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Premium member Presentation Transcript Antibiotic Sensitivity testing Minimum inhibitory concentration skill based learning: Dr.T.V.Rao MD Antibiotic Sensitivity testing Minimum inhibitory concentration skill based learning Dr.T.V.Rao MD 1Why antibiotic susceptibility testing: The responsibility of the microbiology laboratory includes not only microbial detection and isolation but also the determination of microbial susceptibility to antimicrobial agents . Many bacteria, in particular, have unpredictable susceptibilities to antimicrobial agents, and their susceptibilities can be measured in vitro to help guide the selection of the most appropriate antimicrobial agent. Why antibiotic susceptibility testing Dr.T.V.Rao MD 2What is susceptibility to Antibiotics: What is susceptibility to Antibiotics The term susceptible means that the microorganism is inhibited by a concentration of antimicrobial agent that can be attained in blood with the normally recommended dose of the antimicrobial agent and implies that an infection caused by this microorganism may be appropriately treated with the antimicrobial agent. The term resistant indicates that the microorganism is resistant to concentrations of the antimicrobial agent that can be attained with normal doses and implies that an infection caused by this microorganism could not be successfully treated with this antimicrobial agen t. Dr.T.V.Rao MD 3Antimicrobial resistance results from ….: Antimicrobial resistance results from …. Results from misuse, overuse, under/ inadequate use of antimicrobials Costs money, lives and undermines effectiveness of health delivery programs Threat to global stability and national security WHO Global Strategy for Containment of Antimicrobial Resistance: Intervention framework to slow emergence and reduce the spread of antimicrobial resistant microorganisms Dr.T.V.Rao MD 4Antibiotic resistant infections : Antibiotic resistant infections Diseases Agent Resistances Pneumonia S pneumoniae Penicillin Dysentery S dysenteriae Multiple resistances Typhoid S typhi Multiple resistances Gonorrhea N gonorrhea Penicillin and tetracycline Tuberculosis M tuberculosis Rifampicin and INH Nosocomial infections S aureus Methicillin, vancomycin E species Vancomycin Klebsiella, Pseudomonas Multiple resistancesAntimicrobial susceptibility tests: Antimicrobial susceptibility tests Minimum inhibitory concentration [MIC] The smallest concentration of antibiotic that inhibits the growth of organism Liquid media (dilution) allows MIC estimation Solid media (diffusion) Disk diffusion (Kirby-Bauer) E-tests Allows MIC estimation Beta lactamase production: quick screening method Dr.T.V.Rao MD 6Slide 7: Dr.T.V.Rao MD 7Susceptibility testing: The big gaps: Susceptibility testing: The big gaps MICs help, but hard to standardize Correlations appear possible based on individual isolates. Broad correlations based on multiple isolates are still lacking Understanding this helps a lot when trying to correlate outcome with MIC - Some patients get better despite MICs - Some patients just don’t get better despite MICs No rule when it correlates/not correlates Dr.T.V.Rao MD 8The tube dilution test is standard method: The tube dilution test is standard method The tube dilution test is the standard method for determining levels of microbial resistance to an antimicrobial agent Serial dilutions of the test agent are made in a liquid microbial growth medium which is inoculated with a standardized number of organisms and incubated for a prescribed time. The lowest concentration (highest dilution) of test agent preventing appearance of turbidity (growth) is considered to be the minimal / minimum inhibitory concentration (MIC ). At this dilution the test agent is bacteriostatic. Dr.T.V.Rao MD 9 minimum inhibitory concentration test determines: The MIC or minimum inhibitory concentration test determines antimicrobial activity of a material against a specific bacteria. The most commonly employed methods are the tube dilution method and agar dilution methods. Test products that are not clear or precipitate the growth media are tested by agar dilution methods which is about same as tube dilution method except dilutions are plated on agar minimum inhibitory concentration test determines Dr.T.V.Rao MD 10Minimum Inhibitory Concentration (MIC): The Minimum Inhibitory Concentration (MIC) is the smallest concentration of an antimicrobial agent that inhibits the growth of bacteria. The value is obtained in a highly mechanized fashion, but this procedure only provides interval censored reading. It is often of interest to use data collected from complex experiments to see how the mean MIC is affected by different factors Minimum Inhibitory Concentration ( MIC) Dr.T.V.Rao MD 11The minimal bactericidal concentration: The minimal bactericidal concentration (MBC) or the minimum lethal concentration (MLC) of an antibacterial which is defined as the maximum dilution of the product that will kill a test organism can be determined by sub culturing last clear MIC tube onto growth medium and examining for bacterial growth. Serial dilutions are made of the products in bacterial growth media. The minimal bactericidal concentration Dr.T.V.Rao MD 12A minimum bactericidal concentration test: A minimum bactericidal concentration test Figure 10.12 Dr.T.V.Rao MD 13Minimum inhibitory concentration (MIC) Practice the exercise for skills: Minimum inhibitory concentration (MIC ) Practice the exercise for skills 1. Grow cultures up overnight. 