Clinical Bacteriology Lab - 09 Antimicrobial Susceptibility Testing

CLINICAL BACTERIOLOGY LABORATORY Antimicrobial Susceptibility Testing OUTLINE • Introduction • Types of Antibiotic Susceptibility Testing o Broth Dilution Test o Antimicrobial Gradient Method o Disk Diffusion Test INTRODUCTION • An important task of the clinical microbiology laboratory is the performance of antimicrobial susceptibility testing of significant bacterial isolates. The goals of testing are to detect possible drug resistance in common pathogens and to assure susceptibility to drugs of choice for particular infections. • The most widely used testing methods include broth microdilution or rapid automated instrument methods that use commercially marketed materials and devices. Manual methods that provide flexibility and possible cost savings include the disk diffusion and gradient diffusion methods. Each method has strengths and weaknesses, including organisms that may be accurately tested by the method. Some methods provide quantitative results (eg, minimum inhibitory concentration), and all provide qualitative assessments using the categories susceptible, intermediate, or resistant. In general, current testing methods provide accurate detection of common antimicrobial resistance mechanisms. However, newer or emerging mechanisms of resistance require constant vigilance regarding the ability of each test method to accurately detect resistance. • Antibiotics are widely distributed in nature, where they play an important role in regulating the microbial population of the soil, water. Sewage and compost. They differ markedly both chemically and in their mechanism of action. There is thus little or no relation between the antibiotics other than their ability to affect adversely the life process of certain microorganism. There are many known purified antimicrobial agent, but only few have been sufficiently non-toxic to the living host cells. • Antibiotic is a chemical substance produced by various species of microorganism or a similar substance produced wholly or partially by chemical synthesis which in low concentration, inhibits the growth of other microorganism. Although originally produced from the Genus Streptomyces, Penicillium, Bacillus, Actinomyces and Cephalosporium, it is now produced completely by a synthetic process. Penicillins are prime examples of chemical modification by altering the pharmacokinetics, antibacterial spectrum and its potential by influencing its basic chemical structure. • An ideal antimicrobial agent exhibits selective toxicity. This terms implies that the drug is harmful to the pathogenic microbes without or minimally affecting the host. The mechanism of action of most antibiotics can be categorized into: (1) inhibition of the cell wall synthesis, (2) inhibition of the cell wall function (3) inhibition of microbial protein synthesis and (4) inhibition of nucleic acid synthesis. TYPES OF ANTIBIOTIC SUSCEPTIBILITY TESTING BROTH DILUTION TEST • One of the earliest antimicrobial susceptibility testing methods was the macrobroth or tube-dilution method. This procedure involved preparing two-fold dilutions of antibiotics (eg, 1, 2, 4, 8, and 16 µg/mL) in a liquid growth medium dispensed in test tubes. The antibiotic-containing tubes were inoculated with a standardized bacterial suspension of 1 –5×105CFU/mL. Following overnight incubation at 35°C, the tubes were examined for visible bacterial growth as evidenced by turbidity. The lowest concentration of antibiotic that prevented growth represented the minimal inhibitory concentration (MIC). The precision of this method was considered to be plus or minus 1 two-fold concentration, due in large part to the practice of manually preparing serial dilutions of the antibiotics. The advantage of this technique was the generation of a quantitative result (ie, the MIC). The principal disadvantages of the macrodilution method were the tedious, manual task of preparing the antibiotic solutions for each test, the possibility of errors in preparation of the antibiotic solutions, and the relatively large amount of reagents and space required for each test. • The miniaturization and mechanization of the test by use of small, disposable, plastic “microdilution” trays (Figure 1) has made broth dilution testing practical and popular. Standard trays contain 96 wells, each containing a volume of 0.1 mL that allows approximately 12 antibiotics to be tested in a range of 8 two-fold dilutions in a single tray. Microdilution panels are typically prepared using dispensing instruments that aliquot precise volumes of preweighed and diluted antibiotics in broth into the individual wells of trays from large volume vessels. Hundreds of identical trays can be prepared from a single master set of dilutions in a relatively brief period. Few clinical microbiology laboratories prepare their own panels; instead frozen or dried microdilution panels are purchased from one of several commercial suppliers. Inoculation of panels with the standard 5×105CFU/mL is accomplished using a disposable device that transfers 0.01 to 0.05 mL of standardized bacterial suspension into each well of the microdilution tray or by use of a mechanized dispenser. Following incubation, MICs are determined using a manual or automated viewing device for inspection of each of the panel wells for growth. • The advantages of the microdilution procedure include the generation of MICs, the reproducibility and convenience of having preprepared panels, and the economy of reagents and space that occurs due to the miniaturization of the test. There is also assistance in generating computerized reports if an automated panel reader is used. The main disadvantage of the microdilution method is some inflexibility of drug selections available in standard commercial panels.

