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Antimicrobial susceptibility test and assay bls 206

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Antimicrobial susceptibility test and assay bls 206

  1. 1. Antimicrobial Susceptibility Test and Assay Hoza, A.S BLS 206
  2. 2. Aims• be able to describe: – The methods of antimicrobial susceptibility testing – Factors affecting antimicrobial activity – Quality assurance of antibiotic susceptibility testing
  3. 3. contents• Introduction• Antimicrobial Susceptibility Test and Assay – Dilution methods – Disc diffusion method – Factors affecting size of zone of inhibition• Quality Assurance in Antibiotic Susceptibility Testing
  4. 4. Introduction• Susceptibility test, main purposes: – As a guide for treatment • Sensitivity of a given micro-organism to known conc. of drugs • Its concentration in body fluids or tissues – As an epidemiological tool • The emergence of resistant strains of major pathogens (e. g. Shigellae, Salmonella typhi, Mycobactrium tuberculosis) • Continued surveillance of the susceptibility pattern of the prevalent strains (e. g. Staphylococci, Mycobactrium tuberculosis, Gram-negative bacilli)
  5. 5. Introduction• Methods for antimicrobial susceptibility testing – Indirect method • cultured plate from pure culture – Direct method • Pathological specimen • e.g. urine, a positive blood culture, or a swab of pus
  6. 6. Introduction Antimicrobial agents commonly used to treat systemic infection
  7. 7. Introduction• Inoculum preparation• - Number of test organisms can be determined using different methods: – Direct count (Microscopic examination) – The optical density (OD) at 600 nm (Spectrophotometry) – Plate count: making dilution first – Turbidity standard (McFarland)
  8. 8. Introduction Drugs for routine susceptibility tests:  Set 1: the drugs that are available in most hospitals and for which routine testing should be carried out for every strain  Set 2: the drugs that are tested only: ▪ at the special request of the physician/ veterinarian ▪ or when the causative organism is resistant to the first- choice drugs ▪ or when other reasons (allergy to a drug, or its unavailability) make further testing justifiable
  9. 9. Table 1: Basic sets of drugs for routine susceptibility tests ( Set 1 Set 2Staphylococcus Benzyl penicillin Gentamicin Oxacillin Amikacin Erythromycin Co-trimoxazole Tetracycline Clindamycin ChloramphenicolIntestinal Ampicillin Norfloxacin Chloramphenicol Co-trimoxazole Nalidixic acid TetracyclineEnterobacteriaceae Sulfonamide NorfloxacinUrinary Trimethoprim Chloramphenicol Co-trimoxazole Gentamicin Ampicillin Nitrofurantoin Nalidixic acid TetracyclineBlood and tissues Ampicillin Cefuroxime Chloramphenicol Ceftriaxone Cotrimoxazole Ciprofloxacin Tetracycline Piperacillin Gentamicin AmikacinPseudomonas aeruginosa Piperacillin Amikacin Gentamicin Tobramycin
  10. 10. Antimicrobial Susceptibility Testing• Dilution method – vary amount of antimicrobial substances incorporated into liquid or solid media – followed by inoculation of test bacteria• Diffusion method – 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
  11. 11. Dilution Method• Broth dilution/ Agar dilution methods• Permit quantitative results: – Indicating amount of a given drug necessary to inhibit (bacteriostatic activity) or kill (bactericidal activity) the microorganisms tested• Minimum Inhibition Concentration (MIC)• Minimum Bactericidal Concentration (MBC)
  12. 12. Dilution Method• Minimum Inhibition Concentration (MIC) – The lowest concentration of antimicrobial agent that inhibits bacterial growth/ multiplication• Minimum Bactericidal Concentration (MBC) or Minimum Lethal Concentration (MLC) – The lowest concentration of antimicrobial agent that allows less than 0.1% of the original inoculum to survive
  13. 13. Broth Dilution Method• Procedure – Making dilutions (2-fold) of antibiotic in broth • Mueller-Hinton, Tryptic Soy Broth – Inoculation of bacterial inoculum, incubation, overnight • Controls: no inoculum, no antibiotic – Turbidity visualization  MIC – Subculturing of non-turbid tubes, overnight – Growth (bacterial count)  MBC
