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