Antimicrobial susceptibility test and assay bls 206
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Transcript of Antimicrobial susceptibility test and assay bls 206
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Antimicrobial Susceptibility Test and Assay
Hoza, A.S
BLS 206
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Aims
• be able to describe:– The methods of antimicrobial susceptibility testing
– Factors affecting antimicrobial activity
– Quality assurance of antibiotic susceptibility testing
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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
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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)
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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
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Introduction
Antimicrobial agents commonly used to treat systemic infection
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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)
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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 itsunavailability) make further testing justifiable
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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
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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
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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)
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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
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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
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Broth Dilution Method
128 64 32 16 8 4 2 C1 C2
64 32 16 8 4 2 1 C1 C2
Day 1
Add 1 ml of test
bacteria (1*106
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*105 CFU/ml
Incubate 35 oC, over night
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Broth Dilution Method
64 32 16 8 4 2 1 C1 C2
0.01 ml (spread plate), Incubate 35 oC, o/n
64 32 16
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*105 CFU/ml
MBC = 32 mg/ml
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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
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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
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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
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Microbroth Dilution Method
• Visualize turbidity
– Light box/ mirror reader
– Automated reader
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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
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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
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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
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Disc Diffusion Method
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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
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Disc Diffusion Method
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Disc Diffusion Method
• 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
Bacterial growth
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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
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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
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Come on, come on, it’s
either one or the other.
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Factors Affecting Size of Zone of
Inhibition
See Table
• Inoculum density
• Timing of disc application
• Temperature of incubation
• Incubation time
• Larger zones with light inoculum and vice versa
• If after application of disc, the plate is kept for longer time at room temperature, small zones may form
• Larger zones are seen with temperatures < 35 oC
• Ideal 16-18 hours; less time does not give reliable results
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Factors Affecting Size of Zone of
Inhibition
• Size of the plate
• Depth of the agar medium (4 mm)
• Proper spacing of the discs (2.5 cm)
• Smaller plates accommodate less number of discs
• Thin media yield excessively large inhibition zones and vice versa
• Avoids overlapping of zones
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Factors Affecting Size of Zone of
Inhibition
• Potency of antibiotic discs
• Composition of medium
• Acidic pH of medium
• Alkaline pH of medium
• Reading of zones
• Deterioration in contents leads to reduced size
• Affects rate of growth, diffusion of antibiotics and activity of antibiotics
• Tetracycline, novobiocin, methicillin zones are larger
• Aminoglycosides, erythromycin zones are larger
• Subjective errors in determining the clear edge
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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
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Quality Assurance in Antibiotic Susceptibility Test
• Media recommended for test of fastidious bacteria
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Quality Assurance in Antibiotic Susceptibility Test
• Media recommended for test of fastidious bacteria
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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)
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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
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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
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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
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Quality Assurance in Antibiotic Susceptibility Test
• Frequency of quality control test (Fig 1.)
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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
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• E-Test
– MIC = The point (read from scale) where the zone
of inhibition intersect the strip
MIC
Antimicrobial Gradient Strip
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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
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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
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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
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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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