Antimicrobial susceptibility test and assay bls 209


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Antimicrobial susceptibility test and assay bls 209

  1. 1. Antimicrobial Susceptibility Test and Assay Hoza, A.S BLS 206
  2. 2. Aims <ul><li>be able to describe: </li></ul><ul><ul><li>The methods of a nti microbial s usceptibility t est ing </li></ul></ul><ul><ul><li>Factors affecting antimicrobial activity </li></ul></ul><ul><ul><li>Quality assurance of antibiotic s usceptibility t est ing </li></ul></ul>
  3. 3. contents <ul><li>Introduction </li></ul><ul><li>Anti microbial Susceptibility Test and Assay </li></ul><ul><ul><li>Dilution methods </li></ul></ul><ul><ul><li>Disc diffusion method </li></ul></ul><ul><ul><li>Factors affecting size of zone of inhibition </li></ul></ul><ul><li>Quality Assurance in Antibiotic Susceptibility Testing </li></ul>
  4. 4. Introduction <ul><li>Susceptibility test, main purposes: </li></ul><ul><ul><li>As a guide for treatment </li></ul></ul><ul><ul><ul><li>Sensitivity of a given micro-organism to known conc. of drugs </li></ul></ul></ul><ul><ul><ul><li>Its concentration in body fluids or tissues </li></ul></ul></ul><ul><ul><li>As an epidemiological tool </li></ul></ul><ul><ul><ul><li>The emergence of resistant strains of major pathogens (e. g. Shigellae, Salmonella typhi, Mycobactrium tuberculosi s) </li></ul></ul></ul><ul><ul><ul><li>Continued surveillance of the susceptibility pattern of the prevalent strains (e. g. Staphylococci, Mycobactrium tuberculosi s, Gram-negative bacilli) </li></ul></ul></ul>
  5. 5. Introduction <ul><li>Methods for antimicrobial susceptibility testing </li></ul><ul><ul><li>Indirect method </li></ul></ul><ul><ul><ul><li>cultured plate from pure culture </li></ul></ul></ul><ul><ul><li>Direct method </li></ul></ul><ul><ul><ul><li>Pathological specimen </li></ul></ul></ul><ul><ul><ul><li>e.g. urine, a positive blood culture, or a swab of pus </li></ul></ul></ul>
  6. 6. Introduction <ul><li>Antimicrobial agents commonly used to treat systemic infection </li></ul>
  7. 7. Introduction <ul><li>Inoculum preparation </li></ul><ul><li>- Number of test organisms can be determined using different methods: </li></ul><ul><ul><li>Direct count (Microscopic examination) </li></ul></ul><ul><ul><li>The optical density (OD) at 600 nm (Spectrophotometry) </li></ul></ul><ul><ul><li>Plate count: making dilution first </li></ul></ul><ul><ul><li>Turbidity standard (McFarland) </li></ul></ul>
  8. 8. Introduction <ul><li>D rugs for routine susceptibility tests : </li></ul><ul><ul><li>Set 1 : the drugs that are available in most hospitals and for which routine testing should be carried out for every strain </li></ul></ul><ul><ul><li>S et 2 : the drugs that are tested only : </li></ul></ul><ul><ul><ul><li>at the special request of the physician / veterinarian </li></ul></ul></ul><ul><ul><ul><li>or when the causative organism is resistant to the first-choice drugs </li></ul></ul></ul><ul><ul><ul><li>or when other reasons ( allergy to a drug, or its unavailability ) make further testing justifi able </li></ul></ul></ul>
  9. 9. Table 1: Basic sets of drugs for routine susceptibility tests (http :// w3 . whosea . org / ) Set 1 Set 2 Staphylococcus Benzyl penicillin Oxacillin Erythromycin Tetracycline Chloramphenicol Gentamicin Amikacin Co-trimoxazole Clindamycin Intestinal Ampicillin Chloramphenicol Co-trimoxazole Nalidixic acid Tetracycline Norfloxacin Enterobacteriaceae Urinary Sulfonamide Trimethoprim Co-trimoxazole Ampicillin Nitrofurantoin Nalidixic acid Tetracycline Norfloxacin Chloramphenicol Gentamicin Blood and tissues Ampicillin Chloramphenicol Cotrimoxazole Tetracycline Gentamicin Cefuroxime Ceftriaxone Ciprofloxacin Piperacillin Amikacin Pseudomonas aeruginosa Piperacillin Gentamicin Tobramycin Amikacin
