T. DiPiro, Robert L. Talbert, Gary C. Yee , Gary R. Matzke, Barbara G. Wells, L. Michael Posey. Pharmacotherapy : A pathophysiologic approach.12th edition. https://accesspharmacy-mhmedical- com.proxy.lib.uwaterloo.ca/content.aspx?bookid=3097&sectionid=263143621 Direct examination Cultures Rapid diagnostic technologies (you need to have an understanding of the benefits of such technologies) Quantitative antimicrobial susceptibility testing – Macrodilution and microdilution minimal inhibitory concentrations advantages & limitations Qualitative antimicrobial susceptibility test methods - disk diffusion assay versus quantitative susceptibility testing of microorganisms Other susceptibility tests - Epsilometer test Automated antimicrobial susceptibility testing (Give examples of automated tests and advantages and disadvantages of such tests)* Minimal Bactericidal Concentration *Note: You need to know the advantage and disadvantages of the various antibiotic sensitivity testing. For the automated systems, you do not have to know the differences between the various methods, i.e. microscan, Vitek and BD systems, but you just need to know the advantages that these tests have over conventional techniques and the main disadvantages of the fully automated techniques. 9/25/2024 Additional Useful References Betty A.Forbes, Daniel F. Sahm, Alice S. Weissfeld. Bailey & Scott’s Diagnostic Microbiology 2007, 12th edition. Jawetz, Melnick and Adelberg’s Medical Microbiology 2019. 28th Edition. McGraw-Hill companies Robert W. Bauman. 2012. Microbiology with Diseases by Body System. 2nd edition. Pearson- Benjamin Cummings Publishing Company. http://cdc.gov/ http://www.microrao.com/antibiotics.htm 9/25/2024