  14. 14. Broth Dilution Method Day 1 Add 1 ml of test128 64 32 16 8 4 2 C1 C2 bacteria (1*106 CFU/ml) to tubes containing 1 ml broth and concentration of antibiotic (mg/l) Controls:64 32 16 8 4 2 1 C1 C2 C1 = No antibiotic, check viability on agar plates immediately Bacterial conc.= 5*105 CFU/ml Incubate 35 oC, over night C2 = No test bacteria
  15. 15. Broth Dilution Method Day 264 32 16 8 4 2 1 C1 C2 Record visual turbidity Subculture non-turbid tubes to agar plates (use 0.01 ml standard loop)0.01 ml (spread plate), Incubate35 oC, o/n MIC = 16 mg/ml Day 3 Determine CFU on plates: At 16 mg/ = 700 CFU/ml > 64 32 16 0.1% of 5*105 CFU/ml MBC = 32 mg/ml
  16. 16. Broth Dilution Method• 100% of original bacterial conc. – = 5*105 CFU/ml• 0.1% – = [(5*105)*0.1]/100 CFU/ml – = 500 CFU/ml• The bacteria count should be less than 5 CFU on agar plate subcultured with 0.01 ml – 500*0.01 = 5 CFU
  17. 17. Broth Dilution Method• Disadvantages : – Only one antibiotic & one organism can be tested each time – Time-consuming• Solutions?? – Agar dilution method – Disc diffusion method – Microbroth dilution method
  18. 18. Microbroth Dilution Method• Microdilution plates: – “Microdilution/ Microbroth dilutions” – 96 wells/ plate: simultaneously performed with many tests organisms/ specimens, less reagent required• Manually prepared• Commercially prepared – Frozen or Dried/ lyophilized – Consistent performance but high cost – May suffer from degradation of antibiotic during shipping and storage
  19. 19. Microbroth Dilution Method• Visualize turbidity – Light box/ mirror reader – Automated reader
  20. 20. Agar Dilution Method• Procedure – Making dilutions of antimicrobial agent in melted media and pouring plates • One concentration of antibiotic/ plate • Possible for several different strains/plate 64 ug/ml 32 ug/ml 16 ug/ml
  21. 21. Agar Dilution Method• Procedure – Inoculation of bacterial inoculum (McFarland No. 0.5) • Using a replicating inoculator device called “A Steers- Foltz replicator” • Delivers 0.001 ml of bacterial inoculum – Incubation – Spot of growth MIC
  22. 22. Diffusion Method• Disc diffusion method : The Kirby-Bauer test – Antibiotic-impregnated filter disc* – Susceptibility test against more than one antibiotics by measuring size of “inhibition zone ” – 1949: Bondi and colleagues paper disks – 1966: Kirby, Bauer, Sherris, and Tuck  filter paper disks • Demonstrated that the qualitative results of filter disk diffusion assay correlated well with quantitative results from MIC tests
  23. 23. Disc Diffusion Method
  24. 24. Disc Diffusion Method• Procedure (Modified Kirby-Bauer method: National Committee for Clinical Laboratory Standards. NCCLS) – Prepare applx. 108 CFU/ml bacterial inoculum in a saline or tryptic soy broth tube (TSB) or Mueller- Hinton broth (5 ml) • Pick 3-5 isolated colonies from plate • Adjust the turbidity to the same as the McFarland No. 0.5 standard.* – Streak the swab on the surface of the Mueller-Hinton agar (3 times in 3 quadrants) – Leave 5-10 min to dry the surface of agar
  25. 25. Disc Diffusion Method
  26. 26. Disc Diffusion Method Bacterial growth• Procedure (cont.) – Place the appropriate drug- impregnated disc on the surface of the inoculated agar plate – Invert the plates and incubate them at 35 oC, o/n (18-24 h) – Measure the diameters of inhibition zone in mm
  27. 27. Disc Diffusion Method• Measurement of the diameters of inhibition zone – Measure from the edge where the growth starts, BUT there are three exceptions • With sulfonamides and co-trimoxazole, ignore slight growth within the zone • Certain Proteus spp. may swarm into the area of inhibition • 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
  28. 28. Disc Diffusion Method• Interpretation of results – By comparing with the diameters with “standard tables” – Susceptible – Intermediate susceptible • Low toxic antibiotics: Moderate susceptible • High toxic antibiotics: buffer zone btw resistant and susceptible – Resistant
  29. 29. Come on, come on, it’seither one or the other.