  10. 10. Antimicrobial Susceptibility Testing <ul><li>Dilution method </li></ul><ul><ul><li>vary amount of antimicrobial substances incorporated into liquid or solid media </li></ul></ul><ul><ul><li>followed by inoculation of test bacteria </li></ul></ul><ul><li>Diffusion method </li></ul><ul><ul><li>Put a filter disc, or a porous cup/a bottomless cylinder containing measured quantity of drugs on the a solid medium that has been seeded with test bacteria </li></ul></ul>
  11. 11. Dilution Method <ul><li>Broth dilution/ Agar dilution methods </li></ul><ul><li>Permit quantitative results: </li></ul><ul><ul><li>Indicating amount of a given drug necessary to inhibit ( bacteriostatic activity ) or kill ( bactericidal activity ) the microorganisms tested </li></ul></ul><ul><li>M inimum I nhibition C oncentration (MIC) </li></ul><ul><li>M inimum B actericidal C oncentration (MBC) </li></ul>
  12. 12. Dilution Method <ul><li>Minimum Inhibition Concentration (MIC) </li></ul><ul><ul><li>The lowest concentration of antimicrobial agent that inhibits bacterial growth/ multiplication </li></ul></ul><ul><li>Minimum Bactericidal Concentration (MBC) or Minimum Lethal Concentration (MLC) </li></ul><ul><ul><li>The lowest concentration of antimicrobial agent that allows less than 0.1% of the original inoculum to survive </li></ul></ul>
  13. 13. Broth Dilution Method <ul><li>Procedure </li></ul><ul><ul><li>Making dilutions (2-fold) of antibiotic in broth </li></ul></ul><ul><ul><ul><li>Mueller-Hinton, Tryptic Soy Broth </li></ul></ul></ul><ul><ul><li>Inoculation of bacterial inoculum, incubation, overnight </li></ul></ul><ul><ul><ul><li>Controls: no inoculum, no antibiotic </li></ul></ul></ul><ul><ul><li>Turbidity visualization  MIC </li></ul></ul><ul><ul><li>Subculturing of non-turbid tubes, overnight </li></ul></ul><ul><ul><li>Growth (bacterial count)  MBC </li></ul></ul>
  14. 14. Broth Dilution Method Day 1 Add 1 ml of test bacteria ( 1*10 6 CFU/ml ) to tubes containing 1 ml broth and concentration of antibiotic (mg/l) Controls: C1 = No antibiotic, check viability on agar plates immediately C2 = No test bacteria Bacterial conc.= 5*10 5 CFU/ml Incubate 35 o C, over night 128 64 32 16 8 4 2 C1 C2 64 32 16 8 4 2 1 C1 C2
  15. 15. Broth Dilution Method 0.01 ml (spread plate), Incubate 35 o C, o/n Day 2 Record visual turbidity Subculture non-turbid tubes to agar plates (use 0.01 ml standard loop) MIC = 16 mg/ml Day 3 Determine CFU on plates: At 16 mg/ = 700 CFU/ml > 0.1% of 5*10 5 CFU/ml MBC = 32 mg/ml 64 32 16 8 4 2 1 C1 C2 64 32 16
  16. 16. Broth Dilution Method <ul><li>100% of original bacterial conc. </li></ul><ul><ul><li>= 5*10 5 CFU/ml </li></ul></ul><ul><li>0.1% </li></ul><ul><ul><li>= [(5*10 5 )*0.1]/100 CFU/ml </li></ul></ul><ul><ul><li>= 500 CFU/ml </li></ul></ul><ul><li>The bacteria count should be less than 5 CFU on agar plate subcultured with 0.01 ml </li></ul><ul><ul><li>500*0.01 = 5 CFU </li></ul></ul>
  17. 17. Broth Dilution Method <ul><li>Disadvantages : </li></ul><ul><ul><li>Only one antibiotic & one organism can be tested each time </li></ul></ul><ul><ul><li>Time-consuming </li></ul></ul><ul><li>Solutions?? </li></ul><ul><ul><li>Agar dilution method </li></ul></ul><ul><ul><li>Disc diffusion method </li></ul></ul><ul><ul><li>Microbroth dilution method </li></ul></ul>
  18. 18. Microbroth Dilution Method <ul><li>Microdilution plates: </li></ul><ul><ul><li>“ Microdilution/ Microbroth dilutions” </li></ul></ul><ul><ul><li>96 wells/ plate: simultaneously performed with many tests organisms/ specimens, less reagent required </li></ul></ul><ul><li>Manually prepared </li></ul><ul><li>Commercially prepared </li></ul><ul><ul><li>Frozen or Dried/ lyophilized </li></ul></ul><ul><ul><li>Consistent performance but high cost </li></ul></ul><ul><ul><li>May suffer from degradation of antibiotic during shipping and storage </li></ul></ul>
