1. health and fitness
2. therapy

Dr.T.V.Rao MD
Dr.T.V.Rao MD
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Uses of Antibiotic Sensitivity
Testing

Antibiotic sensitivity test: A laboratory
test which determines how effective
antibiotic therapy is against a bacterial
infections.
Antibiotic sensitivity testing will control
the use of Antibiotics in clinical practice
Testing will assist the clinicians in the
choice of drugs for the treatment of
infections.
Dr.T.V.Rao MD
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What is the goal of Antibiotic
Sensitivity testing?

 The goal of antimicrobial susceptibility testing is to
predict the in vivo success or failure of antibiotic
therapy. Tests are performed in vitro, and measure
the growth response of an isolated organism to a
particular drug or drugs. The tests are performed
under standardized conditions so that the results are
reproducible. The test results should be used to
guide antibiotic choice. The results of antimicrobial
susceptibility testing should be combined with
clinical information and experience when selecting
the most appropriate antibiotic for our patients.
Dr.T.V.Rao MD
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Components of Antibiotic
Sensitivity Testing

1.The identification of relevant pathogens in
exudates and body fluids collected from
patients
2. Sensitivity tests done to determine the
degree of sensitivity or resistance of
pathogens isolated from patient to an
appropriate range of antimicrobial drugs
3. Assay of the concentration of an
administered drug in the blood or body fluid
of patient required to control the schedule of
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dosage.
4
Why Need Continues for Testing
Antibiotic Sensitivity
 Bacteria have the ability
to develop resistance
following repeated or
subclinical (insufficient)
doses, so more advanced
antibiotics and synthetic
antimicrobials are
continually required to
overcome them.
 Antibiotic sensitivity
testing is essential part of
Medical Care
Dr.T.V.Rao MD
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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. Shigella, Salmonella typhi)
 Continued surveillance of the susceptibility pattern
of the prevalent strains (e. g. Staphylococci, Gramnegative bacilli)
Dr.T.V.Rao MD
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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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What Does the Laboratory Need to Know
about Antimicrobial Susceptibility Testing (AST) ?

Which organisms to test?
What methods to use?
What antibiotics to test?
How to report results?
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Routine Susceptibility Tests
Disk diffusion
(Kirby Bauer)

Broth microdilution MIC
 NCCLS reference
method
Etest
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Preparing for Testing

 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) routinely
performed.
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Choosing the Appropriate
Antibiotic

 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
 or when the causative organism is resistant to the firstchoice drugs
 or when other reasons (allergy to a drug, or its
unavailability) 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
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

 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
 vary amount of antimicrobial substances incorporated
into liquid or solid media
 followed by inoculation of test bacteria
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Susceptibility Testing Methods

Inoculate
MH plate
Place disks
on agar plate
Incubate plate
18-24 hr, 35 C
Measure and
record zone of
inhibition around
each disk
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

Procedure (Modified Kirby-Bauer method: National
Committee for Clinical Laboratory Standards. NCCLS)
 Prepare approximately. 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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Examining purity of plate
Select the Colonies from Pure Isolates

Reflect
ed light
Dr.T.V.Rao MD
Transmitted light
17
Disk Diffusion
Test

Prepare inoculum
suspension
Prepare inoculum
Select
colonies
suspension
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Prepare the Material for
Inoculation

Standardize inoculum
Suspension as per Mac farland
standard
Mix well
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Swab the plate with optimal
sample

Remove sample
Swab plate
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Select the Disks and Apply

Select disks
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Incubate Overnight

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Disc Diffusion Method


Place the
appropriate drugimpregnated 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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Read the Results with
Precision

Transmitted
Light
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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
Dr.T.V.Rao MD
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Look at the Charts for establishing
the zones of Sensitivity

 The zone sizes are looked
up on a standardized
chart to give a result of
sensitive, resistant, or
intermediate. Many
charts have a
corresponding column
that also gives the MIC
(minimal inhibitory
concentration) for that
drug.
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Disc Diffusion Method
Reporting the Results
 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

 Larger zones with light
 Inoculum density
inoculum and vice versa
 Timing of disc application
 Temperature of incubation
 Incubation time
 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
Dr.T.V.Rao MD
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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
 Deterioration in contents leads
 Composition of
 Affects rate of growth,
 Acidic pH of medium
 Tetracycline, novobiocin,
discs
medium
 Alkaline pH of
medium
 Reading of zones
to reduced size
diffusion of antibiotics and
activity of antibiotics
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 Testing

 Visit - WHO-Regional Office for South East Asia
website
 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 Testing with Control
strains

 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
2785 )
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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
Dr.T.V.Rao MD
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Need for Modified Methods

Modified Methods in Disc diffusion for
Antibiotic sensitivity testing to be used for
detections of following bacterial isolates
1 MRSA
2 ESBL
3 Enterobacteriaceae and Gram negative
bacteria and Carbapenems resistant using
Modified Hodge test
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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
35
Antimicrobial susceptibility
testing using micro-broth
dilutions

ug/ml
64 32 16 8 4
•
•
•
•
•
•
•
•
96 well microtiter plate
•
•
•
•
•
2
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
 Sub culturing of non-turbid tubes, overnight
 Growth (bacterial count)  MBC
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Creating Dilutions