2. The following day, inoculate a fresh culture with a 1:10-20 dilution and grow up to OD600 0.4. 3. Dilute to OD600 0.0005 (1:800) in THB (or relevant media). 4. Dilute this 1:200 and drop about 25µl on THA (or relevant agar plates) to confirm equal starting inoculums. 5. Put 50µl of the 0.0005 cultures in a 96 well plate in triplicates. 6. Add 50µl per well of the substance you want to test (ie. antimicrobial peptide, antibiotic) to each well, keeping in mind the final concentration will be half the original concentration you are adding. It is good to test 1:2 dilutions (in THB, or relevant media) of this substance. 7. Incubate overnight and check by eye or OD600 24 hou rs later to determine what concentration inhibited the growth of the different strains of bacteria. Dr.T.V.Rao MD 14Dilution in liquid broth: Dilution in liquid broth Tubes containing increasing antibiotic concentrations Incubation during 18 hr at 37°C Tedious 0 ( Control) 0,25 0,50 1 2 4 8 mg/l MIC Bacterial growth Inhibition Dr.T.V.Rao MD 15Slide 16: Dr.T.V.Rao MD 16Slide 17: Dr.T.V.Rao MD 17Standard strains for quality assurance: Standard strains for quality assurance Precision and accuracy ensured through control strains Known susceptibility to antimicrobial agents Standard strains include Staphylococcus aureus ATCC 25923 Escherichia coli ATCC 25922 Pseudomonas aeruginosa ATCC 27853 Dr.T.V.Rao MD 18Different standards CLSI Standards followed all over the world: Use standardized reference National Committee for Clinical Laboratory Standard s (USA) Other norms Canadian Chinese National Do not confuse the different tables Choose one for everything Different standards CLSI Standards followed all over the world Dr.T.V.Rao MD 19Critical points in quality assurance- Organise your Laboratory with: Culture media: Muller-Hinton Reagents: disks Size of the inoculums Incubation condition Control with reference strains Reading inhibition diameters (accurate measurement) Knowledge of staff Critical points in quality assurance- Organise your Laboratory with Dr.T.V.Rao MD 20Principles of Minimum inhibitory concentration: Principles of Minimum inhibitory concentration In minimum inhibitory concentration dilute on a log 2 scale each antimicrobial agent in broth to provide a range of concentrations and to inoculate each tube or, if a micro plate is used, each well containing the antimicrobial agent in broth with a standardized suspension of the microorganism to be tested. The lowest concentration of antimicrobial agent that inhibits the growth of the microorganism is the minimal inhibitory concentration (MIC). The MIC and the zone diameter of inhibition are inversely correlated . In other words, the more susceptible the microorganism is to the antimicrobial agent, the lower the MIC and the larger the zone of inhibition. Conversely, the more resistant the microorganism, the higher the MIC and the smaller the zone of inhibition. Dr.T.V.Rao MD 21Slide 22: Minimum Inhibitory Concentration (MIC) : Principle: The tube dilution test is the standard method for determining levels of resistance to an antibiotic. Serial dilutions of the antibiotic are made in a liquid medium which is inoculated with a standardized number of organisms and incubated for a prescribed time. The lowest concentration of antibiotic preventing appearance of turbidity is considered to be the minimal inhibitory concentration (MIC). Dr.T.V.Rao MD 22Slide 23: Different concentrations of Gentamycin in Nutrient broth: Conc. in mcg/ml 0.1 0.2 0.4 0.8 1.6 3.1 Gentamicin, generally considered a bacteriocidal antibiotic, for this bacterium, has an MIC of 0.8 mcg/ml Dr.T.V.Rao MD 23Slide 24: Different concentrations of Tetracycline in Nutrient broth : Conc. in mcg/ml 0.1 0.2 0.4 0.8 1.6 3.1 6.3 12.5 Tetracycline, generally considered a bacteriostatic antibiotic, for this bacterium, has an MIC of 1.6 mcg/ml Dr.T.V.Rao MD 24Minimum inhibitory concentration test: Minimum inhibitory concentration test Figure 10.10 Dr.T.V.Rao MD 25Interpretation: The main concept is the “clinical categorisation" Strains are sorted according to level of Minimal Inhibitory Concentration (MIC) versus reference breakpoints c and C are the minor and major breakpoints Susceptible Intermediate Resistant MIC < c ≤ MIC < C ≤ MIC Interpretation Dr.T.V.Rao MD 26Understanding breakpoints: Understanding breakpoints Words of laboratory specialists It is not possible to work alone Breakpoints are the expression of a consensus among the scientific community at a given time in a country Breakpoints are determined using two approaches Pharmacological concept Epidemiological concept Dr.T.V.Rao MD 27The epidemiological concept for breakpoints : MIC c Wild type Inherited resistance mechanism C The epidemiological concept for breakpoints Dr.T.V.Rao MD 28The pharmacological concept for breakpoints: The pharmacological concept for breakpoints The concentration range tested for a drug and the interpretative