  19. 19. Microbroth Dilution Method <ul><li>Visualize turbidity </li></ul><ul><ul><li>Light box/ mirror reader </li></ul></ul><ul><ul><li>Automated reader </li></ul></ul>
  20. 20. Agar Dilution Method <ul><li>Procedure </li></ul><ul><ul><li>Making dilutions of antimicrobial agent in melted media and pouring plates </li></ul></ul><ul><ul><ul><li>One concentration of antibiotic/ plate </li></ul></ul></ul><ul><ul><ul><li>Possible for several different strains/plate </li></ul></ul></ul>64 ug/ml 32 ug/ml 16 ug/ml
  21. 21. Agar Dilution Method <ul><li>Procedure </li></ul><ul><ul><li>Inoculation of bacterial inoculum (McFarland No. 0.5) </li></ul></ul><ul><ul><ul><li>Using a replicating inoculator device called “A Steers-Foltz replicator” </li></ul></ul></ul><ul><ul><ul><li>Delivers 0.001 ml of bacterial inoculum </li></ul></ul></ul><ul><ul><li>Incubation </li></ul></ul><ul><ul><li>Spot of growth </li></ul></ul>MIC
  22. 22. Diffusion Method <ul><li>Disc diffusion method : The Kirby-Bauer test </li></ul><ul><ul><li>Antibiotic-impregnated filter disc* </li></ul></ul><ul><ul><li>Susceptibility test against more than one antibiotics by measuring size of “ inhibition zone ” </li></ul></ul><ul><ul><li>1949: Bondi and colleagues  paper disks </li></ul></ul><ul><ul><li>1966: Kirby, Bauer, Sherris, and Tuck  filter paper disks </li></ul></ul><ul><ul><ul><li>Demonstrated that the qualitative results of filter disk diffusion assay correlated well with quantitative results from MIC tests </li></ul></ul></ul>
  23. 23. Disc Diffusion Method
  24. 24. Disc Diffusion Method <ul><li>Procedure (Modified Kirby-Bauer method: National Committee f or Clinical Laboratory Standards. NCCLS) </li></ul><ul><ul><li>Prepare applx. 10 8 CFU/ml bacterial inoculum in a saline or tryptic soy broth tube (TSB) or Mueller-Hinton broth (5 m l) </li></ul></ul><ul><ul><ul><li>Pick 3-5 isolated colonies from plate </li></ul></ul></ul><ul><ul><ul><li>Adjust the turbidity to the same as the McFarland No. 0.5 standard.* </li></ul></ul></ul><ul><ul><li>S treak the s wab on the surface of the Mueller-Hinton agar (3 times in 3 quadrants) </li></ul></ul><ul><ul><li>Leave 5-10 min to dry the surface of agar </li></ul></ul>
  25. 25. Disc Diffusion Method
  26. 26. Disc Diffusion Method <ul><li>Procedure (cont.) </li></ul><ul><ul><li>P lace the appropriate drug-impregnated dis c on the surface of the inoculated agar plate </li></ul></ul><ul><ul><li>Invert the plates and incubate them at 35 o C , o/n (18-24 h) </li></ul></ul><ul><ul><li>Measure the diameters of inhibition zone in mm </li></ul></ul>Bacterial growth
  27. 27. Disc Diffusion Method <ul><li>Measurement of the diameters of inhibition zone </li></ul><ul><ul><li>Measure from the edge where the growth starts, BUT there are three exceptions </li></ul></ul><ul><ul><ul><li>With sulfonamides and co-trimoxazole, ignore slight growth within the zone </li></ul></ul></ul><ul><ul><ul><li>Certain Proteus spp. may swarm into the area of inhibition </li></ul></ul></ul><ul><ul><ul><li>When beta-lactamase producing Streptococci are tested, zone of inhibition are produced with a heaped-up, clearly defined edge, regardless of the size of the inhibition zone, they should be reported as resistant </li></ul></ul></ul>
  28. 28. Disc Diffusion Method <ul><li>Interpretation of results </li></ul><ul><ul><li>By comparing with the diameters with “standard tables” </li></ul></ul><ul><ul><li>Susceptible </li></ul></ul><ul><ul><li>Intermediate susceptible </li></ul></ul><ul><ul><ul><li>Low toxic antibiotics: Moderate susceptible </li></ul></ul></ul><ul><ul><ul><li>High toxic antibiotics: buffer zone btw resistant and susceptible </li></ul></ul></ul><ul><ul><li>Resistant </li></ul></ul>
  29. 29. Come on, come on, it’s either one or the other.