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Broth Dilution Method

128 64
64
32
32
16
16
8
8
4
4
2
2 C1 C2
1 C1 C2
Bacterial conc.= 5*105 CFU/ml
Incubate 35 oC, o/n
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
39
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
64
32
16
MIC = 16 mg/l
Day 3
Determine CFU on plates:
At 16 mg/ = 700 CFU/ml >
0.1% of 5*105 CFU/ml
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 are
Technically Difficult

Disadvantages :
Solutions??
Only one
antibiotic & one
organism can be
tested each time
Time-consuming
Dr.T.V.Rao MD
 Agar dilution
method
 Disc diffusion
method
 Micro broth dilution
method
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Micro broth Dilution
Method
 Micro dilution plates:
 “Micro dilution/ Micro broth 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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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 uGu/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 SteersFoltz replicator”
Delivers 0.001 ml of bacterial inoculum
Incubation
Spot of growth
MIC
32 ug/ml
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Minimal inhibitory concentration

The lowest
concentration of
antimicrobial agent
that inhibits the
growth of a
bacterium
Interpret:
 Susceptible
 Intermediate
 Resistant
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Clinical Conditions when MICs are
Useful

Endocarditis
Meningitis
Septicemia
Osteomyelitis
Immunosuppressed patients (HIV, cancer,
etc.)
Prosthetic devices
Patients not responding despite “S” Reports
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Inoculum Preparation
MIC Testing

(NCCLS Reference Method)
Standardize
inoculum
suspension
Final inoculum
concentration
3 – 5 x 105 CFU/ml
(3 – 5 x 104
CFU/well)
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Select Micro titration plate and
prepare optimal inoculum

Micro dilution
MIC tray
Prepare inoculum
suspension
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Dilute & mix inoculum
suspension

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Pour inoculum into reservoir and
inoculate MIC tray

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Incubate overnight
Do not forget to check the purity of Inoculum

Inoculate
purity plate
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Optimal Use of Purity Plates

Sub final test suspension to non-selective medium
(after inoculating MIC test)
Streak for isolation (avoid several specimens per
plate - may not reveal contaminants if no isolated
colonies)
Examine before reading MIC (usually at 16-20 h)
Re-incubate if Antibiograms questionable
Read
MICs
- +
0.51
2
4
8
16
32
64
>6
4
>6
4
The gradient technique,
Etest®

Etest is a well established
AST method in
microbiology laboratories
around the world. The Etest
technique comprises a
predefined gradient of
antibiotic concentrations on
a plastic strip, and can be
used to determine the
Minimum Inhibitory
Concentration (MIC) of
antibiotics, antifungal agents
and antimycobacterial
agents.
Dr.T.V.Rao MD
55
E test – MIC Reports are helpful in
Critical management decisions

 Quantitative MIC data
is a prerequisite for the
management of critical
infections, including
sepsis, especially
among critical care
patients. Etest is
particularly valuable in
such situations, when
on-scale MICs are
needed for treatment
Dr.T.V.Rao MD
decisions.
56
Antimicrobial Gradient Testing
E-test®

Read plates
after
recommended
Incubation
Read MIC
where elipse
intersects
scale
MIC of the Bacteria can be read
Directly

Dr.T.V.Rao MD
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MIC on a strip
abbiodisk.com
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
Technically Demanding
5-Jan-06
Chiang Mai University
60
Antibiotic Sensitivity testing
can be done with automation

Dr.T.V.Rao MD
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VITEK 2 Automates Reporting
of Resistance

 Integrated in the VITEK 2
system is the Advanced
Expert System (AES™), a
software which validates
and interprets susceptibility
test results, and detects
antibiotic resistance
mechanisms. The AES
Expert System is the most
developed software system
in this field, and is capable
of identifying even
emerging and low-level
resistance.
Dr.T.V.Rao MD
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What is the Role of
Microbiology Departments

 Each laboratory should have a staff member
with the time, interest, and expertise to provide
leadership in antibiotic testing and resistance.
This person would read relevant publications,
network with other laboratories, and evaluate
potentially useful tests to detect new forms of
resistance before new CLSI-recommended
tests become available”
 - Ken Thomson, Emerging Infect. Dis., 2001
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References

1Usanee Anukool (Ph.D.) Clinical Microbiology,AMS,
Chiang Mai University
2National Committee For Clinical Laboratory Standards. 1998.
NCCLS document M100 - S8 . Performance Standards for
Antimicrobial Susceptibility Testing. 8th edition, NCCLS, Waynae,
Pa.
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
Created by Dr.T.V.Rao MD for ‘e’
learning resources for Microbiologists
in Developing World
 Email
 [email protected]
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