Dr.T.V.

Rao MD

Dr.T.V.Rao MD 1
 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 2
 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 3
 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
Dr.T.V.Rao MD 4
dosage.
 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 5
 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, Gram-
negative bacilli)

Dr.T.V.Rao MD 6
 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

Dr.T.V.Rao MD 7
 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?

Dr.T.V.Rao MD 8
Routine Susceptibility Tests

Disk diffusion
(Kirby Bauer)
Broth micro-
dilution MIC
 NCCLS reference
method

Etest
Dr.T.V.Rao MD 9
 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.
Dr.T.V.Rao MD 10
 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 first-
choice drugs
 or when other reasons (allergy to a drug, or its
unavailability) make further testing justified

Dr.T.V.Rao MD 11
 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

Dr.T.V.Rao MD 12
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

Dr.T.V.Rao MD 13
 Susceptibility Testing Methods


Incubate plate
Inoculate Place disks
18-24 hr, 35 C
MH plate on agar plate 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
Dr.T.V.Rao MD 15
 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

Dr.T.V.Rao MD 16
 Examining purity of plate
Select the Colonies from Pure Isolates

Reflect Transmitted light

ed light

Dr.T.V.Rao MD 17
 Disk Diffusion
Test

Prepare inoculum
suspension
Prepare inoculum
Select colonies
suspension

Dr.T.V.Rao MD 18
 Prepare the Material for
Inoculation

Standardize inoculum
Suspension as per Mac farland Mix well
standard

Dr.T.V.Rao MD 19
 Swab the plate with optimal
sample


Remove sample Swab plate

Dr.T.V.Rao MD 20
 Select the Disks and Apply


Select disks

Dr.T.V.Rao MD 21
Incubate Overnight


Dr.T.V.Rao MD 22
 Disc Diffusion Method

 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

Dr.T.V.Rao MD 23
Read the Results with
Precision

Transmitted
Light

Dr.T.V.Rao MD 24
 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 25
 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.
Dr.T.V.Rao MD 26
 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
Dr.T.V.Rao MD 27
 Factors Affecting Size of Zone
of Inhibition

 Larger zones with light
 Inoculum density inoculum and vice versa

 If after application of disc, the

 Timing of disc application plate is kept for longer time at
room temperature, small zones
may form

 Temperature of incubation  Larger zones are seen with

temperatures < 35 oC
 Incubation time
 Ideal 16-18 hours; less time
does not give reliable results
Dr.T.V.Rao MD 28
 Factors Affecting Size of Zone of
Inhibition

 Size of the plate

 Smaller plates
accommodate less
number of discs
 Depth of the agar
medium (4 mm)  Thin media yield
excessively large
inhibition zones and vice
versa

 Proper spacing of  Avoids overlapping of

the discs (2.5 cm) zones

Dr.T.V.Rao MD 29
Factors Affecting Size of Zone of
Inhibition
 Potency of antibiotic  Deterioration in contents leads
discs to reduced size

 Composition of  Affects rate of growth,

medium diffusion of antibiotics and
activity of antibiotics

 Acidic pH of medium  Tetracycline, novobiocin,

methicillin zones are larger

 Aminoglycosides,
 Alkaline pH of erythromycin zones are larger
medium
 Subjective errors in
 Reading of zones determining the clear edge

Dr.T.V.Rao MD 30
 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

Dr.T.V.Rao MD 31
 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 )

Dr.T.V.Rao MD 32
 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 33
 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
Dr.T.V.Rao MD 34
 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 2

•
• •
•
•
• • •
• • •
• •
96 well microtiter plate
 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

37
Creating Dilutions


Dr.T.V.Rao MD 38
 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
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
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

40
 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

41
 Broth Dilution Method are
Technically Difficult

Disadvantages : Solutions??
Only one  Agar dilution
antibiotic & one method
organism can be  Disc diffusion
method
tested each time
 Micro broth dilution
Time-consuming method

Dr.T.V.Rao MD 42
 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

43
 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

44
 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
32 ug/ml 45
 Minimal inhibitory concentration


The lowest
concentration of
antimicrobial agent
that inhibits the
growth of a
bacterium
Interpret:
 Susceptible
 Intermediate
 Resistant
Dr.T.V.Rao MD 46
Clinical Conditions when MICs are
Useful
Endocarditis

Meningitis
Septicemia
Osteomyelitis
Immunosuppressed patients (HIV, cancer,
etc.)
Prosthetic devices
Patients not responding despite “S” Reports
Dr.T.V.Rao MD 47
 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)
Dr.T.V.Rao MD 48
Select Micro titration plate and
prepare optimal inoculum

Prepare inoculum
suspension
Micro dilution
MIC tray

Dr.T.V.Rao MD 49
Dilute & mix inoculum
suspension


Dr.T.V.Rao MD 50
Pour inoculum into reservoir and
inoculate MIC tray


Dr.T.V.Rao MD 51
 Incubate overnight
Do not forget to check the purity of Inoculum


Inoculate
purity plate

Dr.T.V.Rao MD 52
 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 >6
4 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 56
decisions.
 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 58
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 61
 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 62
 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

Dr.T.V.Rao MD 63
 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.

Dr.T.V.Rao MD 64
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Dr.T.V.Rao MD 65
 
Created by Dr.T.V.Rao MD for „e‟
learning resources for Microbiologists
in Developing World
 Email
 doctortvrao@gmail.com

Dr.T.V.Rao MD 66