  30. 30. Factors Affecting Size of Zone of InhibitionSee Table• Inoculum density • Larger zones with light inoculum and vice versa • If after application of disc,• Timing of disc the plate is kept for longer application time at room temperature, small zones may form• Temperature of • Larger zones are seen incubation with temperatures < 35 oC• Incubation time • Ideal 16-18 hours; less time does not give reliable results
  31. 31. Factors Affecting Size of Zone of Inhibition• Size of the plate • Smaller plates accommodate less number of discs• Depth of the agar • Thin media yield medium (4 mm) excessively large inhibition zones and vice versa• Proper spacing of the discs (2.5 • Avoids overlapping of cm) zones
  32. 32. Factors Affecting Size of Zone of Inhibition• Potency of • Deterioration in contents antibiotic discs leads to reduced size• Composition of • Affects rate of growth, medium diffusion of antibiotics and activity of antibiotics• Acidic pH of • Tetracycline, novobiocin, medium methicillin zones are larger• Alkaline pH of • Aminoglycosides, medium erythromycin zones are larger• Reading of zones • Subjective errors in determining the clear edge
  33. 33. Quality Assurance in Antibiotic Susceptibility Test– Medium: Mueller-Hinton agar plates • Enterococcus faecalis (ATCC 29212 or 33l86) and a disc of co-trimoxazole 20 mm in diameter of the inhibition zone– Procedure: Modified Kirby-Bauer method recommended by National Committee on Clinical Laboratory Services (NCCLS)– Susceptibility test with quality control strains
  34. 34. Quality Assurance in Antibiotic Susceptibility Test• Media recommended for test of fastidious bacteria
  35. 35. Quality Assurance in Antibiotic Susceptibility Test• Media recommended for test of fastidious bacteria
  36. 36. Quality Assurance in Antibiotic Susceptibility Test• Susceptibility test with quality control strains• for every new batch of Mueller-Hinton agar – Staphylococcus aureus (ATCC 25923) – Escherichia coli (ATCC 25922) – Pseudomonas aeruginosa (ATCC 27853)
  37. 37. Quality Assurance in Antibiotic Susceptibility Test• Salient features of quality control – Use antibiotic discs of 6 mm diameter – Use correct content of antimicrobial agent per disc – Store supply of antimicrobial discs at -20 oC – Use Mueller-Hinton medium for antibiotic sensitivity determination – Use appropriate control cultures – Use standard methodology for the test
  38. 38. Quality Assurance in Antibiotic Susceptibility Test• Salient features of quality control – Use coded strains from time to time for internal quality control – Keep the antibiotic discs at room temperature for one hour before use – Incubate the sensitivity plates for 16-18 hours before reporting – Incubate the sensitivity plates at 35oC – Space the antibiotic discs properly to avoid overlapping of inhibition zone
  39. 39. Quality Assurance in Antibiotic Susceptibility Test• Salient features of quality control – Use inoculum size that produces ‘near confluent’ growth – Ensure even contact of the antibiotic disc with the inoculated medium – Measure zone sizes precisely – Interpret zone sizes by referring to standard charts
  40. 40. Quality Assurance in Antibiotic Susceptibility Test• Frequency of quality control test (Fig 1.)
  41. 41. Antimicrobial Gradient Strip• E-Test – Antibiotic was applied to one side – Interpretive scale printed on another side – The strip is placed on the surface of agar that has been inoculated with a lawn of test bacteria
  42. 42. Antimicrobial Gradient Strip• E-Test – MIC = The point (read from scale) where the zone of inhibition intersect the strip MIC
  43. 43. Serum Susceptibility Tests• To determine drug concentration in the patient’s serum = MIC*SIT – The Serum Inhibitory Titer (SIT) • The highest dilution of patient’s serum that inhibit bacteria• To determine the ability of drug in the patient’s serum to kill bacteria – The Serum Bactericidal Level (SBL) • The lowest dilution of patient’s serum that kills bacteria
  44. 44. Activity of Combined Drugs• The combination of drugs used when: – Serious infection – Organisms with high rate of resistance • E.g. Mycobacterium tuberculosis – In immunosuppressive patients• “Synergistic” – Additive effect: increase in activity level• “Antagonistic” – Interfere effect: reduce activity level
  45. 45. Activity of Combined Drugs• “Synergistic” – E.g. aminoglycosides and penicillins• “Antagonistic” – e. g. Penicillins and bacteriostatic drugs such as tetracyclines are antagonistic, since penicillins require actively growing cells
  46. 46. Antibiotic resistant bacteria• Nosocomial infection / Hospital-acquired – ESBL (Extended beta-lactamase) – MRSA (Methicillin resistant Staphylococcus aureus) Oxacillin – PRSP (Penicillin resistant Streptococcus pneumoniae)  Oxacillin •Combined drug assay •Amoxicillin/ Clavulanic acid (AMC) •ESBL producing strain
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