  30. 30. Factors Affecting Size of Zone of Inhibition <ul><li>See Table </li></ul><ul><li>Inoculum density </li></ul><ul><li>Timing of disc application </li></ul><ul><li>Temperature of incubation </li></ul><ul><li>Incubation time </li></ul><ul><li>Larger zones with light inoculum and vice versa </li></ul><ul><li>If after application of disc, the plate is kept for longer time at room temperature, small zones may form </li></ul><ul><li>Larger zones are seen with temperatures < 35 o C </li></ul><ul><li>Ideal 16-18 hours; less time does not give reliable results </li></ul>
  31. 31. Factors Affecting Size of Zone of Inhibition <ul><li>Size of the plate </li></ul><ul><li>Depth of the agar medium (4 mm) </li></ul><ul><li>Proper spacing of the discs (2.5 cm) </li></ul><ul><li>Smaller plates accommodate less number of discs </li></ul><ul><li>Thin media yield excessively large inhibition zones and vice versa </li></ul><ul><li>Avoids overlapping of zones </li></ul>
  32. 32. Factors Affecting Size of Zone of Inhibition <ul><li>Potency of antibiotic discs </li></ul><ul><li>Composition of medium </li></ul><ul><li>Acidic pH of medium </li></ul><ul><li>Alkaline pH of medium </li></ul><ul><li>Reading of zones </li></ul><ul><li>Deterioration in contents leads to reduced size </li></ul><ul><li>Affects rate of growth, diffusion of antibiotics and activity of antibiotics </li></ul><ul><li>Tetracycline, novobiocin, methicillin zones are larger </li></ul><ul><li>Aminoglycosides, erythromycin zones are larger </li></ul><ul><li>Subjective errors in determining the clear edge </li></ul>
  33. 33. Quality Assurance in Antibiotic Susceptibility Test <ul><ul><li>Medium: Mueller-Hinton agar plates </li></ul></ul><ul><ul><ul><li>Enterococcus faecalis (ATCC 29212 or 33l86) and a disc of co-trimoxazole  20 mm in diameter of the inhibition zone </li></ul></ul></ul><ul><ul><li>Procedure: Modified Kirby-Bauer method recommended by National Committee on Clinical Laboratory Services (NCCLS) </li></ul></ul><ul><ul><li>Susceptibility test with quality control strains </li></ul></ul>
  34. 34. Quality Assurance in Antibiotic Susceptibility Test <ul><li>Media recommended for test of fastidious bacteria </li></ul>
  35. 35. Quality Assurance in Antibiotic Susceptibility Test <ul><li>Media recommended for test of fastidious bacteria </li></ul>
  36. 36. Quality Assurance in Antibiotic Susceptibility Test <ul><li>Susceptibility test with quality control strains </li></ul><ul><li>for every new batch of Mueller-Hinton agar </li></ul><ul><ul><li>Staphylococcus aureus (ATCC 25923) </li></ul></ul><ul><ul><li>Escherichia coli (ATCC 25922) </li></ul></ul><ul><ul><li>Pseudomonas aeruginosa (ATCC 27853 ) </li></ul></ul>
  37. 37. Quality Assurance in Antibiotic Susceptibility Test <ul><li>Salient features of quality control </li></ul><ul><ul><li>Use antibiotic discs of 6 mm diameter </li></ul></ul><ul><ul><li>Use correct content of antimicrobial agent per disc </li></ul></ul><ul><ul><li>Store supply of antimicrobial discs at -20 o C </li></ul></ul><ul><ul><li>Use Mueller-Hinton medium for antibiotic sensitivity determination </li></ul></ul><ul><ul><li>Use appropriate control cultures </li></ul></ul><ul><ul><li>Use standard methodology for the test </li></ul></ul>
