Antimicrobial Susceptibility

Test and Assay

Usanee Anukool (Ph.D.)

Clinical Microbiology, AMS,
Chiang Mai University
5 Jan 2006
Aims
 After class, students will be able to describe:
 The methods of antimicrobial susceptibility
testing

 Factors affecting antimicrobial activity

 Quality assurance of antibiotic susceptibility te

sting

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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 m.o. 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)
 Continued surveillance of the susceptibility
pattern of the prevalent strains (e. g.
Staphylococci, 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 a
nd 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
 or when the causative organism is resistant to th
e first-choice drugs
 or when other reasons (allergy to a drug, or its u
navailability) make further testing justified
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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 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

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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

Day 1

128 64 32 16 8 4 2 C1 C2
Add 1 ml of test bacteria
(1*106 CFU/ml) to tubes
containing 1 ml broth and
concentration of antibiotic
(mg/l)

64 32 16 8 4 2 1 C1 C2 Controls:
C1 = No antibiotic, check
Bacterial conc.= 5*105 CFU/ml viability on agar plates
immediately
Incubate 35 oC, o/n
C2 = No test bacteria
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 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/l

Day 3
Determine CFU on plates:
At 16 mg/ = 700 CFU/ml >
0.1% of 5*105 CFU/ml
64 32 16
MBC = 32 mg/l

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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 Committe
e 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 a
gar (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.)
Bacterial growth
 Place the appropriate drug-im
pregnated 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

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Disc Diffusion Method
 Measurement of the diameters of inhibition
zone
 Measure from the edge where the growth
stats, 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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Factors Affecting Size of Zone of
Inhibition
See Table 2.
 Inoculum density  Larger zones with light inoculu
m and vice versa

 If after application of disc, the pl

 Timing of disc applic
ation ate is kept for longer time at roo
m temperature, small zones ma
y form

 Temperature of incu  Larger zones are seen with tem

bation peratures < 35 oC

 Incubation time  Ideal 16-18 hours; less time do

es not give reliable results

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Factors Affecting Size of Zone of
Inhibition
 Size of the plate  Smaller plates accommodate le
ss number of discs

 Depth of the agar me  Thin media yield excessively la

dium (4 mm) rge inhibition zones and vice ve
rsa

 Proper spacing of th  Avoids overlapping of zones

e discs (2.5 cm)

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Factors Affecting Size of Zone of
Inhibition
 Potency of antibiotic  Deterioration in contents leads t
discs o reduced size

 Composition of medi  Affects rate of growth, diffusion

um of antibiotics and activity of anti
biotics

 Acidic pH of medium  Tetracycline, novobiocin, methi

cillin zones are larger

 Aminoglycosides, erythromycin
 Alkaline pH of mediu zones are larger
m
 Subjective errors in determinin
 Reading of zones g the clear edge

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Quality Assurance in Antibiotic Susce
ptibility Test
 WHO-Regional Office for South East Asia
website
 Medium: Mueller-Hinton agar plates
 Enterococcus faecalis (ATCC 29212 or 33l86) an
d 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 Susce
ptibility Test
 Media recommended for test of fastidious bacteria

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Quality Assurance in Antibiotic Susce
ptibility 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

 See Table 3.

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Quality Assurance in Antibiotic Susce
ptibility 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 sensi
tivity determination
 Use appropriate control cultures
 Use standard methodology for the test

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Quality Assurance in Antibiotic Susce
ptibility Test
 Salient features of quality control
 Use coded strains from time to time for interna
l quality control
 Keep the antibiotic discs at room temperature f
or 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 ov
erlapping of inhibition zone
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Quality Assurance in Antibiotic Susce
ptibility Test
 Salient features of quality control
 Use inoculum size that produces ‘near conflue
nt’ growth
 Ensure even contact of the antibiotic disc with
the inoculated medium
 Measure zone sizes precisely
 Interpret zone sizes by referring to standard ch
arts

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Quality Assurance in Antibiotic Susce
ptibility 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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Antimicrobial Gradient Strip
 E-Test
 MIC = The point (read from scale) where the zone of
inhibition intersect the strip

MIC

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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 รื ดำแรนื
 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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References
 Brooks, G.F., J.S. Butel, S. A. Morse. 1998. Jawetz,
Melnick & Adelberg’s Medical Microbiology, 21st ed.,
Prentice Hall International Inc. USA.
 http://w3.whosea.org/en/section10/section17/
section53/section482_1787.htm
 http://w3.whosea.org/en/Section10/Section17/
Section53/Section375_1185.htm
 National Committee For Clinical Laboratory Standard
s. 1998. NCCLS document M100 - S8 . Performance
Standards for Antimicrobial Susceptibility Testing. 8th
edition, NCCLS, Waynae, Pa.

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