Antimicrobial Susceptibility Testing (AST) Lab PDF

ANTIMICROBIAL SUSCEPTIBILITY TESTING (AST) LAB Aim to introduce the students to the ways in which the activity of antibiotics can be evaluated. Objectives 1) Understand different methods to evaluate antibiotic activity. 2) Understand the disk diffusion method for measuring zones of...

ANTIMICROBIAL SUSCEPTIBILITY TESTING (AST) LAB Aim to introduce the students to the ways in which the activity of antibiotics can be evaluated. Objectives 1) Understand different methods to evaluate antibiotic activity. 2) Understand the disk diffusion method for measuring zones of inhibition. 3) Assign bacteria as sensitive, intermediate, or resistant to antibiotics. 4) Determine the minimum inhibitory concentration (MIC) of an antibiotic. 5) Determine the minimum bactericidal concentration (MBC) of an antibiotic. Working beside the flame Call us to open the flame. NEVER leave the flame unattended. Work close to the flame in the aseptic area. Close the flame using the beaker provided or call us to help you close it. If something happened call lab specialist to come over and help you. PART 1 – Antimicrobial susceptibility assessment using “disc diffusion testing” You are provided with: 1) Cultures of bacterial strains (Salmonella spp. (B17), Staphylococcus aureus (B3), Escherichia coli (B285), E. coli (G651), Pseudomonas aeruginosa (B287), Klebsiella spp. (B14), and Clostridium perfringens (SDB 2) 2) Agar cultures were used to prepare a 0.5 McFarland suspension of each bacterium in sterile saline and these suspensions were diluted according to BSAC guidance (1:100 for Gram negative bacteria or 1:10 for Gram positive bacteria). PART 1 –Antimicrobial susceptibility assessment using “disc diffusion testing” Procedure: 1) Inoculate 100 μl of diluted bacterial suspension onto the surface of ISO plates using a sterile swab and allow to dry for 2 minutes. 2) Dispense antibiotic-impregnated plastic discs (4 different discs) onto the inoculated ISO agar using a disc dispenser (Oxoid, Cambridge, UK). 3) Incubate agar plates overnight at 37˚C in air, or anaerobically for C. perfringens. The inoculum on the agar plates should give semi-confluent growth of colonies after overnight incubation. A denser inoculum will result in reduced zones of inhibition and a lighter inoculum will have the opposite effect. 4) After incubation, measure the zone of inhibition (mm). Using the table below, determine whether the bacterial isolates are sensitive (S), intermediate (I), or resistant (R) to each of the antimicrobial agents. PART 1 –Antimicrobial susceptibility assessment using “disc diffusion testing” Table 1. Interpretative ranges for zone diameters (mm) Table 2. Interpretative ranges for zone diameters (mm) of Gram negative bacterial isolates for BSAC disc of Gram positive bacterial isolates for BSAC disc diffusion susceptibility testing. diffusion susceptibility testing. Gram negative bacteria Gram positive bacteria Disc Sensitive Intermediate Resistant Disc Sensitive Resistant Antibiotic Antibiotic Intermediate (I) content (S) ≥ (I) (R) ≤ content (S) ≥ (R) ≤ Gentamicin 10 μg 20 17-19 16 Gentamicin 10 μg 20 - 19 (CN) (CN) Amoxicillin 10 μg 15 - 14 Penicillin 1 unit 25 - 24 (AML) (P) Tetracycline 10 μg 20 - 19 Tetracycline 10 μg 20 - 19 (TE)* (TE) Meropenem 10 μg 27 20-26 19 Erythromycin 5 μg 20 17-19 16 (MEM) (E) Ciprofloxacin Ciprofloxacin 1 μg 14 - 13 1 μg 20 17-19 16 (CIP) (CIP) Vancomycin 5 μg 13 - 12 Vancomycin 5 μg 13 - 12 (VA) (VA)* Metronidazole 5 μg 18 - 17 Metronidazole 5 μg 18 - 17 (MTZ) (MTZ) *Adapted from Enterococcus spp. range. PART 1 –Antimicrobial susceptibility assessment using “disc diffusion testing” PART 1 –Antimicrobial susceptibility assessment using “disc diffusion testing” RESULTS Measure the zone diameters for each antibiotic (mm) using a ruler and record them in the tables below. Determine whether each bacterial isolate is susceptible, intermediate, or resistant to each antimicrobial agent Record the results in the table below alongside the corresponding zone diameter. ANTIBIOTIC STRAIN S. AUREUS C. PERFRINGENS ENTEROCOCCUS SPP.  