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Antimicrobial Susceptibility Test and AssayHoza, A.SBLS 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
IntroductionAntimicrobial 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-ch
oice drugs▪ or when other reasons (allergy to a drug, or its unavailabi
lity) make further testing justifiable
Table 1: Basic sets of drugs for routine susceptibility tests (http://w3.whosea.org/)
Set 1 Set 2
Staphylococcus Benzyl penicillinOxacillinErythromycinTetracyclineChloramphenicol
GentamicinAmikacinCo-trimoxazoleClindamycin
Intestinal AmpicillinChloramphenicolCo-trimoxazoleNalidixic acidTetracycline
Norfloxacin
Enterobacteriaceae
Urinary
SulfonamideTrimethoprimCo-trimoxazoleAmpicillinNitrofurantoinNalidixic acidTetracycline
NorfloxacinChloramphenicolGentamicin
Blood and tissues AmpicillinChloramphenicolCotrimoxazoleTetracyclineGentamicin
CefuroximeCeftriaxoneCiprofloxacinPiperacillinAmikacin
Pseudomonas aeruginosa PiperacillinGentamicinTobramycin
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 bacteriaBacterial 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 3Determine 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 st
andard.* – 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 sensitivit
y 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 qua
lity control– Keep the antibiotic discs at room temperature for o
ne hour before use– Incubate the sensitivity plates for 16-18 hours befor
e reporting– Incubate the sensitivity plates at 35oC– Space the antibiotic discs properly to avoid overlap
ping of inhibition zone
Quality Assurance in Antibiotic Susceptibility Test
• Salient features of quality control – Use inoculum size that produces ‘near confluent’ g
rowth– Ensure even contact of the antibiotic disc with the
inoculated medium– Measure zone sizes precisely– Interpret zone sizes by referring to standard chart
s
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