  38. 38. Quality Assurance in Antibiotic Susceptibility Test <ul><li>Salient features of quality control </li></ul><ul><ul><li>Use coded strains from time to time for internal quality control </li></ul></ul><ul><ul><li>Keep the antibiotic discs at room temperature for one hour before use </li></ul></ul><ul><ul><li>Incubate the sensitivity plates for 16-18 hours before reporting </li></ul></ul><ul><ul><li>Incubate the sensitivity plates at 35 o C </li></ul></ul><ul><ul><li>Space the antibiotic discs properly to avoid overlapping of inhibition zone </li></ul></ul>
  39. 39. Quality Assurance in Antibiotic Susceptibility Test <ul><li>Salient features of quality control </li></ul><ul><ul><li>Use inoculum size that produces ‘near confluent’ growth </li></ul></ul><ul><ul><li>Ensure even contact of the antibiotic disc with the inoculated medium </li></ul></ul><ul><ul><li>Measure zone sizes precisely </li></ul></ul><ul><ul><li>Interpret zone sizes by referring to standard charts </li></ul></ul>
  40. 40. Quality Assurance in Antibiotic Susceptibility Test <ul><li>Frequency of quality control test (Fig 1.) </li></ul>
  41. 41. Antimicrobial Gradient Strip <ul><li>E-Test </li></ul><ul><ul><li>Antibiotic was applied to one side </li></ul></ul><ul><ul><li>Interpretive scale printed on another side </li></ul></ul><ul><ul><li>The strip is placed on the surface of agar that has been inoculated with a lawn of test bacteria </li></ul></ul>
  42. 42. <ul><li>E-Test </li></ul><ul><ul><li>MIC = The point (read from scale) where the zone of inhibition intersect the strip </li></ul></ul>MIC Antimicrobial Gradient Strip
  43. 43. Serum Susceptibility Tests <ul><li>To determine drug concentration in the patient’s serum = MIC*SIT </li></ul><ul><ul><li>The Serum Inhibitory Titer ( SIT ) </li></ul></ul><ul><ul><ul><li>The highest dilution of patient’s serum that inhibit bacteria </li></ul></ul></ul><ul><li>To determine the ability of drug in the patient’s serum to kill bacteria </li></ul><ul><ul><li>The Serum Bactericidal Level ( SBL ) </li></ul></ul><ul><ul><ul><li>The lowest dilution of patient’s serum that kills bacteria </li></ul></ul></ul>
  44. 44. Activity of Combined Drugs <ul><li>The combination of drugs used when: </li></ul><ul><ul><li>Serious infection </li></ul></ul><ul><ul><li>Organisms with high rate of resistance </li></ul></ul><ul><ul><ul><li>E.g. Mycobacterium tuberculosis </li></ul></ul></ul><ul><ul><li>In immunosuppressive patients </li></ul></ul><ul><li>“ Synergistic” </li></ul><ul><ul><li>Additive effect: increase in activity level </li></ul></ul><ul><li>“ Antagonistic” </li></ul><ul><ul><li>Interfere effect: reduce activity level </li></ul></ul>
  45. 45. Activity of Combined Drugs <ul><li>“ Synergistic” </li></ul><ul><ul><li>E.g. aminoglycosides and penicillins </li></ul></ul><ul><li>“ Antagonistic” </li></ul><ul><ul><li>e. g. Penicillins and bacteriostatic drugs such as tetracyclines are antagonistic, since penicillins require actively growing cells </li></ul></ul>
  46. 46. Antibiotic resistant bacteria <ul><li>Nosocomial infection / Hospital-acquired </li></ul><ul><ul><li>ESBL (Extended beta-lactamase) </li></ul></ul><ul><ul><li>MRSA (Methicillin resistant Staphylococcus aureus )  Oxacillin </li></ul></ul><ul><ul><li>PRSP (Penicillin resistant Streptococcus pneumoniae )  Oxacillin </li></ul></ul><ul><li>Combined drug assay </li></ul><ul><ul><li>Amoxicillin/ Clavulanic acid (AMC) </li></ul></ul><ul><ul><li>ESBL producing strain </li></ul></ul>