PART 2 - Minimum Inhibitory Concentration (MIC) Testing MIC: the minimum concentration of the antimicrobial which is able to inhibit the growth of a bacterial pathogen. Gradient testing: E-testing is a susceptibility testing methodology which comprises an antibiotic-impregnated test strip, which when placed on an agar plate inoculated with the test organism, allows the diffusion of antimicrobial agent into the agar and an elliptical pattern of inhibition of growth. The MIC is read as the lowest concentration of antimicrobial agent on the strip where the growth of the organism intersects the strip. PART 2 - Minimum Inhibitory Concentration (MIC) Testing Broth dilution MIC testing: Procedure: 1) Prepare a series of test tubes containing ISO broth, S. aureus cells, and range of concentrations of the ciprofloxacin 2) Also prepare two control test tubes to validate the experiment: i. Culture inoculated into drug-free ISO broth (‘control’, C+) ii. Drug free ISO broth only (C-) 3) Incubate overnight at 37˚C in air. PART 2 - Minimum Inhibitory Concentration (MIC) Testing Broth dilution MIC testing: RESULTS Determine the ciprofloxacin MIC against each strain of S. aureus. The MIC is the lowest concentration of ciprofloxacin that resulted in no visible growth. Record your results in your logbook. Record the presence of visible growth in the broth dilution assays in the table below, ‘+’ for visible growth and ‘-‘ for no visible growth. Ciprofloxacin concentration (mg/L) Strain C- 0.03 0.06 0.125 0.25 0.5 1 2 C+ B3 B7 PART 3 – Minimum Bactericidal Concentration (MBC) testing MBC is defined as the lowest drug concentration which is capable of reducing bacterial viability by ≥99.9%. MBC is typically determined by sub-culturing the MIC tubes that do not show any visible growth and then calculating the viability by inoculating these cultures onto solid agar media. PART 3 – Minimum Bactericidal Concentration (MBC) testing Procedure: 1. Using aseptic technique, inoculate 0.5 mL of ISO broth from the tubes lacking growth after the MIC test into 4.5 mL of fresh ISO broth (10-1 dilution factor) 2. Repeat step 1 to produce a 10-2 dilution. 3. Using the spread plate technique, transfer 50 μl of the undiluted culture (‘neat’) and each of the dilutions onto separate ISO agar plates. 4. Incubate the inoculated agar plates overnight at 37˚C. 5. Count the number of colony forming units on each of the three plates and determine the total viable count (TVC). 6. Using the initial viable count for each S. aureus from the MIC experiment (‘control’, C+), calculate the percentage kill for each concentration of ciprofloxacin using the following equation: % Kill = 100 - [(TVC from antibiotic containing tube/TVC from control) *100] 7. Determine the MBC for ciprofloxacin for each S. aureus strain. MIC vs MBC Figure 1. Minimum inhibitory concentration (MIC) versus minimum bactericidal concentration (MBC) (Karaman et al. 2017). Karaman, D. Ş., Manner, S., Fallarero, A., & Rosenholm, J. M. (2017). Current approaches for exploration of nanoparticles as antibacterial agents. Antibacterial agents, 61. COURSEWORK ASSESSMENT AST Abstract (25% of the module mark) Students are to write 300-350 words scientific abstract on the antimicrobial susceptibility tests they have conducted in the lab. The abstract should be structured with the following main sections to be considered: Background, Objectives, Methods, Results and Conclusion. Using the data generated in the practical lab and the datasets provided, write a scientific abstract of 300-350 WORDS that details the Background/Objectives, Methods, Results, and Conclusions. The abstract should contain quantitative data from the experiments and be written in a passive and scientific style. FIVE keywords should also be selected and included after the abstract. For guidance on the style and structure of the abstract, see publications in the Journal of