criteria for various categories are based on extensive studies that correlate with Serum achievable levels for each antimicrobial agent Particular resistance mechanisms Successful therapeutic outcome In practice situations the entire range may not be used for decision making and therefore the concept of breakpoint concentration Dr.T.V.Rao MD 29From breakpoints to interpretation: From breakpoints to interpretation Measuring antimicrobial sensitivity of a strain isolated from a patient, to determine its status as S, I or R is an individual problem Defining the status of a bacterial species or genus is an epidemiological problem distributed across time and space that requires monitoring MIC ≤ c Sensitive strain MIC > C Intermediate strain c < MIC ≤ C Resistant strain Dr.T.V.Rao MD 30Interpreting intermediate resistance : Sometime the agent can still be used Higher doses required to ensure efficacy Agent may be efficacious if concentrated in vivo in an infected body fluid (e.g., urine) Sometimes there is uncertainty Intermediate resistance may represent a “buffer” zone that prevents strains with borderline susceptibility from being incorrectly categorized as resistant Interpreting intermediate resistance Dr.T.V.Rao MD 31Common interpretation problems: Common interpretation problems Results depends on the technique used Many factors influence results Lack of standardization of the inoculums Thickness and quality of the culture media Quality and conservation of the disks Quality control with standardized strains Condition and duration of incubation Dr.T.V.Rao MD 32What is e-test: E-test is based on arraying a concentration gradient of each antibiotic on a polymer strip. Concentration values are marked on the other side of the strip so that one can easily locate corresponding concentrations. E-strips, also known as “epsilometers”, are commercially prepared by micro dispersing robotic machines that can deliver Nano liter volumes of antibiotic concentration along the strip. What is e-test Dr.T.V.Rao MD 33E-test: E-test Plastic strips with a predefined gradient of One antibiotic One antifungal Only one manufacturer One strip per antibiotic Wide range of antibiotics Easy to use Storage at -20°C Short shelf life, expensiveAn E-test combines aspects of Kirby-Bauer and MIC tests: An E-test combines aspects of Kirby-Bauer and MIC tests Figure 10.11 Dr.T.V.Rao MD 35Reading E-tests: Reading E-tests Susceptible < 1 Resistant > 4 ug/ml Ciprofloxacin for Yersinia pestis Intermediate 1-4 ug/ml Upper reading Dr.T.V.Rao MD 36Common interpretation problems: Problems with E-test reading Common interpretation problems Dr.T.V.Rao MD 37New Liofilchem MIC Test Strips : The Liofilchem MIC Test Strip is a quantitative assay for determining the Minimum Inhibitory Concentration (MIC) of antimicrobial agents against microorganisms to indicate appropriate patient treatment and for identifying resistance patterns New Liofilchem MIC Test Strips Dr.T.V.Rao MD 38New Liofilchem MIC Test Strips : When the Liofilchem MIC Test Strip is applied onto an inoculated agar surface, the preformed exponential gradient of antimicrobial agent is transferred into the agar matrix. After 18 hours incubation or longer, a symmetrical inhibition ellipse centered along the strip is formed. The MIC is read directly from the scale in terms of µg/mL, at the point where the edge of the inhibition ellipse intersects with the MIC Test Strip. New Liofilchem MIC Test Strips Dr.T.V.Rao MD 39M.I.C.Evaluators (M.I.C.E.) Simple, Convenient Method for Accurate MIC Values: Oxoid has l improved M.I.C.Evaluator™ (M.I.C.E.™) strips , a product range for the accurate determination of minimum inhibitory concentration (MIC) values. The new distinctive gradient format of M.I.C.E. strips provides an excellent contrast with agar and the increased font size makes reading easier. M.I.C.Evaluators (M.I.C.E.) Simple , Convenient Method for Accurate MIC Values Dr.T.V.Rao MD 40Role of NCDC ( INDIA ) in containment of Antimicrobial resistance (AMR): Role of NCDC ( INDIA ) in containment of Antimicrobial resistance (AMR) Antimicrobial resistance in pathogens causing important communicable diseases has become a matter of great public health concern globally including our country. Resistance has emerged even to newer, more potent antimicrobial agents like carbapenems. The factors responsible for this are widespread use and availability of practically all the antimicrobials across the counter meant for human, animal and industrial consumption. There are definite policies /guidelines for appropriate use of antimicrobials at national level in specific national health programme being run in the country Dr.T.V.Rao MD 41Implementation of WHONET CAN HELP TO MONITOR RESISTANCE: Dr.T.V.Rao MD 42 Implementation of WHONET CAN HELP TO MONITOR RESISTANCE Legacy computer systems, quality improvement teams, and strategies for optimizing antibiotic use have the potential to stabilize resistance and reduce costs by encouraging heterogeneous prescribing patterns and use of local susceptibility patterns to inform empiric treatment.Antibiotic Resistance a concern to humanity ???: Antibiotic Resistance a concern to humanity ??? 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