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 (http://w3.whosea.org/)
Set 1 Set 2
Staphylococcus Benzyl penicillin Gentamicin
Oxacillin Amikacin
Erythromycin Co-trimoxazole
Tetracycline Clindamycin
Chloramphenicol
Intestinal Ampicillin Norfloxacin
Chloramphenicol
Co-trimoxazole
Nalidixic acid
Tetracycline
Enterobacteriaceae Sulfonamide Norfloxacin
Urinary Trimethoprim Chloramphenicol
Co-trimoxazole Gentamicin
Ampicillin
Nitrofurantoin
Nalidixic acid
Tetracycline
Blood and tissues Ampicillin Cefuroxime
Chloramphenicol Ceftriaxone
Cotrimoxazole Ciprofloxacin
Tetracycline Piperacillin
Gentamicin Amikacin
Pseudomonas 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 test
128 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 2
64 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), Incubate
35 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’s
either one or the other.

30. Factors Affecting Size of Zone of
Inhibition
See 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