Antimicrobial Chemotherapy. An example abstract is provided in the lab manual. Your abstract does not need to cover all aspects of the practical. Your abstract should not have citations/references. Use credible and scientific resources to do your research such as journal articles, review articles and book chapters. Format: font 12, Times New Roman, single space, Justify text, normal margins Abstract structure: Title Title which reflects the study (descriptive) Title which reflects the selected range of data Title which reflects the drawn conclusions Abstract structure: Introduction & Objectives Background to the study (context, purpose). Do NOT copy the introduction from the practical manual (academic integrity). What is/are the primary aim(s)/objective(s) of the study. Use credible resources (review articles, book chapters) BUT do not cite or put references section. Abstract structure: Methods A clear and concise outline of the 1. Disc diffusion testing method main experiments. 2. MIC (Broth dilution method) What was the experiment. 3. MBC (sub-culturing) Why it was conducted (one sentence). How it was carried out (a brief summary NOT every single detail). Description of experiments should be clear enough for the reader to understand and follow the work. No need to mention MBC equations Abstract structure: Results The MAIN (NOT necessarily ALL) 1. Disc diffusion testing results/outcomes based on the experiments method (sensitive, described in the methods section – depth intermediate, resistant) and breadth are important – (evidence of understanding and analysis). 2. Broth dilution MIC and MBC (for the different Should NOT describe a method or explain its purpose/process (that should be in the strains) methods section). Numbers (percentage, values, units etc) Usually the longest part of the Abstract Abstract structure: Conclusions Conclusions relevant to the described results (evidence of critical analysis, synthesis and reflection). Conclusions MUST answer the objective set in the introduction. Wider perspective beyond the experiments (specific  general broader context) (Take home message) Abstract structure: Key words FIVE Key words which reflect the critical aspects of the study ASSESSMENT CRITERIA FOR AST ABSTRACTS Assessment category, description and % weighting Synthesis, Presentation, Content/Knowledge Evaluation and/or Structure and Style reflection Logical development of the Relevance; accuracy; addressed to Critical analysis; integration of argument; fluency; logical the topic; appropriate evidence; drawing of structure; appropriate academic depth/breadth; evidence of conclusions. style; ‘ease of reading’; spelling; understanding grammar. 20% 40% 40% Data Set SAMPLE: PLEURAL FLUID Gram positive bacteria Antibiotic (zone of Disc Sensitive Intermediate Resistant Patient: 79 years old man inhibition in mm) content (S) ≥ (I) (R) ≤ Co-administered medications: Insulin Fucidic acid 10 μg 20 17-19 16 Disease/condition: Diabetes, aminoglycosides (18 mm) hypersensitivity Ampicillin 1 μg 20 17-19 16 RESULTS (32 mm) Table 1. Interpretative ranges for zone Amoxicillin 1 μg 20 17-19 16 (37 mm) diameters (mm) of Gram-positive bacterial isolates Azithromycin for BSAC disc diffusion susceptibility testing. 5 μg 13 - 12 (12 mm) Data set Table 2. Minimum Inhibitory concentration experiment (mg/L). Strain C- 0. 1 0. 5 1 4 20 40 C+ Streptococcus - + + + - - - + pneumonia Streptococcus - + + - - - - + agalactiae ‘+’ for visible growth and ‘-‘ for no visible growth. Using these observations determine the MIC of norfloxacin against each bacterial isolate. Data Set Table 3 Minimum Bactericidal concentration (MBC) experiment Total viable count (TVC) (CFU/ml) TVC from antibiotic containing Strain TVC from + Control tube Streptococcus 500 9000 pneumonia Streptococcus 35 5000000 agalactiae  Use the equation below to determine the MBC of norfloxacin antibiotic for the provided bacterial strains. % Kill = 100 - [(TVC from antibiotic containing tube/TVC from control) *100] Any Questions?