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Antimicrobial susceptibility test and assay bls 206


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  • 1. Antimicrobial Susceptibility Test and Assay Hoza, A.S BLS 206
  • 2. Aims• be able to describe: – The methods of antimicrobial susceptibility testing – Factors affecting antimicrobial activity – Quality assurance of antibiotic susceptibility testing
  • 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. 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. 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. Introduction Antimicrobial agents commonly used to treat systemic infection
  • 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. Microbroth Dilution Method• Visualize turbidity – Light box/ mirror reader – Automated reader
  • 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. 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. 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. Disc Diffusion Method
  • 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. Disc Diffusion Method
  • 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. 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. 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. Come on, come on, it’seither one or the other.
  • 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. 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. 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. 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. Quality Assurance in Antibiotic Susceptibility Test• Media recommended for test of fastidious bacteria
  • 35. Quality Assurance in Antibiotic Susceptibility Test• Media recommended for test of fastidious bacteria
  • 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. 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. 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. 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. Quality Assurance in Antibiotic Susceptibility Test• Frequency of quality control test (Fig 1.)
  • 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. Antimicrobial Gradient Strip• E-Test – MIC = The point (read from scale) where the zone of inhibition intersect the strip MIC
  • 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. 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. 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. 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