• A broth microdilution susceptibility panel containing 98 reagent wells and a disposable tray inoculator ANTIMICROBIAL GRADIENT METHOD (E-TEST) • Epsilometer test (E- test ) is an ‘exponential gradient’ method of determination of antimicrobial resistance. The E-test has been developed to provide a direct quantification of antimicrobial susceptibility of microorganisms.This is a quantitative method that applies both the dilution of antibiotic and diffusion of antibiotic into the medium. • The device consists of a predefined, continuous, and exponential gradient of antibiotic concentrations immobilized along a rectangular plastic test strip. After 48 hours incubation a drop-shaped inhibition zone intersects the graded test strip at the inhibitory concentration (IC) of the antibiotic. HOW IT WORKS? • A predefined stable antimicrobial gradient is present on a thin inert non-porous plastic carrier strip 5mm wide, 60 mm long known as E-test strip. • When this E test strip is applied onto an inoculated agar plate, there is an immediate release of the drug and establishment of an antimicrobial concentration gradient in an agar medium. • After overnight incubation, the tests are read by viewing the strips from the top of the plate, a symmetrical inhibition ellipse is produced. • The intersection of the lower part of the ellipse shaped growth inhibition area with the test strip indicates the MIC value. MATERIALS • MEDIA: Mueller Hinton agar (MHA plates (uniform depth of 4 mm) • E-test strips (of desired antibiotic) • McFarland standard 0.5 • Forceps • Sterile cotton swabs • Sterile normal saline, 4 ml volumes in tubes • Bacterial strains o Non-fastidious cultures plated out for single colonies. o Strain for quality control: Escherichia coli ATCC 25922 PROCEDURES • Inoculum Preparation 1. Remove the E-test package from the freezer (-20°C) at least 30 minutes before required. 2. Emulsify 3 or 4 individual test strain colonies and transfer to a tube of saline. 3. Compare turbidity to that in 0.5 McFarland standards. Adjust turbidity of inoculum to match that standard. • Inoculation in Muller Hinton Agar 1. Dip a sterile cotton swab into the inoculums and pulling out slightly, rotate the swab several times against the inside of the tube above the fluid level to remove excess liquid. 2. Streak the swab over the entire surface of the agar plate by rotating the plate approximately 60o. Complete inoculation by running the swab around the rim of the agar. 3. Leave the lid of the plate ajar for 5 minutes (no more than 15 minutes) to allow any excess moisture to be absorbed before applying strips. o NOTE: Swab plate within 15 minutes of preparing the adjusted inoculums • Application of E-test strips : 1. Open E-test package by cutting package along the broken line. Apply strips to agar surface using forceps (or E-test applicator if available). 2. Place the strip with the ‘E end’ at the edge of the plate and with the scale visible (i.e. facing upwards). 3. If strips stick together, they may be pulled apart by handling the section marked E. Do not touch any other area of the strip. 4. Use templates to position 4 to 6 strips onto a 150 mm plate or one (seldom two) strips onto a 90 mm plate. Do not remove or replace a strip once it has touched the agar. 5. Repeat the entire procedure also for Quality control Strain (E. coli ATCC 25922) 6. Incubate Plates at 37°C for 18-24 hrs. RESULTS AND INTERPRETATION 1. Read MIC at the point where ellipse intersects the scale. If a MIC value between two twofold dilutions is seen, always round up to the highest value. 2. Read the MIC value at complete inhibition of all growth. 3. If the intersect differs on either side of the strip, read the MIC as the greater value. Ignore any growth at edge of strip. • MIC values of the bacteria should be interpreted as S (Susceptible), I (Intermediate) or R (Resistant) by comparing the breakpoint values of each antibiotic with the criteria recommended by CLSI. • A Staphylococcus aureus isolate tested by the Etest gradient diffusion method

