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    1. Antibiotic Sensitivity testing Minimum inhibitory concentrationskill based learning Dr.T.V.Rao MD Dr.T.V.Rao MD

    2. Why 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. Dr.T.V.Rao MD

    3. 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 agent. Dr.T.V.Rao MD

    4. 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 Antimicrobial resistance results from …. Dr.T.V.Rao MD

    5. Antibiotic resistant infections

    6. 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 Antimicrobial susceptibility tests Dr.T.V.Rao MD

    7. Dr.T.V.Rao MD

    8. 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 Susceptibility testing: The big gaps Dr.T.V.Rao MD

    9. The tube dilution test is standard method • The tube dilutiontest 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

    10. minimum inhibitory concentrationtest 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 Dr.T.V.Rao MD

    11. Minimum 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 Dr.T.V.Rao MD

    12. The 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. Dr.T.V.Rao MD

    13. A minimum bactericidal concentration test Dr.T.V.Rao MD Figure 10.12

    14. 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 hours later to determine what concentration inhibited the growth of the different strains of bacteria. Dr.T.V.Rao MD

    15. Tubes containing increasing antibiotic concentrations Incubation during 18 hr at 37°C Tedious MIC Dilution in liquid broth Bacterial growth Inhibition 0 (Control) 0,25 0,50 1 2 4 8 mg/l Dr.T.V.Rao MD

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    18. 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 Standard strains for quality assurance Dr.T.V.Rao MD

    19. Different standardsCLSI Standards followed all over the world Use standardized reference National Committee for Clinical Laboratory Standards (USA) Other norms • Canadian • Chinese • National Do not confuse the different tables Choose one for everything Dr.T.V.Rao MD

    20. Critical 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 Dr.T.V.Rao MD

    21. Principles of Minimum inhibitory concentration • In minimum inhibitory concentration dilute on a log2 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

    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

    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

    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

    25. Minimum inhibitory concentration test Figure 10.10 Dr.T.V.Rao MD

    26. 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 Interpretation Dr.T.V.Rao MD

    27. 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 Understanding breakpoints Dr.T.V.Rao MD

    28. The epidemiological concept for breakpoints Inherited resistance mechanism Wild type c C MIC Dr.T.V.Rao MD

    29. 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 The pharmacological concept for breakpoints Dr.T.V.Rao MD

    30. 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 From breakpoints to interpretation Dr.T.V.Rao MD

    31. Interpreting 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 Dr.T.V.Rao MD

    32. 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 Common interpretation problems Dr.T.V.Rao MD

    33. What 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. Dr.T.V.Rao MD

    34. 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, expensive

    35. An E-test combines aspects of Kirby-Bauer and MIC tests Dr.T.V.Rao MD Figure 10.11

    36. Reading E-tests Ciprofloxacin for Yersinia pestis Resistant > 4 ug/ml Intermediate 1-4 ug/ml Susceptible < 1 Upper reading Dr.T.V.Rao MD

    37. Problems with E-test reading Common interpretation problems Dr.T.V.Rao MD

    38. New 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 Dr.T.V.Rao MD

    39. New 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. Dr.T.V.Rao MD

    40. M.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. Dr.T.V.Rao MD

    41. 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

    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. Dr.T.V.Rao MD

    43. Antibiotic Resistance a concern to humanity ??? Dr.T.V.Rao MD

    44. Follow me for Articles of Interest on Microbiology .. Dr.T.V.Rao MD

    45. Created by Dr.T.V.Rao MD for ‘ e ‘ learning resources for Medical Microbiologists in the Developing World • Email • doctortvrao@gmail.com Dr.T.V.Rao MD