DISK DIFFUSION METHOD PURPOSE AND SCOPE • The Kirby-Bauer test, known as the disk-diffusion method, is the most widely used antibiotic susceptibility test in determining what choice of antibiotics should be used when treating an infection. This method relies on the inhibition of bacterial growth measured under standard conditions. For this test, a culture medium, specifically the Mueller-Hinton agar, is uniformly and aseptically inoculated with the test organism and then filter paper discs, which are impregnated with a specific concentration of a particular antibiotic, are placed on the medium. The organism will grow on the agar plate while the antibiotic “works” to inhibit the growth. If the organism is susceptible to a specific antibiotic, there will be no growth around the disc containing the antibiotic. Thus, a “zone of inhibition” can be observed and measured to determine the susceptibility to an antibiotic for that particular organism. The measurement is compared to the criteria set by the Clinical and Laboratory Standards Institute (CLSI). Based on the criteria, the organism can be classified as being Resistant (R), Intermediate (I) or Susceptible (S). PRINCIPLE OF THE METHOD • The media used in this test has to be the Mueller-Hinton (15x150mm) agar because it is an agar that is thoroughly tested for its composition and its pH level. Also, using this agar ensures that zones of inhibitions can be reproduced from the same organism, and this agar does not inhibit sulfonamides. The agar itself must also only be 4mm deep. This further ensures standardization and reproducibility. • The size of the inoculated organism must also be standardized (using the MCFarland 0.5 Latex standard with the Wickersham Card. The reasons are because if the size of the inoculum is too small, the zone of inhibition will be larger than what it is supp osed to be (“the antibiotics will have a distinct advantage”) and if the inoculum is too large, the zone of inhibition will be smaller. MATERIALS • Specimen Requirement : o Well isolated colonies from BAP agar plate only. • Reagents and Supplies: o Mueller Hinton 50x150 mm agar plates o Hardy Diagnostics Susceptibility Disks (various antimicrobials as in QC above) o 37 C Incubator o Sterile polyester or cotton swabs o Hardy Diagnostic Saline tubes o McFarland Latex 0.5 Standard and Wickerham Card o Calipers, ruler, or template for measuring the diameters of inhibitory zones. PROCEDURE • Preparation of Bacterial Suspension 1. Remove a Hardy Diagnostic Saline 0.85%, 1.8mL tube from the box, label with the patient name and place in a test tube rack. 2. Using a 1ul loop, pick several isolated colonies from the agar surface. 3. Immerse the loop in a labeled saline tube. Vortex. 4. Using a Wickerham Card and a vortexed McFarland Latex 0.5 standard, compare the turbidity of the inoculated saline tube with the Standard. If the turbidity is comparable, proceed with the inoculation of the Mueller Hinton Plate. If not, adjust the turbidity by adding more isolated colonies in the same manner if the turbidity is less than the standard or more saline if the turbidity is greater. Once the turbidity is comparable to the standard, proceed with the inoculation of the labeled Mueller Hinton plate. 5. The bacterial suspension should be used within 6 h of preparation. If not used immediately after preparation, shake vigorously to resuspend the bacteria just prior to use. • Inoculation of Mueller Hinton Agar o Allow plates to come to room temperature before use. 1. Dip a sterile cotton swab into the bacterial suspension. To remove excess liquid, rotate the swab several times with a firm pressure on the inside wall of the tube above the fluid level. 2. Using the swab, streak the Mueller-Hinton agar (Links to an external site.) plate to form a bacterial lawn. 3. To obtain uniform growth, streak the plate with the swab in one direction, rotate the plate 90° and streak the plate again in that direction. 4. Repeat this rotation 3 times. 5. Allow the plate to dry for approximately 5 minutes. 6. Use an Antibiotic Disc Dispenser to dispense disks containing specific antibiotics onto the plate. 7. Using sterile sticks or loops, gently press each disc to the agar to ensure that the disc is attached to the agar. 8. Plates should be incubated overnight at an incubation (Links to an external site.) temperature of 37°C. • Reading and Interpreting Zone Sizes 1. After overnight incubation measure the zone sizes (area of no growth around the disk) in millimeters using a ruler or template. 2. Enter the zone sizes into the Kirby Bauer sensitivities log along with the patients information. 3. Interpret the results as Resistant, Intermediate or Sensitive for each antimicrobial according to the ranges listed on the log for Enteric gram negative rods. 4. Enter the results (R, I or S) into the LIS. 5. For Staph species that are Cefoxitin (Oxacillin) Resistant, Penicillin and Cefazolin will be reported as "not effective for MRSA". LIMITATIONS • This system is only set up for Enteric gram negative rods or a Staph aureus gram positive cocci. Therefore make sure the gram negative organism is lactose fermenting,and oxidase negative before setting up. • Non-enterics, streptococci/enterococci, gram positive rods and gram negative cocci must be sent out to Quest if sensitivities are needed.